Patent Publication Number: US-2023139800-A1

Title: Car t cell therapies with enhanced efficacy

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of U.S. patent application Ser. No. 15/760,519 filed Mar. 15, 2018, which is a U.S. national phase application and claims the benefit of priority under 35 U.S.C. § 371 of International Application No. PCT/US2016/052260, filed Sep. 16, 2016, which claims priority to U.S. Application Ser. No. 62/220,196, filed Sep. 17, 2015, the contents of each of which are incorporated herein by reference in their entireties. 
    
    
     SEQUENCE LISTING 
     The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Sep. 19, 2022, is named N2067-709820_SL.xml and is 1,914,447 bytes in size. 
     FIELD OF THE INVENTION 
     The present invention relates generally to the use of immune effector cells (e.g., T cells, NK cells) engineered to express a Chimeric Antigen Receptor (CAR) to treat a disease associated with expression of a tumor antigen. 
     BACKGROUND OF THE INVENTION 
     Adoptive cell transfer (ACT) therapy with autologous T-cells, especially with T-cells transduced with Chimeric Antigen Receptors (CARs), has shown promise in hematologic cancer trials. There is a medical need for T cell therapies, especially CAR T cell therapies with improved efficacy. 
     SUMMARY OF THE INVENTION 
     The present invention provides compositions and methods that disrupt methylcytosine dioxygenase genes (e.g., Tet1, Tet2, Tet3), and uses of such compositions and methods for increasing the functional activities of engineered cells (e.g., gene-modified antigen-specific T cells, such as CAR T cells). In particular, the present invention provides methods and compositions for bolstering the therapeutic efficacy of chimeric antigen receptor (CAR) T cells. While not to be bound by the theory, disruption of a single allele of a Tet gene (e.g., a Tet1, Tet2, or Tet3) leads to decreased total levels of 5-hydroxymethylcytosine in association with enhanced proliferation, regulation of effector cytokine production and degranulation, and thereby increases CAR T cell proliferation and/or function. 
     Accordingly, the present invention provides a cell (e.g., a population of cells, such as a population of immune effector cells) engineered to express a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, and wherein expression and/or function of Tet1, Tet2 and/or Tet3 in said cell has been reduced or eliminated. In one embodiment, the expression and/or function of Tet2 in said cell has been reduced or eliminated. In some embodiments, the antigen-binding domain binds to a tumor antigen is selected from a group consisting of: TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, CD20, Folate receptor alpha, ERBB2 (Her2/neu), MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1/Galectin 8, MelanA/MART1, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor, Cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, and IGLL1. In one embodiment, the tumor antigen is CD19. In some embodiments, the antigen-binding domain is an antibody or antibody fragment as described in, e.g., WO2012/079000 or WO2014/153270. 
     In one aspect, the present invention provides a cell (e.g., a population of cells, such as a population of immune effector cells) engineered to express a CAR, and wherein expression and/or function of Tet1, Tet2 and/or Tet3 in said cell has been reduced or eliminated. In one embodiment, the expression and/or function of Tet2 in said cell has been reduced or eliminated. In some embodiments, the transmembrane domain of said CAR comprises: (i) an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications of an amino acid sequence of SEQ ID NO: 12, or a sequence with 95-99% identity to an amino acid sequence of SEQ ID NO: 12; or (ii) the sequence of SEQ ID NO: 12. 
     In one aspect, the present invention provides a cell (e.g., a population of cells, such as a population of immune effector cells) engineered to express a CAR, and wherein expression and/or function of Tet1, Tet2 and/or Tet3 in said cell has been reduced or eliminated. In one embodiment, the antigen binding domain of said CAR is connected to the transmembrane domain by a hinge region, wherein said hinge region comprises SEQ ID NO: 2 or SEQ ID NO: 6, or a sequence with 95-99% identity thereof. In some embodiments, the intracellular signaling domain of said CAR comprises a primary signaling domain and/or a costimulatory signaling domain, wherein the primary signaling domain comprises a functional signaling domain of a protein chosen from CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, common FcR gamma (FCER1G), FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fcgamma RIIa, DAP10, or DAP12. 
     In some embodiments, the primary signaling domain of said CAR comprises: (i) an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications of an amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 20, or a sequence with 95-99% identity to an amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 20; or (ii) the amino acid sequence of SEQ ID NO:18 or SEQ ID NO: 20. In some embodiments, the intracellular signaling domain of said CAR comprises a costimulatory signaling domain, or a primary signaling domain and a costimulatory signaling domain, wherein the costimulatory signaling domain comprises a functional signaling domain of a protein selected from the group consisting of CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, and NKG2D. 
     In some embodiments, the costimulatory signaling domain of said CAR comprises an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications of an amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 16, or a sequence with 95-99% identity to an amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 16. In some embodiments, the intracellular domain of said CAR comprises the sequence of SEQ ID NO: 14 or SEQ ID NO: 16, and the sequence of SEQ ID NO: 18 or SEQ ID NO: 20, wherein the sequences comprising the intracellular signaling domain are expressed in the same frame and as a single polypeptide chain. In some embodiments, the CAR of the present invention further comprises a leader sequence comprises the sequence of SEQ ID NO: 2. 
     In some embodiments, the immune effector cell of the present invention is a T cell or an NK cell. In some embodiments, the T cell is a CD4+ T cell, a CD8+ T cell, or a combination thereof. In one aspect, the cells of the present invention are human cells. In one aspect, the cells (e.g., engineered immune effector cells, e.g., CAR T cells) of the present invention comprise an inhibitor of Tet1, Tet2, and/or Tet3. In some embodiments, the cells of the present invention comprise a CAR, and an inhibitor of Tet1, Tet2 and/or Tet3, wherein said inhibitor is (1) a gene editing system targeted to one or more sites within the gene encoding Tet1, Tet2 and/or Tet3, or its regulatory elements, e.g., Tet2, or its regulatory elements; (2) nucleic acid encoding one or more components of said gene editing system; or (3) combinations thereof. 
     In some embodiments, the cells of the present invention comprise a CAR, and an inhibitor of Tet1, Tet2 and/or Tet3, wherein said inhibitor is a gene editing system targeted to one or more sites within the gene encoding Tet1, Tet2 and/or Tet3, or its regulatory elements, e.g., Tet2, or its regulatory elements, and wherein the gene editing system is selected from the group consisting of: a CRISPR/Cas9 system, a zinc finger nuclease system, a TALEN system and a meganuclease system. 
     In some embodiments, the cells of the present invention comprise a CAR, and an inhibitor of Tet1, Tet2 and/or Tet3, wherein said inhibitor is a gene editing system targeted to one or more sites within the gene encoding Tet1, Tet2 and/or Tet3, or its regulatory elements, e.g., Tet2, or its regulatory elements, and wherein the gene editing system binds to a target sequence in an early exon or intron of a gene encoding Tet1, Tet2 and/or Tet3, e.g., Tet2. 
     In some embodiments, the cells of the present invention comprise a CAR, and an inhibitor of Tet1, Tet2 and/or Tet3, wherein said inhibitor is a gene editing system targeted to one or more sites within the gene encoding Tet1, Tet2 and/or Tet3, or its regulatory elements, e.g., Tet2, or its regulatory elements, and wherein the gene editing system binds a target sequence of a gene encoding tet2, and the target sequence is upstream of exon 4, e.g., in exon1, exon2, or exon3, e.g. in exon 3. 
     In some embodiments, the cells of the present invention comprise a CAR, and an inhibitor of Tet1, Tet2 and/or Tet3, wherein said inhibitor is a gene editing system targeted to one or more sites within the gene encoding Tet1, Tet2 and/or Tet3, or its regulatory elements, e.g., Tet2, or its regulatory elements, and wherein the gene editing system binds to a target sequence in a late exon or intron of a gene encoding Tet1, Tet2 and/or Tet3, e.g., Tet2. 
     In some embodiments, the cells of the present invention comprise a CAR, and an inhibitor of Tet1, Tet2 and/or Tet3, wherein said inhibitor is a gene editing system targeted to one or more sites within the gene encoding Tet1, Tet2 and/or Tet3, or its regulatory elements, e.g., Tet2, or its regulatory elements, and wherein the gene editing system binds a target sequence of a gene encoding tet2, and the target sequence is downstream of exon 8, e.g., is in exon9, exon10, or exon11, e.g. is in exon 9. 
     In some embodiments, the cells of the present invention comprise a CAR, and an inhibitor of Tet1, Tet2 and/or Tet3, wherein said inhibitor is a gene editing system targeted to one or more sites within the gene encoding Tet1, Tet2 and/or Tet3, or its regulatory elements, e.g., Tet2, or its regulatory elements, and wherein the gene editing system is a CRISPR/Cas system comprising a gRNA molecule comprising a targeting sequence which hybridize to a target sequence of a Tet2 gene. In some embodiments, the targeting sequence is a targeting sequence listed in Table 3. In some embodiments, the target sequence is a targeting sequence listed in Table 5. 
     In some embodiments, the cells of the present invention comprise a CAR, and an inhibitor of Tet1, Tet2 and/or Tet3, wherein said inhibitor is an siRNA or shRNA specific for Tet1, Tet2, Tet3, or nucleic acid encoding said siRNA or shRNA. In some embodiments, the siRNA or shRNA comprises a sequence complementary to a sequence of a Tet2 mRNA, e.g., comprises a target sequence of shRNA listed in Table 4. 
     In some embodiments, the cells of the present invention comprise a CAR, and an inhibitor of Tet1, Tet2 and/or Tet3, wherein said inhibitor a small molecule. 
     In some embodiments, the cells of the present invention comprise a CAR, and an inhibitor of Tet1, Tet2 and/or Tet3, wherein the inhibitor is a protein, e.g., is a dominant negative binding partner of Tet1, Tet2, and/or Tet3 (e.g., a histone deacetylase (HDAC) that interacts with Tet1, Tet2, and/or Tet3), or nucleic acid encoding said dominant negative binding partner of Tet1, Tet2, and Tet3. 
     In some embodiments, the cells of the present invention comprise a CAR, and an inhibitor of Tet1, Tet2 and/or Tet3, wherein the inhibitor is a protein, e.g., is a dominant negative (e.g., catalytically inactive) Tet1, Tet2, or Tet3, or nucleic acid encoding said dominant negative Tet1, Tet2, or Tet3. 
     In one aspect, the present invention provides a method of increasing the therapeutic efficacy of a CAR-expressing cell, e.g., a cell of any of the previous claims, e.g., a CAR19-expressing cell (e.g., CTL019), comprising a step of decreasing the level of 5-hydroxymethylcytosine in said cell. In some embodiments, said step comprises contacting said cells with a Tet (e.g., Tet1, Tet2, and/or Tet3) inhibitor. In some embodiments, said Tet inhibitor is a Tet2 inhibitor. In some embodiments, a Tet (e.g., Tet1, Tet2, and/or Tet3) inhibitor of the present invention is selected from the group consisting of: (1) a gene editing system targeted to one or more sites within the gene encoding Tet1, Tet2, or Tet3, or its corresponding regulatory elements; (2) a nucleic acid (e.g., an siRNA or shRNA) that inhibits expression of Tet1, Tet2, or Tet3; (3) a protein (e.g., a dominant negative, e.g., catalytically inactive) Tet1, Tet2, or Tet3, or a binding partner of Tet1, Tet2, or Tet3; (4) a small molecule that inhibits expression and/or function of Tet1, Tet2, or Tet3; (5) a nucleic acid encoding any of (1)-(3); and (6) any combination of (1)-(5). In some embodiments, the Tet inhibitor of the present invention is a Tet2 inhibitor. 
     In one aspect, the present invention provides a method of increasing the therapeutic efficacy of a CAR-expressing cell, e.g., a cell of any of the previous claims, e.g., a CAR19-expressing cell (e.g., CTL019), comprising a step of decreasing the level of 5-hydroxymethylcytosine in said cell. In some embodiments, said step comprises contacting said cells with a Tet (e.g., Tet1, Tet2, and/or Tet3) inhibitor. In some embodiments, said contacting occurs ex vivo. In some embodiments, said contacting occurs in vivo. In some embodiments, said contacting occurs in vivo prior to delivery of nucleic acid encoding a CAR into the cell. In some embodiments, said contacting occurs in vivo after the cells have been administered to a subject in need thereof. 
     In one aspect, the present invention provides a method of increasing the therapeutic efficacy of a CAR-expressed cell, e.g., a cell of any of the previous claims, e.g., a CAR19-expressing cell (e.g., CTL019), comprising a step of contacting said cell with a Tet inhibitor, e.g., a Tet1, Tet2 and/or Tet3 inhibitor. In some embodiments, said Tet inhibitor is a Tet2 inhibitor. In some embodiments, a Tet (e.g., Tet1, Tet2, and/or Tet3) inhibitor of the present invention is selected from the group consisting of. (1) a gene editing system targeted to one or more sites within the gene encoding Tet1, Tet2, or Tet3, or its corresponding regulatory elements; (2) a nucleic acid (e.g., an siRNA or shRNA) that inhibits expression of Tet1, Tet2, or Tet3; (3) a protein (e.g., a dominant negative, e.g., catalytically inactive) Tet1, Tet2, or Tet3, or a binding partner of Tet1, Tet2, or Tet3; (4) a small molecule that inhibits expression and/or function of Tet1, Tet2, or Tet3; (5) a nucleic acid encoding any of (1)-(3); and (6) any combination of (1)-(5). In some embodiments, the Tet inhibitor of the present invention is a Tet2 inhibitor. 
     In one aspect, the present invention provides a method of increasing the therapeutic efficacy of a CAR-expressed cell, e.g., a cell of any of the previous claims, e.g., a CAR19-expressing cell (e.g., CTL019), comprising a step of contacting said cell with a Tet inhibitor, e.g., a Tet1, Tet2 and/or Tet3 inhibitor. In some embodiments, said step comprises contacting said cells with a Tet (e.g., Tet1, Tet2, and/or Tet3) inhibitor. In some embodiments, said contacting occurs ex vivo. In some embodiments, said contacting occurs in vivo. In some embodiments, said contacting occurs in vivo prior to delivery of nucleic acid encoding a CAR into the cell. In some embodiments, said contacting occurs in vivo after the cells have been administered to a subject in need thereof. 
     In one aspects, the present invention provides a method of treating a subject in need thereof, comprising administering to said subject an effective amount of the cells as described herein, e.g., an immune effector cell (e.g., T cell or NK cell) comprising a CAR, and, optionally, administering to said subject a Tet1, Tet2, and/or Tet3 inhibitor. In some embodiments, the subject receives a pre-treatment of the Tet1, Tet2 and/or Tet3 inhibitor, and prior to the initiation of the CAR-expressing cell therapy. In some embodiments, the subject receives concurrent treatment with a Tet1, Tet2, and/or Tet3 inhibitor and the CAR expressing cell therapy. In some embodiments, the subject receives treatment with a Tet1, Tet2, and/or Tet3 inhibitor post-CAR-expressing cell therapy. In some embodiments, the subject has a disease associated with expression of a tumor antigen, e.g., a proliferative disease, a precancerous condition, a cancer, and a non-cancer related indication associated with expression of the tumor antigen. In some embodiments, the subject has a hematologic cancer chosen from one or more of chronic lymphocytic leukemia (CLL), acute leukemias, acute lymphoid leukemia (ALL), B-cell acute lymphoid leukemia (B-ALL), T-cell acute lymphoid leukemia (T-ALL), chronic myelogenous leukemia (CML), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt&#39;s lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin&#39;s lymphoma, Hodgkin&#39;s lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, or preleukemia. 
     The present invention provides uses of the compositions and/or methods described here for treatment of cancer, wherein the cancer is selected from the group consisting of colon cancer, rectal cancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine, cancer of the esophagus, melanoma, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin&#39;s Disease, non-Hodgkin&#39;s lymphoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi&#39;s sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers, combinations of said cancers, and metastatic lesions of said cancers. 
     The present invention provides Tet1, Tet2 and/or Tet3 inhibitors for use in the treatment of a subject, wherein said subject has received, is receiving, or is about to receive therapy comprising a CAR-expressing cell. 
     The present invention further provides a method of manufacturing a CAR-expressing cell, comprising introducing nucleic acid encoding a CAR into a cell such that said nucleic acid (or CAR-encoding portion thereof) integrates into the genome of the cell within a Tet1, Tet2 and/or Tet3 gene (e.g., within an intron or exon of a Tet1, Tet2 and/or Tet3 gene), such that Tet1, Tet2 and/or Tet3 expression and/or function is reduced or eliminated. 
     The present invention further provides a method of manufacturing a CAR-expressing cell, comprising contacting said CAR-expressing cell ex vivo with a Tet1, Tet2 and/or Tet3 inhibitor. In some embodiments, the inhibitor is a Tet2 inhibitor. 
     The present invention further provides a vector comprising sequence encoding a CAR and sequence encoding a Tet inhibitor, e.g., a Tet1, Tet2, and/or Tet3 inhibitor. In some embodiments, the Tet inhibitor is a (1) a gene editing system targeted to one or more sites within the gene encoding Tet1, Tet2, or Tet3, or its corresponding regulatory elements; (2) a nucleic acid (e.g., an siRNA or shRNA) that inhibits expression of Tet1, Tet2, or Tet3; (3) a protein (e.g., a dominant negative, e.g., catalytically inactive) Tet1, Tet2, or Tet3, or a binding partner of Tet1, Tet2, or Tet3; and (4) a nucleic acid encoding any of (1)-(3), or combinations thereof. In some embodiments, the sequence encoding a CAR and the sequence encoding a Tet inhibitor are separated by a 2A site. 
     The present invention further provides a gene editing system that is specific for a sequence of a Tet gene or its regulatory elements, e.g., a Tet1, Tet2 or Tet3 gene or its regulatory elements. In some embodiments, the gene editing system is specific for a sequence of a Tet2 gene. In some embodiments, the gene editing system is (1) a CRISPR/Cas gene editing system, (2) a zinc finger nuclease system, a TALEN system and a meganuclease system. In some embodiments, the gene editing system is a CRISPR/Cas gene editing system. In some embodiments, the gene editing system comprises: a gRNA molecule comprising a targeting sequence specific to a sequence of a Tet2 gene or its regulatory elements, and a Cas9 protein; a gRNA molecule comprising a targeting sequence specific to a sequence of a Tet2 gene or its regulatory elements, and a nucleic acid encoding a Cas9 protein; a nucleic acid encoding a gRNA molecule comprising a targeting sequence specific to a sequence of a Tet2 gene or its regulatory elements, and a Cas9 protein; or a nucleic acid encoding a gRNA molecule comprising a targeting sequence specific to a sequence of a Tet2 gene or its regulatory elements, and a nucleic acid encoding a Cas9 protein. In some embodiments, the gene editing system further comprises a template DNA. In some embodiments, the template DNA comprises nucleic acid sequence encoding a CAR, e.g., a CAR as described herein. 
     The present invention further provides a composition for the ex vivo manufacture of a CAR-expressing cell, comprising a Tet inhibitor, e.g., a Tet1, Tet2, and/or Tet3 inhibitor, e.g., a Tet2 inhibitor. In some embodiments, the Tet inhibitor is selected from N-[3-[7-(2,5-dimethyl-2H-pyrazol-3-ylamino)-1-methyl-2-oxo-1,4-dihydro-2H-pyrimido[4,5-d]pyrimidin-3-yl]-4-methylphenyl]-3-trifluoromethyl-benzamide, 2-[(2,6-dichloro-3-methylphenyl)amino]benzoic acid and 2-hydroxyglutarate. 
     The present invention further provides a population of cells comprising one or more cells described herein, wherein the population of cells comprises a higher percentage of Tscm cells (e.g., CD45RA+CD62L+CCR7+CD27+CD95+ T cells) than a population of cells which does not comprise one or more cells in which expression and/or function of Tet1, Tet2 and/or Tet3 in said cell has been reduced or eliminated. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG.  1   : CD19-expressing CART cells were administered to a patient (UPCC04409-10) for the treatment of CLL. CART cells in patient UPCC04409-10 were monitored over time by sampling blood. The amount of BBZ expression in cells was determined (red). The number of copies of sequence from the Vbeta5.1 TCR family was determined (blue). Both measurements were made from samples collected on the indicated days after the second infusion of CART cells. 
         FIGS.  2 A and  2 B : The T-cell receptor repertoire from patient UPCC04409-10 was determined from a sample collected on day 28 ( FIG.  2 A ) or day 51 ( FIG.  2 B ) after CART infusion. This demonstrates the abundance of the TCRBV05-01 family of T-cell receptors at day 51 indicating clonal expansion over time. 
         FIG.  3   : The T-cells isolated from patient UPCC04409-10 were analyzed for the simultaneous expression of CAR19 and 2 different TCR family genes over time (day 50 and day 51) and compared to the input dosed material (product): upper panel is TCR family Vb13.1; the lower panel shows TCR family Vb5.1. The data demonstrate that the CAR19 positive cells contain a single TCR family gene (Vb5.1) that becomes rapidly enriched between days 50 and 51. 
         FIG.  4   : The T-cell receptor repertoire of CD8 positive cells from patient UPCC04409-10 was determined from a sample collected on day 51 after CART infusion. This demonstrates the abundance of the TCRBV05-01 family of T-cell receptors at day 51 indicating clonal expansion of CD8 positive cells over time. 
         FIG.  5   : The T-cell receptor from patient UPCC04409-10 was sequenced and the sequence of the alpha and beta chains are shown (Amino Acid sequences disclosed as SEQ ID NOS: 1297-1298 and Nucleotide sequences disclosed as SEQ ID NOS: 1299-1301, all respectively, in order of appearance). 
         FIG.  6   : Sonically fragmented DNA was generated from T-cells from Patient UPCC04409-10. This material was used to amplify genomic sequences adjacent to the CAR19 insertion. The genes indicated were identified as being enriched relative to the infused product (DO) adjacent to CAR19 in the genome. At the different time points after CART infusion indicated (d=day; m=month), a different relative abundance of adjacent genes was seen, with Tet2 abundance peaking in both peripheral blood (PBMC) and CAR+CD8+ T-cells samples at day 51. 
         FIG.  7   : The site of insertion of the CAR19 gene was mapped to the Tet2 gene. More specifically, the insertion occurred between exons 9 and 10 of the Tet2 gene. The catalytic domain for Tet2 resides in exon 11. The insertion at this location may lead to expression of aberrant mRNA transcripts or decrease the expression of functional (wild-type) Tet2. 
         FIG.  8   : Transcripts of the Tet2 gene from mRNA isolated from patient UPCC04409-10 were evaluated by RTPCR using primers spanning the indicated regions of Tet2 or CAR19 or both as indicated in the right hand side of the figure. Rxn 3 contains primers designed to amplify the region of the Tet2 transcript spanning exons 9 and 10. Rxn, 6, 7, 8, 9, and 10 are primers designed to amplify the indicated portions of the CAR19 lentivirus. Rxn 12-16 are pairs of primers that contain exon 9 sequence of the Tet2 transcript as well as sequence from the CAR19 lentiviral construct. These data show that transcripts are made from the Tet2 locus that contains both Tet2 sequence as well as CAR19 sequence. 
         FIG.  9   : A schematic representation of the transcripts derived from the Tet2 locus discovered in  FIGS.  10 A and  10 B  is shown. This figure indicates splice variants of this Tet2/CAR19 fusion that were detected in the patient sample. This analysis has revealed that the CAR19 insertion into Tet2 has resulted in transcripts containing stop codons upstream of exon 11. Exon 11 has been demonstrated to be important for Tet2 function. This suggests Tet2 function has been disrupted by the insertion of the CAR19. This also suggests that the disruption of Tet2 function has resulted in favorable expansion of this individual CART clones. 
         FIGS.  10 A and  10 B : The enzymatic activity of Tet2 is schematized ( FIG.  10 A ). Tet family protein convert 5-methylcytosine (5-mc) to 5-hydroxymethylcytosine (5-hmc) and then into 5-formylcytosine (5-fmc) resulting in demethylated cytosine. Methylated DNA is an epigenetic state that is known to affect transcriptional profiles. The methylation state of the T-cells from patient UPCC04409-10 was evaluated ( FIG.  10 B ). The patient&#39;s T-cells were stained for TCRVb5.1 (which contain the CAR19 insertion at Tet2) and the 5-hmc and 5-fmc were evaluated in TCRVb5.1 positive (red) and TCRVb5.1 negative (blue) populations by flow cytometry. This data indicates that the cells containing the insertion of CAR19 in the Tet2 gene are defective in demethylation. 
         FIG.  11   : TET2 shRNAs reduce 5-hmc levels in normal human T cells. TET2 and scramble control shRNA constructs expressing mCherry were introduced into normal human T cells. 5-hmc levels were determined by intracellular staining by FACS on day 6 following expansion with anti-CD3/CD28 beads. Knockdown of TET2 reduced overall 5-hmc levels. 
         FIG.  12   : TET2 shRNAs expand Tscm T cells. TET2 and scramble control shRNA constructs expressing mCherry were introduced into normal human T cells. CD45RA+CD62L+CCR7+CD27+CD95+ Tscm T cells were determined by FACS staining on day 11 following expansion with anti-CD3/CD28 beads. Knockdown of TET2 promoted the expansion of T cells with a Tscm phenotype. 
         FIG.  13 A : Gating strategy for quantification of CAR+ cells. 
         FIG.  13 B : CAR expression levels in cells electroporated with CRISPR/Cas systems targeting Tet2, as compared with untransfected cells. 
         FIG.  14   : Quantitation of CD4+ and CD8+ cells after CAR transduction and Tet2 editing. 
         FIG.  15   : Effect of Tet2 inhibition on CD3/CD28 bead expansion of CAR T cells. 
         FIG.  16   : Effect of Tet2 inhibition on antigen-dependendent interleukin-2 (IL-2) production by CAR T cells. 
         FIG.  17   : Effect of Tet2 inhibition on antigen-dependendent interferon gamma production by CAR T cells. 
         FIG.  18   : Effect of Tet2 inhibition on antigen-driven CAR+ T cell proliferation. 
         FIG.  19   : Effect of Tet2 inhibition on antigen-driven T cell proliferation. 
         FIG.  20   : Effect of Tet2 inhibition on antigen-driven CD4+ T cell proliferation. 
         FIG.  21   : Effect of Tet2 inhibition on antigen-driven CAR+CD4+ T cell proliferation. 
         FIG.  22   : Effect of Tet2 inhibition on antigen-driven CD8+ T cell proliferation. 
         FIG.  23   : Effect of Tet2 inhibition on antigen-driven CAR+CD8+ T cell proliferation. 
         FIG.  24   : % editing, and % frameshift edit by introduction of CRISPR/Cas systems targeting Tet2 as measured by NGS. 
         FIG.  25   : Top 5 most frequent indels observed in T cells after addition of RNP that included the indicated TET2 Exon 3-targeting gRNAs (SEQ ID NOS: 1302-1326, respectively, in order of appearance). Changes from the unmodified wt sequence are shown, with insertions represented with lowercase letters (“a”. “t”, “g” and “c”) and deletions shown with a dash (“—”). Indel frequency is shown in the right-most column. 
         FIG.  26   : Top 5 most frequent indels observed in T cells after addition of RNP that included the indicated TET2 Exon 9-targeting gRNAs (SEQ ID NOS: 1327-1356, respectively, in order of appearance). Changes from the unmodified wt sequence are shown, with insertions represented with lowercase letters (“a,” “t,” “g,” and “c”) and deletions shown with a dash (“—”). Indel frequency is shown in the right-most column. 
         FIG.  27   : Schematic experimental protocol for determination of TET2 knockdown in Jurkat cells in response to lentivirus encoding shRNA TET2 inhibitors. 
         FIG.  28   : RFP expression in shRNA infected Jurkat cells. RFP expression was determined by FACS on day 6 after puromycin treatment. Based on RFP expression, greater than 99% shRNA introduced jurkat cells were selected by puromycin treatment. 
         FIG.  29   : Knockdown efficiency of tet2 in TET2 shRNAs infected Jurkat cells. qRT-PCR experiment was performed. The expression levels of tet1 and tet3 were also measured. 3-actin serves as an internal control to quantify relative gene expression among samples tested. To increase reliability of qRT-PCR, two j-actin primers and one RPLP1 primer were used in this experiment. 
         FIG.  30   : Knockdown of TET2 protein in response to shRNAs in Jurkat cells. A western blot experiment was performed. 
         FIG.  31 A : Venn diagrams of ATAC peaks in the CAR+CD8+ T cells from a patient with a Tet2 disruption compared to CAR-CD8+ T cells from the same patient at the matched time point without the Tet2 disruption. The box plots show differences in ATAC enrichment between the two cell populations. 
         FIG.  31 B : GO terms associated with ATAC peaks more closed in the cell population with the Tet2 disruption, compared to its counterpart. 
         FIG.  32 A : Silencing of Tet2 by shRNA in primary CD8+ T cells from healthy donors as measured by quantitative PCR. Expression (mean, SEM) normalized to GAPDH is presented as fold change relative to non-targeting control shRNA. 
         FIGS.  32 B and  32 C : Relative frequencies of central memory ( FIG.  32 B ) and effector CD8+ T cells ( FIG.  32 C ) at day 14 post-expansion via CD3/CD28 stimulation in the same healthy donors as presented in A. 
     
    
    
     DETAILED DESCRIPTION 
     Definitions 
     Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. 
     The term “a” and “an” refers to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. 
     The term “about” when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of 20% or in some instances ±10%, or in some instances ±5%, or in some instances ±1%, or in some instances ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. 
     The term “Chimeric Antigen Receptor” or alternatively a “CAR” refers to a set of polypeptides, typically two in the simplest embodiments, which when in an immune effector cell, provides the cell with specificity for a target cell, typically a cancer cell, and with intracellular signal generation. In some embodiments, a CAR comprises at least an extracellular antigen binding domain, a transmembrane domain and a cytoplasmic signaling domain (also referred to herein as “an intracellular signaling domain”) comprising a functional signaling domain derived from a stimulatory molecule and/or costimulatory molecule as defined below. In some aspects, the set of polypeptides are contiguous with each other. In some embodiments, the set of polypeptides include a dimerization switch that, upon the presence of a dimerization molecule, can couple the polypeptides to one another, e.g., can couple an antigen binding domain to an intracellular signaling domain. In one aspect, the stimulatory molecule is the zeta chain associated with the T cell receptor complex. In one aspect, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule as defined below. In one aspect, the costimulatory molecule is chosen from the costimulatory molecules described herein, e.g., 4-1BB (i.e., CD137), CD27 and/or CD28. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises an optional leader sequence at the amino-terminus (N-ter) of the CAR fusion protein. In one aspect, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen binding domain, wherein the leader sequence is optionally cleaved from the antigen binding domain (e.g., a scFv) during cellular processing and localization of the CAR to the cellular membrane. 
     A CAR that comprises an antigen binding domain (e.g., a scFv, or TCR) that targets a specific tumor maker X, such as those described herein, is also referred to as XCAR. For example, a CAR that comprises an antigen binding domain that targets CD19 is referred to as CD19CAR. 
     The term “signaling domain” refers to the functional portion of a protein which acts by transmitting information within the cell to regulate cellular activity via defined signaling pathways by generating second messengers or functioning as effectors by responding to such messengers. 
     The term “antibody,” as used herein, refers to a protein, or polypeptide sequence derived from an immunoglobulin molecule which specifically binds with an antigen. Antibodies can be polyclonal or monoclonal, multiple or single chain, or intact immunoglobulins, and may be derived from natural sources or from recombinant sources. Antibodies can be tetramers of immunoglobulin molecules. 
     The term “antibody fragment” refers to at least one portion of an antibody, that retains the ability to specifically interact with (e.g., by binding, steric hinderance, stabilizing/destabilizing, spatial distribution) an epitope of an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab′, F(ab′) 2 , Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), a Fd fragment consisting of the VH and CH1 domains, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid VHH domains, multi-specific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments linked by a disulfide brudge at the hinge region, and an isolated CDR or other epitope binding fragments of an antibody. An antigen binding fragment can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen binding fragments can also be grafted into scaffolds based on polypeptides such as a fibronectin type III (Fn3)(see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide minibodies). 
     The term “scFv” refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked, e.g., via a synthetic linker, e.g., a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL. 
     The portion of the CAR of the invention comprising an antibody or antibody fragment thereof may exist in a variety of forms where the antigen binding domain is expressed as part of a contiguous polypeptide chain including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv), a humanized antibody or bispecific antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In one aspect, the antigen binding domain of a CAR composition of the invention comprises an antibody fragment. In a further aspect, the CAR comprises an antibody fragment that comprises a scFv. The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (“Kabat” numbering scheme), A1-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme), or a combination thereof. 
     As used herein, the term “binding domain” or “antibody molecule” refers to a protein, e.g., an immunoglobulin chain or fragment thereof, comprising at least one immunoglobulin variable domain sequence. The term “binding domain” or “antibody molecule” encompasses antibodies and antibody fragments. In an embodiment, an antibody molecule is a multispecific antibody molecule, e.g., it comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In an embodiment, a multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity for no more than two antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence which has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. 
     The portion of the CAR of the invention comprising an antibody or antibody fragment thereof may exist in a variety of forms where the antigen binding domain is expressed as part of a contiguous polypeptide chain including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv), a humanized antibody, or bispecific antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In one aspect, the antigen binding domain of a CAR composition of the invention comprises an antibody fragment. In a further aspect, the CAR comprises an antibody fragment that comprises a scFv. 
     The term “antibody heavy chain,” refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, and which normally determines the class to which the antibody belongs. 
     The term “antibody light chain,” refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa (κ) and lambda (λ) light chains refer to the two major antibody light chain isotypes. 
     The term “recombinant antibody” refers to an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage or yeast expression system. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequence technology which is available and well known in the art. 
     The term “antigen” or “Ag” refers to a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample, or might be macromolecule besides a polypeptide. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a fluid with other biological components. 
     The term “anti-cancer effect” refers to a biological effect which can be manifested by various means, including but not limited to, e.g., a decrease in tumor volume, a decrease in the number of cancer cells, a decrease in the number of metastases, an increase in life expectancy, decrease in cancer cell proliferation, decrease in cancer cell survival, or amelioration of various physiological symptoms associated with the cancerous condition. An “anti-cancer effect” can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies in prevention of the occurrence of cancer in the first place. The term “anti-tumor effect” refers to a biological effect which can be manifested by various means, including but not limited to, e.g., a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, or a decrease in tumor cell survival. 
     The term “autologous” refers to any material derived from the same individual to whom it is later to be re-introduced into the individual. 
     The term “allogeneic” refers to any material derived from a different animal of the same species as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenically 
     The term “xenogeneic” refers to a graft derived from an animal of a different species. 
     The term “cancer” refers to a disease characterized by the uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein and include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer and the like. The terms “tumor” and “cancer” are used interchangeably herein, e.g., both terms encompass solid and liquid, e.g., diffuse or circulating, tumors. As used herein, the term “cancer” or “tumor” includes premalignant, as well as malignant cancers and tumors. 
     “Derived from” as that term is used herein, indicates a relationship between a first and a second molecule. It generally refers to structural similarity between the first molecule and a second molecule and does not connotate or include a process or source limitation on a first molecule that is derived from a second molecule. For example, in the case of an intracellular signaling domain that is derived from a CD3zeta molecule, the intracellular signaling domain retains sufficient CD3zeta structure such that is has the required function, namely, the ability to generate a signal under the appropriate conditions. It does not connotate or include a limitation to a particular process of producing the intracellular signaling domain, e.g., it does not mean that, to provide the intracellular signaling domain, one must start with a CD3zeta sequence and delete unwanted sequence, or impose mutations, to arrive at the intracellular signaling domain. 
     The phrase “disease associated with expression of a tumor antigen as described herein” includes, but is not limited to, a disease associated with expression of a tumor antigen as described herein or condition associated with cells which express a tumor antigen as described herein including, e.g., proliferative diseases such as a cancer or malignancy or a precancerous condition such as a myelodysplasia, a myelodysplastic syndrome or a preleukemia; or a noncancer related indication associated with cells which express a tumor antigen as described herein. In one aspect, a cancer associated with expression of a tumor antigen as described herein is a hematological cancer. In one aspect, a cancer associated with expression of a tumor antigen as described herein is a solid cancer. Further diseases associated with expression of a tumor antigen described herein include, but not limited to, e.g., atypical and/or non-classical cancers, malignancies, precancerous conditions or proliferative diseases associated with expression of a tumor antigen as described herein. Non-cancer related indications associated with expression of a tumor antigen as described herein include, but are not limited to, e.g., autoimmune disease, (e.g., lupus), inflammatory disorders (allergy and asthma) and transplantation. In some embodiments, the tumor antigen-expressing cells express, or at any time expressed, mRNA encoding the tumor antigen. In an embodiment, the tumor antigen-expressing cells produce the tumor antigen protein (e.g., wild-type or mutant), and the tumor antigen protein may be present at normal levels or reduced levels. In an embodiment, the tumor antigen-expressing cells produced detectable levels of a tumor antigen protein at one point, and subsequently produced substantially no detectable tumor antigen protein. 
     The term “conservative sequence modifications” refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody or antibody fragment of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within a CAR of the invention can be replaced with other amino acid residues from the same side chain family and the altered CAR can be tested using the functional assays described herein. 
     The term “stimulation,” refers to a primary response induced by binding of a stimulatory molecule (e.g., a TCR/CD3 complex or CAR) with its cognate ligand (or tumor antigen in the case of a CAR) thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR/CD3 complex or signal transduction via the appropriate NK receptor or signaling domains of the CAR. Stimulation can mediate altered expression of certain molecules. 
     The term “stimulatory molecule,” refers to a molecule expressed by an immune cell (e.g., T cell, NK cell, B cell) that provides the cytoplasmic signaling sequence(s) that regulate activation of the immune cell in a stimulatory way for at least some aspect of the immune cell signaling pathway. In one aspect, the signal is a primary signal that is initiated by, for instance, binding of a TCR/CD3 complex with an MHC molecule loaded with peptide, and which leads to mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. A primary cytoplasmic signaling sequence (also referred to as a “primary signaling domain”) that acts in a stimulatory manner may contain a signaling motif which is known as immunoreceptor tyrosine-based activation motif or ITAM. Examples of an ITAM containing cytoplasmic signaling sequence that is of particular use in the invention includes, but is not limited to, those derived from CD3 zeta, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc Epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12. In a specific CAR of the invention, the intracellular signaling domain in any one or more CARS of the invention comprises an intracellular signaling sequence, e.g., a primary signaling sequence of CD3-zeta. In a specific CAR of the invention, the primary signaling sequence of CD3-zeta is the sequence provided as SEQ ID NO:18, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like. In a specific CAR of the invention, the primary signaling sequence of CD3-zeta is the sequence as provided in SEQ ID NO: 20, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like. 
     The term “antigen presenting cell” or “APC” refers to an immune system cell such as an accessory cell (e.g., a B-cell, a dendritic cell, and the like) that displays a foreign antigen complexed with major histocompatibility complexes (MHC&#39;s) on its surface. T-cells may recognize these complexes using their T-cell receptors (TCRs). APCs process antigens and present them to T-cells. 
     An “intracellular signaling domain,” as the term is used herein, refers to an intracellular portion of a molecule. The intracellular signaling domain generates a signal that promotes an immune effector function of the CAR containing cell, e.g., a CART cell. Examples of immune effector function, e.g., in a CART cell, include cytolytic activity and helper activity, including the secretion of cytokines. 
     In an embodiment, the intracellular signaling domain can comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from the molecules responsible for primary stimulation, or antigen dependent simulation. In an embodiment, the intracellular signaling domain can comprise a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals, or antigen independent stimulation. For example, in the case of a CART, a primary intracellular signaling domain can comprise a cytoplasmic sequence of a T cell receptor, and a costimulatory intracellular signaling domain can comprise cytoplasmic sequence from co-receptor or costimulatory molecule. 
     A primary intracellular signaling domain can comprise a signaling motif which is known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of ITAM containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 zeta, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc Epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12. 
     The term “zeta” or alternatively “zeta chain”, “CD3-zeta” or “TCR-zeta” is defined as the protein provided as GenBan Acc. No. BAG36664.1, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like, and a “zeta stimulatory domain” or alternatively a “CD3-zeta stimulatory domain” or a “TCR-zeta stimulatory domain” is defined as the amino acid residues from the cytoplasmic domain of the zeta chain, or functional derivatives thereof, that are sufficient to functionally transmit an initial signal necessary for T cell activation. In one aspect the cytoplasmic domain of zeta comprises residues 52 through 164 of GenBank Acc. No. BAG36664.1 or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like, that are functional orthologs thereof. In one aspect, the “zeta stimulatory domain” or a “CD3-zeta stimulatory domain” is the sequence provided as SEQ ID NO: 18. In one aspect, the “zeta stimulatory domain” or a “CD3-zeta stimulatory domain” is the sequence provided as SEQ ID NO: 20. 
     The term a “costimulatory molecule” refers to a cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are contribute to an efficient immune response. Costimulatory molecules include, but are not limited to an MHC class I molecule, BTLA and a Toll ligand receptor, as well as OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), and 4-1BB (CD137). Further examples of such costimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, and a ligand that specifically binds with CD83. 
     A costimulatory intracellular signaling domain can be the intracellular portion of a costimulatory molecule. A costimulatory molecule can be represented in the following protein families: TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and activating NK cell receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CDS, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3, and a ligand that specifically binds with CD83, and the like. 
     The intracellular signaling domain can comprise the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional fragment or derivative thereof. 
     The term “4-1BB” refers to a member of the TNFR superfamily with an amino acid sequence provided as GenBank Acc. No. AAA62478.2, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like; and a “4-1BB costimulatory domain” is defined as amino acid residues 214-255 of GenBank Acc. No. AAA62478.2, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like. In one aspect, the “4-1BB costimulatory domain” is the sequence provided as SEQ ID NO: 14 or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like. 
     “Immune effector cell,” as that term is used herein, refers to a cell that is involved in an immune response, e.g., in the promotion of an immune effector response. Examples of immune effector cells include T cells, e.g., alpha/beta T cells and gamma/delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloic-derived phagocytes. 
     “Immune effector function or immune effector response,” as that term is used herein, refers to function or response, e.g., of an immune effector cell, that enhances or promotes an immune attack of a target cell. E.g., an immune effector function or response refers a property of a T or NK cell that promotes killing or the inhibition of growth or proliferation, of a target cell. In the case of a T cell, primary stimulation and co-stimulation are examples of immune effector function or response. 
     The term “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA. 
     Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or a RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s). 
     The term “effective amount” or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result. 
     The term “endogenous” refers to any material from or produced inside an organism, cell, tissue or system. 
     The term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue or system. 
     The term “expression” refers to the transcription and/or translation of a particular nucleotide sequence driven by a promoter. 
     The term “transfer vector” refers to a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “transfer vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to further include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, a polylysine compound, liposome, and the like. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like. 
     The term “expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide. 
     The term “lentivirus” refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. 
     The term “lentiviral vector” refers to a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentivirus vectors that may be used in the clinic, include but are not limited to, e.g., the LENTIVECTOR® gene delivery technology from Oxford BioMedica, the LENTIMAX™ vector system from Lentigen and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art. 
     The term “homologous” or “identity” refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous or identical at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 of 10), are matched or homologous, the two sequences are 90% homologous. 
     “Humanized” forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies and antibody fragments thereof are human immunoglobulins (recipient antibody or antibody fragment) in which residues from a complementary-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, a humanized antibody/antibody fragment can comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications can further refine and optimize antibody or antibody fragment performance. In general, the humanized antibody or antibody fragment thereof will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or a significant portion of the FR regions are those of a human immunoglobulin sequence. The humanized antibody or antibody fragment can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332: 323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992. 
     “Fully human” refers to an immunoglobulin, such as an antibody or antibody fragment, where the whole molecule is of human origin or consists of an amino acid sequence identical to a human form of the antibody or immunoglobulin. 
     The term “isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell. 
     In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine. 
     The term “operably linked” or “transcriptional control” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be contiguous with each other and, e.g., where necessary to join two protein coding regions, are in the same reading frame. 
     The term “parenteral” administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, intratumoral, or infusion techniques. 
     The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). 
     The terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein&#39;s or peptide&#39;s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof. 
     The term “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence. 
     The term “promoter/regulatory sequence” refers to a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner. 
     The term “constitutive” promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell. 
     The term “inducible” promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell. 
     The term “tissue-specific” promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter. 
     The terms “cancer associated antigen” or “tumor antigen” interchangeably refers to a molecule (typically a protein, carbohydrate or lipid) that is expressed on the surface of a cancer cell, either entirely or as a fragment (e.g., MHC/peptide), and which is useful for the preferential targeting of a pharmacological agent to the cancer cell. In some embodiments, a tumor antigen is a marker expressed by both normal cells and cancer cells, e.g., a lineage marker, e.g., CD19 on B cells. In some embodiments, a tumor antigen is a cell surface molecule that is overexpressed in a cancer cell in comparison to a normal cell, for instance, 1-fold over expression, 2-fold overexpression, 3-fold overexpression or more in comparison to a normal cell. In some embodiments, a tumor antigen is a cell surface molecule that is inappropriately synthesized in the cancer cell, for instance, a molecule that contains deletions, additions or mutations in comparison to the molecule expressed on a normal cell. In some embodiments, a tumor antigen will be expressed exclusively on the cell surface of a cancer cell, entirely or as a fragment (e.g., MHC/peptide), and not synthesized or expressed on the surface of a normal cell. In some embodiments, the CARs of the present invention includes CARs comprising an antigen binding domain (e.g., antibody or antibody fragment) that binds to a MHC presented peptide. Normally, peptides derived from endogenous proteins fill the pockets of Major histocompatibility complex (MHC) class I molecules, and are recognized by T cell receptors (TCRs) on CD8+T lymphocytes. The MHC class I complexes are constitutively expressed by all nucleated cells. In cancer, virus-specific and/or tumor-specific peptide/MHC complexes represent a unique class of cell surface targets for immunotherapy. TCR-like antibodies targeting peptides derived from viral or tumor antigens in the context of human leukocyte antigen (HLA)-A1 or HLA-A2 have been described (see, e.g., Sastry et al., J Virol. 2011 85(5):1935-1942; Sergeeva et al., Blood, 2011 117(16):4262-4272; Verma et al., J Immunol 2010 184(4):2156-2165; Willemsen et al., Gene Ther 2001 8(21):1601-1608; Dao et al., Sci Transl Med 2013 5(176):176ra33; Tassev et al., Cancer Gene Ther 2012 19(2):84-100). For example, TCR-like antibody can be identified from screening a library, such as a human scFv phage displayed library. 
     The term “tumor-supporting antigen” or “cancer-supporting antigen” interchangeably refer to a molecule (typically a protein, carbohydrate or lipid) that is expressed on the surface of a cell that is, itself, not cancerous, but supports the cancer cells, e.g., by promoting their growth or survival e.g., resistance to immune cells. Exemplary cells of this type include stromal cells and myeloid-derived suppressor cells (MDSCs). The tumor-supporting antigen itself need not play a role in supporting the tumor cells so long as the antigen is present on a cell that supports cancer cells. 
     The term “flexible polypeptide linker” or “linker” as used in the context of a scFv refers to a peptide linker that consists of amino acids such as glycine and/or serine residues used alone or in combination, to link variable heavy and variable light chain regions together. In one embodiment, the flexible polypeptide linker is a Gly/Ser linker and comprises the amino acid sequence (Gly-Gly-Gly-Ser)n, where n is a positive integer equal to or greater than 1. For example, n=1, n=2, n=3. n=4, n=5 and n=6, n=7, n=8, n=9 and n=10 (SEQ ID NO:28). In one embodiment, the flexible polypeptide linkers include, but are not limited to, (Gly4 Ser)4 (SEQ ID NO:29) or (Gly4 Ser)3 (SEQ ID NO:30). In another embodiment, the linkers include multiple repeats of (Gly2Ser), (GlySer) or (Gly3Ser) (SEQ ID NO:31). Also included within the scope of the invention are linkers described in WO2012/138475, incorporated herein by reference). 
     As used herein, a 5′ cap (also termed an RNA cap, an RNA 7-methylguanosine cap or an RNA m 7 G cap) is a modified guanine nucleotide that has been added to the “front” or 5′ end of a eukaryotic messenger RNA shortly after the start of transcription. The 5′ cap consists of a terminal group which is linked to the first transcribed nucleotide. Its presence is critical for recognition by the ribosome and protection from RNases. Cap addition is coupled to transcription, and occurs co-transcriptionally, such that each influences the other. Shortly after the start of transcription, the 5′ end of the mRNA being synthesized is bound by a cap-synthesizing complex associated with RNA polymerase. This enzymatic complex catalyzes the chemical reactions that are required for mRNA capping. Synthesis proceeds as a multi-step biochemical reaction. The capping moiety can be modified to modulate functionality of mRNA such as its stability or efficiency of translation. 
     As used herein, “in vitro transcribed RNA” refers to RNA, preferably mRNA, that has been synthesized in vitro. Generally, the in vitro transcribed RNA is generated from an in vitro transcription vector. The in vitro transcription vector comprises a template that is used to generate the in vitro transcribed RNA. 
     As used herein, a “poly(A)” is a series of adenosines attached by polyadenylation to the mRNA. In the preferred embodiment of a construct for transient expression, the polyA is between 50 and 5000 (SEQ ID NO: 34), preferably greater than 64, more preferably greater than 100, most preferably greater than 300 or 400. poly(A) sequences can be modified chemically or enzymatically to modulate mRNA functionality such as localization, stability or efficiency of translation. 
     As used herein, “polyadenylation” refers to the covalent linkage of a polyadenylyl moiety, or its modified variant, to a messenger RNA molecule. In eukaryotic organisms, most messenger RNA (mRNA) molecules are polyadenylated at the 3′ end. The 3′ poly(A) tail is a long sequence of adenine nucleotides (often several hundred) added to the pre-mRNA through the action of an enzyme, polyadenylate polymerase. In higher eukaryotes, the poly(A) tail is added onto transcripts that contain a specific sequence, the polyadenylation signal. The poly(A) tail and the protein bound to it aid in protecting mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, export of the mRNA from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but additionally can also occur later in the cytoplasm. After transcription has been terminated, the mRNA chain is cleaved through the action of an endonuclease complex associated with RNA polymerase. The cleavage site is usually characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA has been cleaved, adenosine residues are added to the free 3′ end at the cleavage site. 
     As used herein, “transient” refers to expression of a non-integrated transgene for a period of hours, days or weeks, wherein the period of time of expression is less than the period of time for expression of the gene if integrated into the genome or contained within a stable plasmid replicon in the host cell. 
     As used herein, the terms “treat”, “treatment” and “treating” refer to the reduction or amelioration of the progression, severity and/or duration of a proliferative disorder, or the amelioration of one or more symptoms (preferably, one or more discernible symptoms) of a proliferative disorder resulting from the administration of one or more therapies (e.g., one or more therapeutic agents such as a CAR of the invention). In specific embodiments, the terms “treat”, “treatment” and “treating” refer to the amelioration of at least one measurable physical parameter of a proliferative disorder, such as growth of a tumor, not necessarily discernible by the patient. In other embodiments the terms “treat”, “treatment” and “treating”-refer to the inhibition of the progression of a proliferative disorder, either physically by, e.g., stabilization of a discernible symptom, physiologically by, e.g., stabilization of a physical parameter, or both. In other embodiments the terms “treat”, “treatment” and “treating” refer to the reduction or stabilization of tumor size or cancerous cell count. 
     The term “signal transduction pathway” refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell. The phrase “cell surface receptor” includes molecules and complexes of molecules capable of receiving a signal and transmitting signal across the membrane of a cell. 
     The term “subject” is intended to include living organisms in which an immune response can be elicited (e.g., mammals, human). 
     The term, a “substantially purified” cell refers to a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell which has been separated from other cell types with which it is normally associated in its naturally occurring state. In some instances, a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to cell that have been separated from the cells with which they are naturally associated in their natural state. In some aspects, the cells are cultured in vitro. In other aspects, the cells are not cultured in vitro. 
     The term “therapeutic” as used herein means a treatment. A therapeutic effect is obtained by reduction, suppression, remission, or eradication of a disease state. 
     The term “prophylaxis” as used herein means the prevention of or protective treatment for a disease or disease state. 
     In the context of the present invention, “tumor antigen” or “hyperproliferative disorder antigen” or “antigen associated with a hyperproliferative disorder” refers to antigens that are common to specific hyperproliferative disorders. In certain aspects, the hyperproliferative disorder antigens of the present invention are derived from, cancers including but not limited to primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, leukemias, uterine cancer, cervical cancer, bladder cancer, kidney cancer and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, and the like. 
     The term “transfected” or “transformed” or “transduced” refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny. 
     The term “specifically binds,” refers to an antibody, or a ligand, which recognizes and binds with a binding partner (e.g., a tumor antigen) protein present in a sample, but which antibody or ligand does not substantially recognize or bind other molecules in the sample. 
     “Regulatable chimeric antigen receptor (RCAR),” as that term is used herein, refers to a set of polypeptides, typically two in the simplest embodiments, which when in a RCARX cell, provides the RCARX cell with specificity for a target cell, typically a cancer cell, and with regulatable intracellular signal generation or proliferation, which can optimize an immune effector property of the RCARX cell. An RCARX cell relies at least in part, on an antigen binding domain to provide specificity to a target cell that comprises the antigen bound by the antigen binding domain. In an embodiment, an RCAR includes a dimerization switch that, upon the presence of a dimerization molecule, can couple an intracellular signaling domain to the antigen binding domain. 
     “Membrane anchor” or “membrane tethering domain”, as that term is used herein, refers to a polypeptide or moiety, e.g., a myristoyl group, sufficient to anchor an extracellular or intracellular domain to the plasma membrane. 
     “Switch domain,” as that term is used herein, e.g., when referring to an RCAR, refers to an entity, typically a polypeptide-based entity, that, in the presence of a dimerization molecule, associates with another switch domain. The association results in a functional coupling of a first entity linked to, e.g., fused to, a first switch domain, and a second entity linked to, e.g., fused to, a second switch domain. A first and second switch domain are collectively referred to as a dimerization switch. In embodiments, the first and second switch domains are the same as one another, e.g., they are polypeptides having the same primary amino acid sequence, and are referred to collectively as a homodimerization switch. In embodiments, the first and second switch domains are different from one another, e.g., they are polypeptides having different primary amino acid sequences, and are referred to collectively as a heterodimerization switch. In embodiments, the switch is intracellular. In embodiments, the switch is extracellular. In embodiments, the switch domain is a polypeptide-based entity, e.g., FKBP or FRB-based, and the dimerization molecule is small molecule, e.g., a rapalogue. In embodiments, the switch domain is a polypeptide-based entity, e.g., an scFv that binds a myc peptide, and the dimerization molecule is a polypeptide, a fragment thereof, or a multimer of a polypeptide, e.g., a myc ligand or multimers of a myc ligand that bind to one or more myc scFvs. In embodiments, the switch domain is a polypeptide-based entity, e.g., myc receptor, and the dimerization molecule is an antibody or fragments thereof, e.g., myc antibody. 
     “Dimerization molecule,” as that term is used herein, e.g., when referring to an RCAR, refers to a molecule that promotes the association of a first switch domain with a second switch domain. In embodiments, the dimerization molecule does not naturally occur in the subject, or does not occur in concentrations that would result in significant dimerization. In embodiments, the dimerization molecule is a small molecule, e.g., rapamycin or a rapalogue, e.g, RAD001. 
     The term “bioequivalent” refers to an amount of an agent other than the reference compound (e.g., RAD001), required to produce an effect equivalent to the effect produced by the reference dose or reference amount of the reference compound (e.g., RAD001). In an embodiment the effect is the level of mTOR inhibition, e.g., as measured by P70 S6 kinase inhibition, e.g., as evaluated in an in vivo or in vitro assay, e.g., as measured by an assay described herein, e.g., the Boulay assay. In an embodiment, the effect is alteration of the ratio of PD-1 positive/PD-1 negative T cells, as measured by cell sorting. In an embodiment a bioequivalent amount or dose of an mTOR inhibitor is the amount or dose that achieves the same level of P70 S6 kinase inhibition as does the reference dose or reference amount of a reference compound. In an embodiment, a bioequivalent amount or dose of an mTOR inhibitor is the amount or dose that achieves the same level of alteration in the ratio of PD-1 positive/PD-1 negative T cells as does the reference dose or reference amount of a reference compound. 
     The term “low, immune enhancing, dose” when used in conjunction with an mTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g., RAD001 or rapamycin, or a catalytic mTOR inhibitor, refers to a dose of mTOR inhibitor that partially, but not fully, inhibits mTOR activity, e.g., as measured by the inhibition of P70 S6 kinase activity. Methods for evaluating mTOR activity, e.g., by inhibition of P70 S6 kinase, are discussed herein. The dose is insufficient to result in complete immune suppression but is sufficient to enhance the immune response. In an embodiment, the low, immune enhancing, dose of mTOR inhibitor results in a decrease in the number of PD-1 positive T cells and/or an increase in the number of PD-1 negative T cells, or an increase in the ratio of PD-1 negative T cells/PD-1 positive T cells. In an embodiment, the low, immune enhancing, dose of mTOR inhibitor results in an increase in the number of naive T cells. In an embodiment, the low, immune enhancing, dose of mTOR inhibitor results in one or more of the following:
         an increase in the expression of one or more of the following markers: CD62 high , CD127 high , CD27 + , and BCL2, e.g., on memory T cells, e.g., memory T cell precursors;   a decrease in the expression of KLRG1, e.g., on memory T cells, e.g., memory T cell precursors; and   an increase in the number of memory T cell precursors, e.g., cells with any one or combination of the following characteristics: increased CD62 high  increased CD127 high  increased CD27+, decreased KLRG1, and increased BCL2;   wherein any of the changes described above occurs, e.g., at least transiently, e.g., as compared to a non-treated subject.       

     “Refractory” as used herein refers to a disease, e.g., cancer, that does not respond to a treatment. In embodiments, a refractory cancer can be resistant to a treatment before or at the beginning of the treatment. In other embodiments, the refractory cancer can become resistant during a treatment. A refractory cancer is also called a resistant cancer. 
     “Relapsed” as used herein refers to the return of a disease (e.g., cancer) or the signs and symptoms of a disease such as cancer after a period of improvement, e.g., after prior treatment of a therapy, e.g., cancer therapy 
     Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity, includes something with 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This applies regardless of the breadth of the range. 
     “Tet” as the term is used herein, refers to the family of genes, and the proteins encoded by said genes, of the ten-eleven translocation methylcytosine dioxygenase family. Tet includes, for example, Tet1, Tet2 and Tet3. 
     “Tet2” as the term is used herein, refers to gene, tet methylcytosine dioxygenase 2, and the protein encoded by said gene, the tet2 methylcytosine dioxygenase, which catalyzes the conversion of methylcytosine to 5-hydroxymethylcytosine. It is sometimes also referred to as “KIAA1546,” “FLJ20032” and “tet oncogene family member 2” The encoded protein is involved in myelopoiesis, and defects in this gene have been associated with several myeloproliferative disorders. In the human genome, TET2 is located on chromosome 4q24. Currently six TET2 isoforms have been described and their Genebank numbers are: NM_001127208.2; XM_005263082.1; XM_006714242.2; NM_017628.4; XM_011532044.1; and XM_011532043.1. 
     An example of the protein sequence of human Tet2 is provided as UniProt accession number Q6N021: 
     
       
         
           
               
               
            
               
                   
                 [SEQ ID NO: 1357] 
               
               
                   
                         10         20         30         40 
               
               
                   
                 MEQDRTNHVE GNRLSPFLIP SPPICQTEPL ATKLQNGSPL 
               
               
                   
                   
               
               
                   
                         50         60         70         80  
               
               
                   
                 PERAHPEVNG DTKWHSFKSY YGIPCMKGSQ NSRVSPDFTQ 
               
               
                   
                   
               
               
                   
                         90        100        110        120 
               
               
                   
                 ESRGYSKCLQ NGGIKRTVSE PSLSGLLQIK KLKQDQKANG 
               
               
                   
                   
               
               
                   
                        130        140        150        160 
               
               
                   
                 ERRNFGVSQE RNPGESSQPN VSDLSDKKES VSSVAQENAV 
               
               
                   
                   
               
               
                   
                        170        180        190        200 
               
               
                   
                 KDFTSFSTHN CSGPENPELQ ILNEQEGKSA NYHDKNIVLL 
               
               
                   
                   
               
               
                   
                        210        220        230        240  
               
               
                   
                 KNKAVLMPNG ATVSASSVEH THGELLEKTL SQYYPDCVSI  
               
               
                   
                   
               
               
                   
                        250        260        270        280 
               
               
                   
                 AVQKTTSHIN AINSQATNEL SCEITHPSHT SGQINSAQTS 
               
               
                   
                   
               
               
                   
                        290        300        310        320 
               
               
                   
                 NSELPPKPAA VVSEACDADD ADNASKLAAM LNTCSFQKPE 
               
               
                   
                   
               
               
                   
                        330        340        350        360 
               
               
                   
                 QLQQQKSVFE ICPSPAENNI QGTTKLASGE EFCSGSSSNL 
               
               
                   
                   
               
               
                   
                        370        380        390        400 
               
               
                   
                 QAPGGSSERY LKQNEMNGAY FKQSSVFTKD SFSATTTPPP 
               
               
                   
                   
               
               
                   
                        410        420        430        440  
               
               
                   
                 PSQLLLSPPP PLPQVPQLPS EGKSTLNGGV LEEHHHYPNQ  
               
               
                   
                   
               
               
                   
                        450        460        470        480  
               
               
                   
                 SNTTLLREVK IEGKPEAPPS QSPNPSTHVC SPSPMLSERP  
               
               
                   
                   
               
               
                   
                        490        500        510        520 
               
               
                   
                 QNNCVNRNDI QTAGTMTVPL CSEKTRPMSE HLKHNPPIFG 
               
               
                   
                   
               
               
                   
                        530        540        550        560 
               
               
                   
                 SSGELQDNCQ QLMRNKEQEI LKGRDKEQTR DLVPPTQHYL 
               
               
                   
                   
               
               
                   
                        570        580        590        600 
               
               
                   
                 KPGWIELKAP RFHQAESHLK RNEASLPSIL QYQPNLSNQM 
               
               
                   
                   
               
               
                   
                        610        620        630        640  
               
               
                   
                 TSKQYTGNSN MPGGLPRQAY TQKTTQLEHK SQMYQVEMNQ  
               
               
                   
                   
               
               
                   
                        650        660        670        680 
               
               
                   
                 GQSQGTVDQH LQFQKPSHQV HFSKTDHLPK AHVQSLCGTR 
               
               
                   
                   
               
               
                   
                        690        700        710        720 
               
               
                   
                 FHFQQRADSQ TEKLMSPVLK QHLNQQASET EPFSNSHLLQ 
               
               
                   
                   
               
               
                   
                        730        740        750        760 
               
               
                   
                 HKPHKQAAQT QPSQSSHLPQ NQQQQQKLQI KNKEEILQTF 
               
               
                   
                   
               
               
                   
                        770        780        790        800 
               
               
                   
                 PHPQSNNDQQ REGSFFGQTK VEECFHGENQ YSKSSEFETH 
               
               
                   
                   
               
               
                   
                        810        820        830        840  
               
               
                   
                 NVQMGLEEVQ NINRRNSPYS QTMKSSACKI QVSCSNNTHL  
               
               
                   
                   
               
               
                   
                        850        860        870        880  
               
               
                   
                 VSENKEQTTH PELFAGNKTQ NLHHMQYFPN NVIPKQDLLH  
               
               
                   
                   
               
               
                   
                        890        900        910        920 
               
               
                   
                 RCFQEQEQKS QQASVLQGYK NRNQDMSGQQ AAQLAQQRYL 
               
               
                   
                   
               
               
                   
                        930        940        950        960 
               
               
                   
                 IHNHANVFPV PDQGGSHTQT PPQKDTQKHA ALRWHLLQKQ 
               
               
                   
                   
               
               
                   
                        970        980        990       1000 
               
               
                   
                 EQQQTQQPQT ESCHSQMHRP IKVEPGCKPH ACMHTAPPEN 
               
               
                   
                   
               
               
                   
                       1010       1020       1030       1040  
               
               
                   
                 KTWKKVTKQE NPPASCDNVQ QKSIIETMEQ HLKQFHAKSL  
               
               
                   
                   
               
               
                   
                       1050       1060       1070       1080  
               
               
                   
                 FDHKALTLKS QKQVKVEMSG PVTVLTRQTT AAELDSHTPA  
               
               
                   
                   
               
               
                   
                       1090       1100       1110       1120 
               
               
                   
                 LEQQTTSSEK TPTKRTAASV LNNFIESPSK LLDTPIKNLL 
               
               
                   
                   
               
               
                   
                       1130       1140       1150       1160 
               
               
                   
                 DTPVKTQYDF PSCRCVEQII EKDEGPFYTH LGAGPNVAAI 
               
               
                   
                   
               
               
                   
                       1170       1180       1190       1200 
               
               
                   
                 REIMEERFGQ KGKAIRIERV IYTGKEGKSS QGCPIAKWVV 
               
               
                   
                   
               
               
                   
                       1210       1220       1230       1240  
               
               
                   
                 RRSSSEEKLL CLVRERAGHT CEAAVIVILI LVWEGIPLSL  
               
               
                   
                   
               
               
                   
                       1250       1260       1270       1280  
               
               
                   
                 ADKLYSELTE TLRKYGTLTN RRCALNEERT CACQGLDPET  
               
               
                   
                   
               
               
                   
                       1290       1300       1310       1320 
               
               
                   
                 CGASFSFGCS WSMYYNGCKF ARSKIPRKFK LLGDDPKEEE 
               
               
                   
                   
               
               
                   
                       1330       1340       1350       1360 
               
               
                   
                 KLESHLQNLS TLMAPTYKKL APDAYNNQIE YEHRAPECRL 
               
               
                   
                   
               
               
                   
                       1370       1380       1390       1400 
               
               
                   
                 GLKEGRPFSG VTACLDFCAH AHRDLHNMQN GSTLVCTLTR 
               
               
                   
                   
               
               
                   
                       1410       1420       1430       1440  
               
               
                   
                 EDNREFGGKP EDEQLHVLPL YKVSDVDEFG SVEAQEEKKR  
               
               
                   
                   
               
               
                   
                       1450       1460       1470       1480  
               
               
                   
                 SGAIQVLSSF RRKVRMLAEP VKTCRQRKLE AKKAAAEKLS  
               
               
                   
                   
               
               
                   
                       1490       1500       1510       1520 
               
               
                   
                 SLENSSNKNE KEKSAPSRTK QTENASQAKQ LAELLRLSGP 
               
               
                   
                   
               
               
                   
                       1530       1540       1550       1560 
               
               
                   
                 VMQQSQQPQP LQKQPPQPQQ QQRPQQQQPH HPQTESVNSY 
               
               
                   
                   
               
               
                   
                       1570       1580       1590       1600 
               
               
                   
                 SASGSTNPYM RRPNPVSPYP NSSHTSDIYG STSPMNFYST 
               
               
                   
                   
               
               
                   
                       1610       1620       1630       1640  
               
               
                   
                 SSQAAGSYLN SSNPMNPYPG LLNQNTQYPS YQCNGNLSVD  
               
               
                   
                   
               
               
                   
                       1650       1660       1670       1680   
               
               
                   
                 NCSPYLGSYS PQSQPMDLYR YPSQDPLSKL SLPPIHTLYQ   
               
               
                   
                   
               
               
                   
                       1690       1700       1710       1720   
               
               
                   
                 PRFGNSQSFT SKYLGYGNQN MQGDGFSSCT IRPNVHHVGK   
               
               
                   
                   
               
               
                   
                       1730       1740       1750       1760  
               
               
                   
                 LPPYPTHEMD GHFMGATSRL PPNLSNPNMD YKNGEHHSPS  
               
               
                   
                   
               
               
                   
                       1770       1780       1790       1800  
               
               
                   
                 HIIHNYSAAP GMFNSSLHAL HLQNKENDML SHTANGLSKM  
               
               
                   
                   
               
               
                   
                       1810       1820       1830       1840  
               
               
                   
                 LPALNHDRTA CVQGGLHKLS DANGQEKQPL ALVQGVASGA  
               
               
                   
                   
               
               
                   
                       1850       1860       1870       1880 
               
               
                   
                 EDNDEVWSDS EQSFLDPDIG GVAVAPTHGS ILIECAKREL 
               
               
                   
                   
               
               
                   
                       1890       1900       1910       1920 
               
               
                   
                 HATTPLKNPN RNHPTRISLV FYQHKSMNEP KHGLALWEAK 
               
               
                   
                   
               
               
                   
                       1930       1940       1950       1960 
               
               
                   
                 MAEKAREKEE ECEKYGPDYV PQKSHGKKVK REPAEPHETS 
               
               
                   
                   
               
               
                   
                       1970       1980       1990       2000 
               
               
                   
                 EPTYLRFIKS LAERTMSVTT DSTVTTSPYA FTRVTGPYNR 
               
               
                   
                   
               
               
                   
                  2002 
               
               
                   
                 YI 
               
            
           
         
       
     
     The tet2 gene is located on chromosome 4, location GRCh38.p2 (GCF_000001405.28) (NC_000004.12 (105145875 to 105279803); Gene TD 54790. 
     Examples of nucleic acid sequences encoding Tet2 are provided below. There are 6 identified isoforms of human Tet2 have been identified. The mRNA sequences are provided below (In embodiments, in each sequence, T may be replaced with U). In embodiments, Tet2 includes the proteins encoded by each of the sequences below: 
     
       
         
           
               
               
               
             
               
                   
               
               
                   
                 NCBI 
                   
               
               
                   
                 Reference 
                   
               
               
                 NName 
                 Sequence 
                 Sequence 
               
               
                   
               
             
            
               
                 H Homo sapiens   
                 NNM_001127208.2 
                 GGCAGTGGCAGCGGCGAGAGCTTGGGCGGCCGCCGCCGCC 
               
               
                 tet 
                   
                 TCCTCGCGAGCGCCGCGCGCCCGGGTCCCG 
               
               
                 methylcytosine 
                   
                 CTCGCATGCAAGTCACGTCCGCCCCCTCGGCGCGGCCGCCC 
               
               
                 dioxygenase 2 
                   
                 CGAGACGCCGGCCCCGCTGAGTGATGAGA 
               
               
                 (TET2), 
                   
                 ACAGACGTCAAACTGCCTTATGAATATTGATGCGGAGGCTA 
               
               
                 transcript 
                   
                 GGCTGCTTTCGTAGAGAAGCAGAAGGAAG 
               
               
                 variant 1, 
                   
                 CAAGATGGCTGCCCTTTAGGATTTGTTAGAAAGGAGACCCG 
               
               
                 mRNA 
                   
                 ACTGCAACTGCTGGATTGCTGCAAGGCTG 
               
               
                 [SEQ ID NO: 
                   
                 AGGGACGAGAACGAGGCTGGCAAACATTCAGCAGCACACC 
               
               
                 1358] 
                   
                 CTCTCAAGATTGTTTACTTGCCTTTGCTCC 
               
               
                   
                   
                 TGTTGAGTTACAACGCTTGGAAGCAGGAGATGGGCTCAGCA 
               
               
                   
                   
                 GCAGCCAATAGGACATGATCCAGGAAGAG 
               
               
                   
                   
                 CAGTAAGGGACTGAGCTGCTGAATTCAACTAGAGGGCAGC 
               
               
                   
                   
                 CTTGTGGATGGCCCCGAAGCAAGCCTGATG 
               
               
                   
                   
                 GAACAGGATAGAACCAACCATGTTGAGGGCAACAGACTAA 
               
               
                   
                   
                 GTCCATTCCTGATACCATCACCTCCCATTT 
               
               
                   
                   
                 GCCAGACAGAACCTCTGGCTACAAAGCTCCAGAATGGAAG 
               
               
                   
                   
                 CCCACTGCCTGAGAGAGCTCATCCAGAAGT 
               
               
                   
                   
                 AAATGGAGACACCAAGTGGCACTCTTTCAAAAGTTATTATG 
               
               
                   
                   
                 GAATACCCTGTATGAAGGGAAGCCAGAAT 
               
               
                   
                   
                 AGTCGTGTGAGTCCTGACTTTACACAAGAAAGTAGAGGGTA 
               
               
                   
                   
                 TTCCAAGTGTTTGCAAAATGGAGGAATAA 
               
               
                   
                   
                 AACGCACAGTTAGTGAACCTTCTCTCTCTGGGCTCCTTCAGA 
               
               
                   
                   
                 TCAAGAAATTGAAACAAGACCAAAAGGC 
               
               
                   
                   
                 TAATGGAGAAAGACGTAACTTCGGGGTAAGCCAAGAAAGA 
               
               
                   
                   
                 AATCCAGGTGAAAGCAGTCAACCAAATGTC 
               
               
                   
                   
                 TCCGATTTGAGTGATAAGAAAGAATCTGTGAGTTCTGTAGC 
               
               
                   
                   
                 CCAAGAAAATGCAGTTAAAGATTTCACCA 
               
               
                   
                   
                 GTTTTTCAACACATAACTGCAGTGGGCCTGAAAATCCAGAG 
               
               
                   
                   
                 CTTCAGATTCTGAATGAGCAGGAGGGGAA 
               
               
                   
                   
                 AAGTGCTAATTACCATGACAAGAACATTGTATTACTTAAAA 
               
               
                   
                   
                 ACAAGGCAGTGCTAATGCCTAATGGTGCT 
               
               
                   
                   
                 ACAGTTTCTGCCTCTTCCGTGGAACACACACATGGTGAACT 
               
               
                   
                   
                 CCTGGAAAAAACACTGTCTCAATATTATC 
               
               
                   
                   
                 CAGATTGTGTTTCCATTGCGGTGCAGAAAACCACATCTCAC 
               
               
                   
                   
                 ATAAATGCCATTAACAGTCAGGCTACTAA 
               
               
                   
                   
                 TGAGTTGTCCTGTGAGATCACTCACCCATCGCATACCTCAG 
               
               
                   
                   
                 GGCAGATCAATTCCGCACAGACCTCTAAC 
               
               
                   
                   
                 TCTGAGCTGCCTCCAAAGCCAGCTGCAGTGGTGAGTGAGGC 
               
               
                   
                   
                 CTGTGATGCTGATGATGCTGATAATGCCA 
               
               
                   
                   
                 GTAAACTAGCTGCAATGCTAAATACCTGTTCCTTTCAGAAA 
               
               
                   
                   
                 CCAGAACAACTACAACAACAAAAATCAGT 
               
               
                   
                   
                 TTTTGAGATATGCCCATCTCCTGCAGAAAATAACATCCAGG 
               
               
                   
                   
                 GAACCACAAAGCTAGCGTCTGGTGAAGAA 
               
               
                   
                   
                 TTCTGTTCAGGTTCCAGCAGCAATTTGCAAGCTCCTGGTGGC 
               
               
                   
                   
                 AGCTCTGAACGGTATTTAAAACAAAATG 
               
               
                   
                   
                 AAATGAATGGTGCTTACTTCAAGCAAAGCTCAGTGTTCACT 
               
               
                   
                   
                 AAGGATTCCTTTTCTGCCACTACCACACC 
               
               
                   
                   
                 ACCACCACCATCACAATTGCTTCTTTCTCCCCCTCCTCCTCT 
               
               
                   
                   
                 TCCACAGGTTCCTCAGCTTCCTTCAGAA 
               
               
                   
                   
                 GGAAAAAGCACTCTGAATGGTGGAGTTTTAGAAGAACACC 
               
               
                   
                   
                 ACCACTACCCCAACCAAAGTAACACAACAC 
               
               
                   
                   
                 TTTTAAGGGAAGTGAAAATAGAGGGTAAACCTGAGGCACC 
               
               
                   
                   
                 ACCTTCCCAGAGTCCTAATCCATCTACACA 
               
               
                   
                   
                 TGTATGCAGCCCTTCTCCGATGCTTTCTGAAAGGCCTCAGA 
               
               
                   
                   
                 ATAATTGTGTGAACAGGAATGACATACAG 
               
               
                   
                   
                 ACTGCAGGGACAATGACTGTTCCATTGTGTTCTGAGAAAAC 
               
               
                   
                   
                 AAGACCAATGTCAGAACACCTCAAGCATA 
               
               
                   
                   
                 ACCCACCAATTTTTGGTAGCAGTGGAGAGCTACAGGACAAC 
               
               
                   
                   
                 TGCCAGCAGTTGATGAGAAACAAAGAGCA 
               
               
                   
                   
                 AGAGATTCTGAAGGGTCGAGACAAGGAGCAAACACGAGAT 
               
               
                   
                   
                 CTTGTGCCCCCAACACAGCACTATCTGAAA 
               
               
                   
                   
                 CCAGGATGGATTGAATTGAAGGCCCCTCGTTTTCACCAAGC 
               
               
                   
                   
                 GGAATCCCATCTAAAACGTAATGAGGCAT 
               
               
                   
                   
                 CACTGCCATCAATTCTTCAGTATCAACCCAATCTCTCCAATC 
               
               
                   
                   
                 AAATGACCTCCAAACAATACACTGGAAA 
               
               
                   
                   
                 TTCCAACATGCCTGGGGGGCTCCCAAGGCAAGCTTACACCC 
               
               
                   
                   
                 AGAAAACAACACAGCTGGAGCACAAGTCA 
               
               
                   
                   
                 CAAATGTACCAAGTTGAAATGAATCAAGGGCAGTCCCAAG 
               
               
                   
                   
                 GTACAGTGGACCAACATCTCCAGTTCCAAA 
               
               
                   
                   
                 AACCCTCACACCAGGTGCACTTCTCCAAAACAGACCATTTA 
               
               
                   
                   
                 CCAAAAGCTCATGTGCAGTCACTGTGTGG 
               
               
                   
                   
                 CACTAGATTTCATTTTCAACAAAGAGCAGATTCCCAAACTG 
               
               
                   
                   
                 AAAAACTTATGTCCCCAGTGTTGAAACAG 
               
               
                   
                   
                 CACTTGAATCAACAGGCTTCAGAGACTGAGCCATTTTCAAA 
               
               
                   
                   
                 CTCACACCTTTTGCAACATAAGCCTCATA 
               
               
                   
                   
                 AACAGGCAGCACAAACACAACCATCCCAGAGTTCACATCTC 
               
               
                   
                   
                 CCTCAAAACCAGCAACAGCAGCAAAAATT 
               
               
                   
                   
                 ACAAATAAAGAATAAAGAGGAAATACTCCAGACTTTTCCTC 
               
               
                   
                   
                 ACCCCCAAAGCAACAATGATCAGCAAAGA 
               
               
                   
                   
                 GAAGGATCATTCTTTGGCCAGACTAAAGTGGAAGAATGTTT 
               
               
                   
                   
                 TCATGGTGAAAATCAGTATTCAAAATCAA 
               
               
                   
                   
                 GCGAGTTCGAGACTCATAATGTCCAAATGGGACTGGAGGA 
               
               
                   
                   
                 AGTACAGAATATAAATCGTAGAAATTCCCC 
               
               
                   
                   
                 TTATAGTCAGACCATGAAATCAAGTGCATGCAAAATACAGG 
               
               
                   
                   
                 TTTCTTGTTCAAACAATACACACCTAGTT 
               
               
                   
                   
                 TCAGAGAATAAAGAACAGACTACACATCCTGAACTTTTTGC 
               
               
                   
                   
                 AGGAAACAAGACCCAAAACTTGCATCACA 
               
               
                   
                   
                 TGCAATATTTTCCAAATAATGTGATCCCAAAGCAAGATCTT 
               
               
                   
                   
                 CTTCACAGGTGCTTTCAAGAACAGGAGCA 
               
               
                   
                   
                 GAAGTCACAACAAGCTTCAGTTCTACAGGGATATAAAAATA 
               
               
                   
                   
                 GAAACCAAGATATGTCTGGTCAACAAGCT 
               
               
                   
                   
                 GCGCAACTTGCTCAGCAAAGGTACTTGATACATAACCATGC 
               
               
                   
                   
                 AAATGTTTTTCCTGTGCCTGACCAGGGAG 
               
               
                   
                   
                 GAAGTCACACTCAGACCCCTCCCCAGAAGGACACTCAAAA 
               
               
                   
                   
                 GCATGCTGCTCTAAGGTGGCATCTCTTACA 
               
               
                   
                   
                 GAAGCAAGAACAGCAGCAAACACAGCAACCCCAAACTGAG 
               
               
                   
                   
                 TCTTGCCATAGTCAGATGCACAGGCCAATT 
               
               
                   
                   
                 AAGGTGGAACCTGGATGCAAGCCACATGCCTGTATGCACAC 
               
               
                   
                   
                 AGCACCACCAGAAAACAAAACATGGAAAA 
               
               
                   
                   
                 AGGTAACTAAGCAAGAGAATCCACCTGCAAGCTGTGATAAT 
               
               
                   
                   
                 GTGCAGCAAAAGAGCATCATTGAGACCAT 
               
               
                   
                   
                 GGAGCAGCATCTGAAGCAGTTTCACGCCAAGTCGTTATTTG 
               
               
                   
                   
                 ACCATAAGGCTCTTACTCTCAAATCACAG 
               
               
                   
                   
                 AAGCAAGTAAAAGTTGAAATGTCAGGGCCAGTCACAGTTTT 
               
               
                   
                   
                 GACTAGACAAACCACTGCTGCAGAACTTG 
               
               
                   
                   
                 ATAGCCACACCCCAGCTTTAGAGCAGCAAACAACTTCTTCA 
               
               
                   
                   
                 GAAAAGACACCAACCAAAAGAACAGCTGC 
               
               
                   
                   
                 TTCTGTTCTCAATAATTTTATAGAGTCACCTTCCAAATTACT 
               
               
                   
                   
                 AGATACTCCTATAAAAAATTTATTGGAT 
               
               
                   
                   
                 ACACCTGTCAAGACTCAATATGATTTCCCATCTTGCAGATGT 
               
               
                   
                   
                 GTAGAGCAAATTATTGAAAAAGATGAAG 
               
               
                   
                   
                 GTCCTTTTTATACCCATCTAGGAGCAGGTCCTAATGTGGCA 
               
               
                   
                   
                 GCTATTAGAGAAATCATGGAAGAAAGGTT 
               
               
                   
                   
                 TGGACAGAAGGGTAAAGCTATTAGGATTGAAAGAGTCATCT 
               
               
                   
                   
                 ATACTGGTAAAGAAGGCAAAAGTTCTCAG 
               
               
                   
                   
                 GGATGTCCTATTGCTAAGTGGGTGGTTCGCAGAAGCAGCAG 
               
               
                   
                   
                 TGAAGAGAAGCTACTGTGTTTGGTGCGGG 
               
               
                   
                   
                 AGCGAGCTGGCCACACCTGTGAGGCTGCAGTGATTGTGATT 
               
               
                   
                   
                 CTCATCCTGGTGTGGGAAGGAATCCCGCT 
               
               
                   
                   
                 GTCTCTGGCTGACAAACTCTACTCGGAGCTTACCGAGACGC 
               
               
                   
                   
                 TGAGGAAATACGGCACGCTCACCAATCGC 
               
               
                   
                   
                 CGGTGTGCCTTGAATGAAGAGAGAACTTGCGCCTGTCAGGG 
               
               
                   
                   
                 GCTGGATCCAGAAACCTGTGGTGCCTCCT 
               
               
                   
                   
                 TCTCTTTTGGTTGTTCATGGAGCATGTACTACAATGGATGTA 
               
               
                   
                   
                 AGTTTGCCAGAAGCAAGATCCCAAGGAA 
               
               
                   
                   
                 GTTTAAGCTGCTTGGGGATGACCCAAAAGAGGAAGAGAAA 
               
               
                   
                   
                 CTGGAGTCTCATTTGCAAAACCTGTCCACT 
               
               
                   
                   
                 CTTATGGCACCAACATATAAGAAACTTGCACCTGATGCATA 
               
               
                   
                   
                 TAATAATCAGATTGAATATGAACACAGAG 
               
               
                   
                   
                 CACCAGAGTGCCGTCTGGGTCTGAAGGAAGGCCGTCCATTC 
               
               
                   
                   
                 TCAGGGGTCACTGCATGTTTGGACTTCTG 
               
               
                   
                   
                 TGCTCATGCCCACAGAGACTTGCACAACATGCAGAATGGCA 
               
               
                   
                   
                 GCACATTGGTATGCACTCTCACTAGAGAA 
               
               
                   
                   
                 GACAATCGAGAATTTGGAGGAAAACCTGAGGATGAGCAGC 
               
               
                   
                   
                 TTCACGTTCTGCCTTTATACAAAGTCTCTG 
               
               
                   
                   
                 ACGTGGATGAGTTTGGGAGTGTGGAAGCTCAGGAGGAGAA 
               
               
                   
                   
                 AAAACGGAGTGGTGCCATTCAGGTACTGAG 
               
               
                   
                   
                 TTCTTTTCGGCGAAAAGTCAGGATGTTAGCAGAGCCAGTCA 
               
               
                   
                   
                 AGACTTGCCGACAAAGGAAACTAGAAGCC 
               
               
                   
                   
                 AAGAAAGCTGCAGCTGAAAAGCTTTCCTCCCTGGAGAACAG 
               
               
                   
                   
                 CTCAAATAAAAATGAAAAGGAAAAGTCAG 
               
               
                   
                   
                 CCCCATCACGTACAAAACAAACTGAAAACGCAAGCCAGGC 
               
               
                   
                   
                 TAAACAGTTGGCAGAACTTTTGCGACTTTC 
               
               
                   
                   
                 AGGACCAGTCATGCAGCAGTCCCAGCAGCCCCAGCCTCTAC 
               
               
                   
                   
                 AGAAGCAGCCACCACAGCCCCAGCAGCAG 
               
               
                   
                   
                 CAGAGACCCCAGCAGCAGCAGCCACATCACCCTCAGACAG 
               
               
                   
                   
                 AGTCTGTCAACTCTTATTCTGCTTCTGGAT 
               
               
                   
                   
                 CCACCAATCCATACATGAGACGGCCCAATCCAGTTAGTCCT 
               
               
                   
                   
                 TATCCAAACTCTTCACACACTTCAGATAT 
               
               
                   
                   
                 CTATGGAAGCACCAGCCCTATGAACTTCTATTCCACCTCATC 
               
               
                   
                   
                 TCAAGCTGCAGGTTCATATTTGAATTCT 
               
               
                   
                   
                 TCTAATCCCATGAACCCTTACCCTGGGCTTTTGAATCAGAAT 
               
               
                   
                   
                 ACCCAATATCCATCATATCAATGCAATG 
               
               
                   
                   
                 GAAACCTATCAGTGGACAACTGCTCCCCATATCTGGGTTCC 
               
               
                   
                   
                 TATTCTCCCCAGTCTCAGCCGATGGATCT 
               
               
                   
                   
                 GTATAGGTATCCAAGCCAAGACCCTCTGTCTAAGCTCAGTC 
               
               
                   
                   
                 TACCACCCATCCATACACTTTACCAGCCA 
               
               
                   
                   
                 AGGTTTGGAAATAGCCAGAGTTTTACATCTAAATACTTAGG 
               
               
                   
                   
                 TTATGGAAACCAAAATATGCAGGGAGATG 
               
               
                   
                   
                 GTTTCAGCAGTTGTACCATTAGACCAAATGTACATCATGTA 
               
               
                   
                   
                 GGGAAATTGCCTCCTTATCCCACTCATGA 
               
               
                   
                   
                 GATGGATGGCCACTTCATGGGAGCCACCTCTAGATTACCAC 
               
               
                   
                   
                 CCAATCTGAGCAATCCAAACATGGACTAT 
               
               
                   
                   
                 AAAAATGGTGAACATCATTCACCTTCTCACATAATCCATAA 
               
               
                   
                   
                 CTACAGTGCAGCTCCGGGCATGTTCAACA 
               
               
                   
                   
                 GCTCTCTTCATGCCCTGCATCTCCAAAACAAGGAGAATGAC 
               
               
                   
                   
                 ATGCTTTCCCACACAGCTAATGGGTTATC 
               
               
                   
                   
                 AAAGATGCTTCCAGCTCTTAACCATGATAGAACTGCTTGTG 
               
               
                   
                   
                 TCCAAGGAGGCTTACACAAATTAAGTGAT 
               
               
                   
                   
                 GCTAATGGTCAGGAAAAGCAGCCATTGGCACTAGTCCAGG 
               
               
                   
                   
                 GTGTGGCTTCTGGTGCAGAGGACAACGATG 
               
               
                   
                   
                 AGGTCTGGTCAGACAGCGAGCAGAGCTTTCTGGATCCTGAC 
               
               
                   
                   
                 ATTGGGGGAGTGGCCGTGGCTCCAACTCA 
               
               
                   
                   
                 TGGGTCAATTCTCATTGAGTGTGCAAAGCGTGAGCTGCATG 
               
               
                   
                   
                 CCACAACCCCTTTAAAGAATCCCAATAGG 
               
               
                   
                   
                 AATCACCCCACCAGGATCTCCCTCGTCTTTTACCAGCATAA 
               
               
                   
                   
                 GAGCATGAATGAGCCAAAACATGGCTTGG 
               
               
                   
                   
                 CTCTTTGGGAAGCCAAAATGGCTGAAAAAGCCCGTGAGAA 
               
               
                   
                   
                 AGAGGAAGAGTGTGAAAAGTATGGCCCAGA 
               
               
                   
                   
                 CTATGTGCCTCAGAAATCCCATGGCAAAAAAGTGAAACGG 
               
               
                   
                   
                 GAGCCTGCTGAGCCACATGAAACTTCAGAG 
               
               
                   
                   
                 CCCACTTACCTGCGTTTCATCAAGTCTCTTGCCGAAAGGACC 
               
               
                   
                   
                 ATGTCCGTGACCACAGACTCCACAGTAA 
               
               
                   
                   
                 CTACATCTCCATATGCCTTCACTCGGGTCACAGGGCCTTACA 
               
               
                   
                   
                 ACAGATATATATGATATCACCCCCTTTT 
               
               
                   
                   
                 GTTGGTTACCTCACTTGAAAAGACCACAACCAACCTGTCAG 
               
               
                   
                   
                 TAGTATAGTTCTCATGACGTGGGCAGTGG 
               
               
                   
                   
                 GGAAAGGTCACAGTATTCATGACAAATGTGGTGGGAAAAA 
               
               
                   
                   
                 CCTCAGCTCACCAGCAACAAAAGAGGTTAT 
               
               
                   
                   
                 CTTACCATAGCACTTAATTTTCACTGGCTCCCAAGTGGTCAC 
               
               
                   
                   
                 AGATGGCATCTAGGAAAAGACCAAAGCA 
               
               
                   
                   
                 TTCTATGCAAAAAGAAGGTGGGGAAGAAAGTGTTCCGCAA 
               
               
                   
                   
                 TTTACATTTTTAAACACTGGTTCTATTATT 
               
               
                   
                   
                 GGACGAGATGATATGTAAATGTGATCCCCCCCCCCCGCTTA 
               
               
                   
                   
                 CAACTCTACACATCTGTGACCACTTTTAA 
               
               
                   
                   
                 TAATATCAAGTTTGCATAGTCATGGAACACAAATCAAACAA 
               
               
                   
                   
                 GTACTGTAGTATTACAGTGACAGGAATCT 
               
               
                   
                   
                 TAAAATACCATCTGGTGCTGAATATATGATGTACTGAAATA 
               
               
                   
                   
                 CTGGAATTATGGCTTTTTGAAATGCAGTT 
               
               
                   
                   
                 TTTACTGTAATCTTAACTTTTATTTATCAAAATAGCTACAGG 
               
               
                   
                   
                 AAACATGAATAGCAGGAAAACACTGAAT 
               
               
                   
                   
                 TTGTTTGGATGTTCTAAGAAATGGTGCTAAGAAAATGGTGT 
               
               
                   
                   
                 CTTTAATAGCTAAAAATTTAATGCCTTTA 
               
               
                   
                   
                 TATCATCAAGATGCTATCAGTGTACTCCAGTGCCCTTGAAT 
               
               
                   
                   
                 AATAGGGGTACCTTTTCATTCAAGTTTTT 
               
               
                   
                   
                 ATCATAATTACCTATTCTTACACAAGCTTAGTTTTTAAAATG 
               
               
                   
                   
                 TGGACATTTTAAAGGCCTCTGGATTTTG 
               
               
                   
                   
                 CTCATCCAGTGAAGTCCTTGTAGGACAATAAACGTATATAT 
               
               
                   
                   
                 GTACATATATACACAAACATGTATATGTG 
               
               
                   
                   
                 CACACACATGTATATGTATAAATATTTTAAATGGTGTTTTAG 
               
               
                   
                   
                 AAGCACTTTGTCTACCTAAGCTTTGACA 
               
               
                   
                   
                 ACTTGAACAATGCTAAGGTACTGAGATGTTTAAAAAACAAG 
               
               
                   
                   
                 TTTACTTTCATTTTAGAATGCAAAGTTGA 
               
               
                   
                   
                 TTTTTTTAAGGAAACAAAGAAAGCTTTTAAAATATTTTTGCT 
               
               
                   
                   
                 TTTAGCCATGCATCTGCTGATGAGCAAT 
               
               
                   
                   
                 TGTGTCCATTTTTAACACAGCCAGTTAAATCCACCATGGGG 
               
               
                   
                   
                 CTTACTGGATTCAAGGGAATACGTTAGTC 
               
               
                   
                   
                 CACAAAACATGTTTTCTGGTGCTCATCTCACATGCTATACTG 
               
               
                   
                   
                 TAAAACAGTTTTATACAAAATTGTATGA 
               
               
                   
                   
                 CAAGTTCATTGCTCAAAAATGTACAGTTTTAAGAATTTTCTA 
               
               
                   
                   
                 TTAACTGCAGGTAATAATTAGCTGCATG 
               
               
                   
                   
                 CTGCAGACTCAACAAAGCTAGTTCACTGAAGCCTATGCTAT 
               
               
                   
                   
                 TTTATGGATCATAGGCTCTTCAGAGAACT 
               
               
                   
                   
                 GAATGGCAGTCTGCCTTTGTGTTGATAATTATGTACATTGTG 
               
               
                   
                   
                 ACGTTGTCATTTCTTAGCTTAAGTGTCC 
               
               
                   
                   
                 TCTTTAACAAGAGGATTGAGCAGACTGATGCCTGCATAAGA 
               
               
                   
                   
                 TGAATAAACAGGGTTAGTTCCATGTGAAT 
               
               
                   
                   
                 CTGTCAGTTAAAAAGAAACAAAAACAGGCAGCTGGTTTGCT 
               
               
                   
                   
                 GTGGTGGTTTTAAATCATTAATTTGTATA 
               
               
                   
                   
                 AAGAAGTGAAAGAGTTGTATAGTAAATTAAATTGTAAACA 
               
               
                   
                   
                 AAACTTTTTTAATGCAATGCTTTAGTATTT 
               
               
                   
                   
                 TAGTACTGTAAAAAAATTAAATATATACATATATATATATA 
               
               
                   
                   
                 TATATATATATATATATATGAGTTTGAAG 
               
               
                   
                   
                 CAGAATTCACATCATGATGGTGCTACTCAGCCTGCTACAAA 
               
               
                   
                   
                 TATATCATAATGTGAGCTAAGAATTCATT 
               
               
                   
                   
                 AAATGTTTGAGTGATGTTCCTACTTGTCATATACCTCAACAC 
               
               
                   
                   
                 TAGTTTGGCAATAGGATATTGAACTGAG 
               
               
                   
                   
                 AGTGAAAGCATTGTGTACCATCATTTTTTTCCAAGTCCTTTT 
               
               
                   
                   
                 TTTTATTGTTAAAAAAAAAAGCATACCT 
               
               
                   
                   
                 TTTTTCAATACTTGATTTCTTAGCAAGTATAACTTGAACTTC 
               
               
                   
                   
                 AACCTTTTTGTTCTAAAAATTCAGGGAT 
               
               
                   
                   
                 ATTTCAGCTCATGCTCTCCCTATGCCAACATGTCACCTGTGT 
               
               
                   
                   
                 TTATGTAAAATTGTTGTAGGTTAATAAA 
               
               
                   
                   
                 TATATTCTTTGTCAGGGATTTAACCCTTTTATTTTGAATCCCT 
               
               
                   
                   
                 TCTATTTTACTTGTACATGTGCTGATG 
               
               
                   
                   
                 TAACTAAAACTAATTTTGTAAATCTGTTGGCTCTTTTTATTG 
               
               
                   
                   
                 TAAAGAAAAGCATTTTAAAAGTTTGAGG 
               
               
                   
                   
                 AATCTTTTGACTGTTTCAAGCAGGAAAAAAAAATTACATGA 
               
               
                   
                   
                 AAATAGAATGCACTGAGTTGATAAAGGGA 
               
               
                   
                   
                 AAAATTGTAAGGCAGGAGTTTGGCAAGTGGCTGTTGGCCAG 
               
               
                   
                   
                 AGACTTACTTGTAACTCTCTAAATGAAGT 
               
               
                   
                   
                 TTTTTTGATCCTGTAATCACTGAAGGTACATACTCCATGTGG 
               
               
                   
                   
                 ACTTCCCTTAAACAGGCAAACACCTACA 
               
               
                   
                   
                 GGTATGGTGTGCAACAGATTGTACAATTACATTTTGGCCTA 
               
               
                   
                   
                 AATACATTTTTGCTTACTAGTATTTAAAA 
               
               
                   
                   
                 TAAATTCTTAATCAGAGGAGGCCTTTGGGTTTTATTGGTCAA 
               
               
                   
                   
                 ATCTTTGTAAGCTGGCTTTTGTCTTTTT 
               
               
                   
                   
                 AAAAAATTTCTTGAATTTGTGGTTGTGTCCAATTTGCAAACA 
               
               
                   
                   
                 TTTCCAAAAATGTTTGCTTTGCTTACAA 
               
               
                   
                   
                 ACCACATGATTTTAATGTTTTTTGTATACCATAATATCTAGC 
               
               
                   
                   
                 CCCAAACATTTGATTACTACATGTGCAT 
               
               
                   
                   
                 TGGTGATTTTGATCATCCATTCTTAATATTTGATTTCTGTGTC 
               
               
                   
                   
                 ACCTACTGTCATTTGTTAAACTGCTGG 
               
               
                   
                   
                 CCAACAAGAACAGGAAGTATAGTTTGGGGGGTTGGGGAGA 
               
               
                   
                   
                 GTTTACATAAGGAAGAGAAGAAATTGAGTG 
               
               
                   
                   
                 GCATATTGTAAATATCAGATCTATAATTGTAAATATAAAAC 
               
               
                   
                   
                 CTGCCTCAGTTAGAATGAATGGAAAGCAG 
               
               
                   
                   
                 ATCTACAATTTGCTAATATAGGAATATCAGGTTGACTATAT 
               
               
                   
                   
                 AGCCATACTTGAAAATGCTTCTGAGTGGT 
               
               
                   
                   
                 GTCAACTTTACTTGAATGAATTTTTCATCTTGATTGACGCAC 
               
               
                   
                   
                 AGTGATGTACAGTTCACTTCTGAAGCTA 
               
               
                   
                   
                 GTGGTTAACTTGTGTAGGAAACTTTTGCAGTTTGACACTAA 
               
               
                   
                   
                 GATAACTTCTGTGTGCATTTTTCTATGCT 
               
               
                   
                   
                 TTTTTAAAAACTAGTTTCATTTCATTTTCATGAGATGTTTGG 
               
               
                   
                   
                 TTTATAAGATCTGAGGATGGTTATAAAT 
               
               
                   
                   
                 ACTGTAAGTATTGTAATGTTATGAATGCAGGTTATTTGAAA 
               
               
                   
                   
                 GCTGTTTATTATTATATCATTCCTGATAA 
               
               
                   
                   
                 TGCTATGTGAGTGTTTTTAATAAAATTTATATTTATTTAATG 
               
               
                   
                   
                 CACTCTAAAAAAAAAAAAAAAAAA 
               
               
                   
               
               
                 PPREDICTED: 
                 XXM_005263082.1 
                 AAGCAGAAGGAAGCAAGATGGCTGCCCTTTAGGATTTGTTA 
               
               
                 
                   Homo sapiens 
                 
                   
                 GAAAGGAGACCCGACTGCAACTGCTGGAT 
               
               
                 tet 
                   
                 TGCTGCAAGGCTGAGGGACGAGAACGAGAATTCAACTAGA 
               
               
                 methylcytosine 
                   
                 GGGCAGCCTTGTGGATGGCCCCGAAGCAAG 
               
               
                 dioxygenase 2 
                   
                 CCTGATGGAACAGGATAGAACCAACCATGTTGAGGGCAAC 
               
               
                 (TET2), 
                   
                 AGACTAAGTCCATTCCTGATACCATCACCT 
               
               
                 transcript 
                   
                 CCCATTTGCCAGACAGAACCTCTGGCTACAAAGCTCCAGAA 
               
               
                 variant X1, 
                   
                 TGGAAGCCCACTGCCTGAGAGAGCTCATC 
               
               
                 mRNA 
                   
                 CAGAAGTAAATGGAGACACCAAGTGGCACTCTTTCAAAAGT 
               
               
                 [SEQ ID NO: 
                   
                 TATTATGGAATACCCTGTATGAAGGGAAG 
               
               
                 1359] 
                   
                 CCAGAATAGTCGTGTGAGTCCTGACTTTACACAAGAAAGTA 
               
               
                   
                   
                 GAGGGTATTCCAAGTGTTTGCAAAATGGA 
               
               
                   
                   
                 GGAATAAAACGCACAGTTAGTGAACCTTCTCTCTCTGGGCT 
               
               
                   
                   
                 CCTTCAGATCAAGAAATTGAAACAAGACC 
               
               
                   
                   
                 AAAAGGCTAATGGAGAAAGACGTAACTTCGGGGTAAGCCA 
               
               
                   
                   
                 AGAAAGAAATCCAGGTGAAAGCAGTCAACC 
               
               
                   
                   
                 AAATGTCTCCGATTTGAGTGATAAGAAAGAATCTGTGAGTT 
               
               
                   
                   
                 CTGTAGCCCAAGAAAATGCAGTTAAAGAT 
               
               
                   
                   
                 TTCACCAGTTTTTCAACACATAACTGCAGTGGGCCTGAAAA 
               
               
                   
                   
                 TCCAGAGCTTCAGATTCTGAATGAGCAGG 
               
               
                   
                   
                 AGGGGAAAAGTGCTAATTACCATGACAAGAACATTGTATTA 
               
               
                   
                   
                 CTTAAAAACAAGGCAGTGCTAATGCCTAA 
               
               
                   
                   
                 TGGTGCTACAGTTTCTGCCTCTTCCGTGGAACACACACATG 
               
               
                   
                   
                 GTGAACTCCTGGAAAAAACACTGTCTCAA 
               
               
                   
                   
                 TATTATCCAGATTGTGTTTCCATTGCGGTGCAGAAAACCAC 
               
               
                   
                   
                 ATCTCACATAAATGCCATTAACAGTCAGG 
               
               
                   
                   
                 CTACTAATGAGTTGTCCTGTGAGATCACTCACCCATCGCAT 
               
               
                   
                   
                 ACCTCAGGGCAGATCAATTCCGCACAGAC 
               
               
                   
                   
                 CTCTAACTCTGAGCTGCCTCCAAAGCCAGCTGCAGTGGTGA 
               
               
                   
                   
                 GTGAGGCCTGTGATGCTGATGATGCTGAT 
               
               
                   
                   
                 AATGCCAGTAAACTAGCTGCAATGCTAAATACCTGTTCCTT 
               
               
                   
                   
                 TCAGAAACCAGAACAACTACAACAACAAA 
               
               
                   
                   
                 AATCAGTTTTTGAGATATGCCCATCTCCTGCAGAAAATAAC 
               
               
                   
                   
                 ATCCAGGGAACCACAAAGCTAGCGTCTGG 
               
               
                   
                   
                 TGAAGAATTCTGTTCAGGTTCCAGCAGCAATTTGCAAGCTC 
               
               
                   
                   
                 CTGGTGGCAGCTCTGAACGGTATTTAAAA 
               
               
                   
                   
                 CAAAATGAAATGAATGGTGCTTACTTCAAGCAAAGCTCAGT 
               
               
                   
                   
                 GTTCACTAAGGATTCCTTTTCTGCCACTA 
               
               
                   
                   
                 CCACACCACCACCACCATCACAATTGCTTCTTTCTCCCCCTC 
               
               
                   
                   
                 CTCCTCTTCCACAGGTTCCTCAGCTTCC 
               
               
                   
                   
                 TTCAGAAGGAAAAAGCACTCTGAATGGTGGAGTTTTAGAAG 
               
               
                   
                   
                 AACACCACCACTACCCCAACCAAAGTAAC 
               
               
                   
                   
                 ACAACACTTTTAAGGGAAGTGAAAATAGAGGGTAAACCTG 
               
               
                   
                   
                 AGGCACCACCTTCCCAGAGTCCTAATCCAT 
               
               
                   
                   
                 CTACACATGTATGCAGCCCTTCTCCGATGCTTTCTGAAAGGC 
               
               
                   
                   
                 CTCAGAATAATTGTGTGAACAGGAATGA 
               
               
                   
                   
                 CATACAGACTGCAGGGACAATGACTGTTCCATTGTGTTCTG 
               
               
                   
                   
                 AGAAAACAAGACCAATGTCAGAACACCTC 
               
               
                   
                   
                 AAGCATAACCCACCAATTTTTGGTAGCAGTGGAGAGCTACA 
               
               
                   
                   
                 GGACAACTGCCAGCAGTTGATGAGAAACA 
               
               
                   
                   
                 AAGAGCAAGAGATTCTGAAGGGTCGAGACAAGGAGCAAAC 
               
               
                   
                   
                 ACGAGATCTTGTGCCCCCAACACAGCACTA 
               
               
                   
                   
                 TCTGAAACCAGGATGGATTGAATTGAAGGCCCCTCGTTTTC 
               
               
                   
                   
                 ACCAAGCGGAATCCCATCTAAAACGTAAT 
               
               
                   
                   
                 GAGGCATCACTGCCATCAATTCTTCAGTATCAACCCAATCT 
               
               
                   
                   
                 CTCCAATCAAATGACCTCCAAACAATACA 
               
               
                   
                   
                 CTGGAAATTCCAACATGCCTGGGGGGCTCCCAAGGCAAGCT 
               
               
                   
                   
                 TACACCCAGAAAACAACACAGCTGGAGCA 
               
               
                   
                   
                 CAAGTCACAAATGTACCAAGTTGAAATGAATCAAGGGCAG 
               
               
                   
                   
                 TCCCAAGGTACAGTGGACCAACATCTCCAG 
               
               
                   
                   
                 TTCCAAAAACCCTCACACCAGGTGCACTTCTCCAAAACAGA 
               
               
                   
                   
                 CCATTTACCAAAAGCTCATGTGCAGTCAC 
               
               
                   
                   
                 TGTGTGGCACTAGATTTCATTTTCAACAAAGAGCAGATTCC 
               
               
                   
                   
                 CAAACTGAAAAACTTATGTCCCCAGTGTT 
               
               
                   
                   
                 GAAACAGCACTTGAATCAACAGGCTTCAGAGACTGAGCCAT 
               
               
                   
                   
                 TTTCAAACTCACACCTTTTGCAACATAAG 
               
               
                   
                   
                 CCTCATAAACAGGCAGCACAAACACAACCATCCCAGAGTTC 
               
               
                   
                   
                 ACATCTCCCTCAAAACCAGCAACAGCAGC 
               
               
                   
                   
                 AAAAATTACAAATAAAGAATAAAGAGGAAATACTCCAGAC 
               
               
                   
                   
                 TTTTCCTCACCCCCAAAGCAACAATGATCA 
               
               
                   
                   
                 GCAAAGAGAAGGATCATTCTTTGGCCAGACTAAAGTGGAA 
               
               
                   
                   
                 GAATGTTTTCATGGTGAAAATCAGTATTCA 
               
               
                   
                   
                 AAATCAAGCGAGTTCGAGACTCATAATGTCCAAATGGGACT 
               
               
                   
                   
                 GGAGGAAGTACAGAATATAAATCGTAGAA 
               
               
                   
                   
                 ATTCCCCTTATAGTCAGACCATGAAATCAAGTGCATGCAAA 
               
               
                   
                   
                 ATACAGGTTTCTTGTTCAAACAATACACA 
               
               
                   
                   
                 CCTAGTTTCAGAGAATAAAGAACAGACTACACATCCTGAAC 
               
               
                   
                   
                 TTTTTGCAGGAAACAAGACCCAAAACTTG 
               
               
                   
                   
                 CATCACATGCAATATTTTCCAAATAATGTGATCCCAAAGCA 
               
               
                   
                   
                 AGATCTTCTTCACAGGTGCTTTCAAGAAC 
               
               
                   
                   
                 AGGAGCAGAAGTCACAACAAGCTTCAGTTCTACAGGGATAT 
               
               
                   
                   
                 AAAAATAGAAACCAAGATATGTCTGGTCA 
               
               
                   
                   
                 ACAAGCTGCGCAACTTGCTCAGCAAAGGTACTTGATACATA 
               
               
                   
                   
                 ACCATGCAAATGTTTTTCCTGTGCCTGAC 
               
               
                   
                   
                 CAGGGAGGAAGTCACACTCAGACCCCTCCCCAGAAGGACA 
               
               
                   
                   
                 CTCAAAAGCATGCTGCTCTAAGGTGGCATC 
               
               
                   
                   
                 TCTTACAGAAGCAAGAACAGCAGCAAACACAGCAACCCCA 
               
               
                   
                   
                 AACTGAGTCTTGCCATAGTCAGATGCACAG 
               
               
                   
                   
                 GCCAATTAAGGTGGAACCTGGATGCAAGCCACATGCCTGTA 
               
               
                   
                   
                 TGCACACAGCACCACCAGAAAACAAAACA 
               
               
                   
                   
                 TGGAAAAAGGTAACTAAGCAAGAGAATCCACCTGCAAGCT 
               
               
                   
                   
                 GTGATAATGTGCAGCAAAAGAGCATCATTG 
               
               
                   
                   
                 AGACCATGGAGCAGCATCTGAAGCAGTTTCACGCCAAGTCG 
               
               
                   
                   
                 TTATTTGACCATAAGGCTCTTACTCTCAA 
               
               
                   
                   
                 ATCACAGAAGCAAGTAAAAGTTGAAATGTCAGGGCCAGTC 
               
               
                   
                   
                 ACAGTTTTGACTAGACAAACCACTGCTGCA 
               
               
                   
                   
                 GAACTTGATAGCCACACCCCAGCTTTAGAGCAGCAAACAAC 
               
               
                   
                   
                 TTCTTCAGAAAAGACACCAACCAAAAGAA 
               
               
                   
                   
                 CAGCTGCTTCTGTTCTCAATAATTTTATAGAGTCACCTTCCA 
               
               
                   
                   
                 AATTACTAGATACTCCTATAAAAAATTT 
               
               
                   
                   
                 ATTGGATACACCTGTCAAGACTCAATATGATTTCCCATCTTG 
               
               
                   
                   
                 CAGATGTGTAGAGCAAATTATTGAAAAA 
               
               
                   
                   
                 GATGAAGGTCCTTTTTATACCCATCTAGGAGCAGGTCCTAA 
               
               
                   
                   
                 TGTGGCAGCTATTAGAGAAATCATGGAAG 
               
               
                   
                   
                 AAAGGTTTGGACAGAAGGGTAAAGCTATTAGGATTGAAAG 
               
               
                   
                   
                 AGTCATCTATACTGGTAAAGAAGGCAAAAG 
               
               
                   
                   
                 TTCTCAGGGATGTCCTATTGCTAAGTGGGTGGTTCGCAGAA 
               
               
                   
                   
                 GCAGCAGTGAAGAGAAGCTACTGTGTTTG 
               
               
                   
                   
                 GTGCGGGAGCGAGCTGGCCACACCTGTGAGGCTGCAGTGAT 
               
               
                   
                   
                 TGTGATTCTCATCCTGGTGTGGGAAGGAA 
               
               
                   
                   
                 TCCCGCTGTCTCTGGCTGACAAACTCTACTCGGAGCTTACCG 
               
               
                   
                   
                 AGACGCTGAGGAAATACGGCACGCTCAC 
               
               
                   
                   
                 CAATCGCCGGTGTGCCTTGAATGAAGAGAGAACTTGCGCCT 
               
               
                   
                   
                 GTCAGGGGCTGGATCCAGAAACCTGTGGT 
               
               
                   
                   
                 GCCTCCTTCTCTTTTGGTTGTTCATGGAGCATGTACTACAAT 
               
               
                   
                   
                 GGATGTAAGTTTGCCAGAAGCAAGATCC 
               
               
                   
                   
                 CAAGGAAGTTTAAGCTGCTTGGGGATGACCCAAAAGAGGA 
               
               
                   
                   
                 AGAGAAACTGGAGTCTCATTTGCAAAACCT 
               
               
                   
                   
                 GTCCACTCTTATGGCACCAACATATAAGAAACTTGCACCTG 
               
               
                   
                   
                 ATGCATATAATAATCAGATTGAATATGAA 
               
               
                   
                   
                 CACAGAGCACCAGAGTGCCGTCTGGGTCTGAAGGAAGGCC 
               
               
                   
                   
                 GTCCATTCTCAGGGGTCACTGCATGTTTGG 
               
               
                   
                   
                 ACTTCTGTGCTCATGCCCACAGAGACTTGCACAACATGCAG 
               
               
                   
                   
                 AATGGCAGCACATTGGTATGCACTCTCAC 
               
               
                   
                   
                 TAGAGAAGACAATCGAGAATTTGGAGGAAAACCTGAGGAT 
               
               
                   
                   
                 GAGCAGCTTCACGTTCTGCCTTTATACAAA 
               
               
                   
                   
                 GTCTCTGACGTGGATGAGTTTGGGAGTGTGGAAGCTCAGGA 
               
               
                   
                   
                 GGAGAAAAAACGGAGTGGTGCCATTCAGG 
               
               
                   
                   
                 TACTGAGTTCTTTTCGGCGAAAAGTCAGGATGTTAGCAGAG 
               
               
                   
                   
                 CCAGTCAAGACTTGCCGACAAAGGAAACT 
               
               
                   
                   
                 AGAAGCCAAGAAAGCTGCAGCTGAAAAGCTTTCCTCCCTGG 
               
               
                   
                   
                 AGAACAGCTCAAATAAAAATGAAAAGGAA 
               
               
                   
                   
                 AAGTCAGCCCCATCACGTACAAAACAAACTGAAAACGCAA 
               
               
                   
                   
                 GCCAGGCTAAACAGTTGGCAGAACTTTTGC 
               
               
                   
                   
                 GACTTTCAGGACCAGTCATGCAGCAGTCCCAGCAGCCCCAG 
               
               
                   
                   
                 CCTCTACAGAAGCAGCCACCACAGCCCCA 
               
               
                   
                   
                 GCAGCAGCAGAGACCCCAGCAGCAGCAGCCACATCACCCT 
               
               
                   
                   
                 CAGACAGAGTCTGTCAACTCTTATTCTGCT 
               
               
                   
                   
                 TCTGGATCCACCAATCCATACATGAGACGGCCCAATCCAGT 
               
               
                   
                   
                 TAGTCCTTATCCAAACTCTTCACACACTT 
               
               
                   
                   
                 CAGATATCTATGGAAGCACCAGCCCTATGAACTTCTATTCC 
               
               
                   
                   
                 ACCTCATCTCAAGCTGCAGGTTCATATTT 
               
               
                   
                   
                 GAATTCTTCTAATCCCATGAACCCTTACCCTGGGCTTTTGAA 
               
               
                   
                   
                 TCAGAATACCCAATATCCATCATATCAA 
               
               
                   
                   
                 TGCAATGGAAACCTATCAGTGGACAACTGCTCCCCATATCT 
               
               
                   
                   
                 GGGTTCCTATTCTCCCCAGTCTCAGCCGA 
               
               
                   
                   
                 TGGATCTGTATAGGTATCCAAGCCAAGACCCTCTGTCTAAG 
               
               
                   
                   
                 CTCAGTCTACCACCCATCCATACACTTTA 
               
               
                   
                   
                 CCAGCCAAGGTTTGGAAATAGCCAGAGTTTTACATCTAAAT 
               
               
                   
                   
                 ACTTAGGTTATGGAAACCAAAATATGCAG 
               
               
                   
                   
                 GGAGATGGTTTCAGCAGTTGTACCATTAGACCAAATGTACA 
               
               
                   
                   
                 TCATGTAGGGAAATTGCCTCCTTATCCCA 
               
               
                   
                   
                 CTCATGAGATGGATGGCCACTTCATGGGAGCCACCTCTAGA 
               
               
                   
                   
                 TTACCACCCAATCTGAGCAATCCAAACAT 
               
               
                   
                   
                 GGACTATAAAAATGGTGAACATCATTCACCTTCTCACATAA 
               
               
                   
                   
                 TCCATAACTACAGTGCAGCTCCGGGCATG 
               
               
                   
                   
                 TTCAACAGCTCTCTTCATGCCCTGCATCTCCAAAACAAGGA 
               
               
                   
                   
                 GAATGACATGCTTTCCCACACAGCTAATG 
               
               
                   
                   
                 GGTTATCAAAGATGCTTCCAGCTCTTAACCATGATAGAACT 
               
               
                   
                   
                 GCTTGTGTCCAAGGAGGCTTACACAAATT 
               
               
                   
                   
                 AAGTGATGCTAATGGTCAGGAAAAGCAGCCATTGGCACTA 
               
               
                   
                   
                 GTCCAGGGTGTGGCTTCTGGTGCAGAGGAC 
               
               
                   
                   
                 AACGATGAGGTCTGGTCAGACAGCGAGCAGAGCTTTCTGGA 
               
               
                   
                   
                 TCCTGACATTGGGGGAGTGGCCGTGGCTC 
               
               
                   
                   
                 CAACTCATGGGTCAATTCTCATTGAGTGTGCAAAGCGTGAG 
               
               
                   
                   
                 CTGCATGCCACAACCCCTTTAAAGAATCC 
               
               
                   
                   
                 CAATAGGAATCACCCCACCAGGATCTCCCTCGTCTTTTACC 
               
               
                   
                   
                 AGCATAAGAGCATGAATGAGCCAAAACAT 
               
               
                   
                   
                 GGCTTGGCTCTTTGGGAAGCCAAAATGGCTGAAAAAGCCCG 
               
               
                   
                   
                 TGAGAAAGAGGAAGAGTGTGAAAAGTATG 
               
               
                   
                   
                 GCCCAGACTATGTGCCTCAGAAATCCCATGGCAAAAAAGTG 
               
               
                   
                   
                 AAACGGGAGCCTGCTGAGCCACATGAAAC 
               
               
                   
                   
                 TTCAGAGCCCACTTACCTGCGTTTCATCAAGTCTCTTGCCGA 
               
               
                   
                   
                 AAGGACCATGTCCGTGACCACAGACTCC 
               
               
                   
                   
                 ACAGTAACTACATCTCCATATGCCTTCACTCGGGTCACAGG 
               
               
                   
                   
                 GCCTTACAACAGATATATATGATATCACC 
               
               
                   
                   
                 CCCTTTTGTTGGTTACCTCACTTGAAAAGACCACAACCAAC 
               
               
                   
                   
                 CTGTCAGTAGTATAGTTCTCATGACGTGG 
               
               
                   
                   
                 GCAGTGGGGAAAGGTCACAGTATTCATGACAAATGTGGTG 
               
               
                   
                   
                 GGAAAAACCTCAGCTCACCAGCAACAAAAG 
               
               
                   
                   
                 AGGTTATCTTACCATAGCACTTAATTTTCACTGGCTCCCAAG 
               
               
                   
                   
                 TGGTCACAGATGGCATCTAGGAAAAGAC 
               
               
                   
                   
                 CAAAGCATTCTATGCAAAAAGAAGGTGGGGAAGAAAGTGT 
               
               
                   
                   
                 TCCGCAATTTACATTTTTAAACACTGGTTC 
               
               
                   
                   
                 TATTATTGGACGAGATGATATGTAAATGTGATCCCCCCCCC 
               
               
                   
                   
                 CCGCTTACAACTCTACACATCTGTGACCA 
               
               
                   
                   
                 CTTTTAATAATATCAAGTTTGCATAGTCATGGAACACAAAT 
               
               
                   
                   
                 CAAACAAGTACTGTAGTATTACAGTGACA 
               
               
                   
                   
                 GGAATCTTAAAATACCATCTGGTGCTGAATATATGATGTAC 
               
               
                   
                   
                 TGAAATACTGGAATTATGGCTTTTTGAAA 
               
               
                   
                   
                 TGCAGTTTTTACTGTAATCTTAACTTTTATTTATCAAAATAG 
               
               
                   
                   
                 CTACAGGAAACATGAATAGCAGGAAAAC 
               
               
                   
                   
                 ACTGAATTTGTTTGGATGTTCTAAGAAATGGTGCTAAGAAA 
               
               
                   
                   
                 ATGGTGTCTTTAATAGCTAAAAATTTAAT 
               
               
                   
                   
                 GCCTTTATATCATCAAGATGCTATCAGTGTACTCCAGTGCCC 
               
               
                   
                   
                 TTGAATAATAGGGGTACCTTTTCATTCA 
               
               
                   
                   
                 AGTTTTTATCATAATTACCTATTCTTACACAAGCTTAGTTTT 
               
               
                   
                   
                 TAAAATGTGGACATTTTAAAGGCCTCTG 
               
               
                   
                   
                 GATTTTGCTCATCCAGTGAAGTCCTTGTAGGACAATAAACG 
               
               
                   
                   
                 TATATATGTACATATATACACAAACATGT 
               
               
                   
                   
                 ATATGTGCACACACATGTATATGTATAAATATTTTAAATGG 
               
               
                   
                   
                 TGTTTTAGAAGCACTTTGTCTACCTAAGC 
               
               
                   
                   
                 TTTGACAACTTGAACAATGCTAAGGTACTGAGATGTTTAAA 
               
               
                   
                   
                 AAACAAGTTTACTTTCATTTTAGAATGCA 
               
               
                   
                   
                 AAGTTGATTTTTTTAAGGAAACAAAGAAAGCTTTTAAAATA 
               
               
                   
                   
                 TTTTTGCTTTTAGCCATGCATCTGCTGAT 
               
               
                   
                   
                 GAGCAATTGTGTCCATTTTTAACACAGCCAGTTAAATCCAC 
               
               
                   
                   
                 CATGGGGCTTACTGGATTCAAGGGAATAC 
               
               
                   
                   
                 GTTAGTCCACAAAACATGTTTTCTGGTGCTCATCTCACATGC 
               
               
                   
                   
                 TATACTGTAAAACAGTTTTATACAAAAT 
               
               
                   
                   
                 TGTATGACAAGTTCATTGCTCAAAAATGTACAGTTTTAAGA 
               
               
                   
                   
                 ATTTTCTATTAACTGCAGGTAATAATTAG 
               
               
                   
                   
                 CTGCATGCTGCAGACTCAACAAAGCTAGTTCACTGAAGCCT 
               
               
                   
                   
                 ATGCTATTTTATGGATCATAGGCTCTTCA 
               
               
                   
                   
                 GAGAACTGAATGGCAGTCTGCCTTTGTGTTGATAATTATGT 
               
               
                   
                   
                 ACATTGTGACGTTGTCATTTCTTAGCTTA 
               
               
                   
                   
                 AGTGTCCTCTTTAACAAGAGGATTGAGCAGACTGATGCCTG 
               
               
                   
                   
                 CATAAGATGAATAAACAGGGTTAGTTCCA 
               
               
                   
                   
                 TGTGAATCTGTCAGTTAAAAAGAAACAAAAACAGGCAGCT 
               
               
                   
                   
                 GGTTTGCTGTGGTGGTTTTAAATCATTAAT 
               
               
                   
                   
                 TTGTATAAAGAAGTGAAAGAGTTGTATAGTAAATTAAATTG 
               
               
                   
                   
                 TAAACAAAACTTTTTTAATGCAATGCTTT 
               
               
                   
                   
                 AGTATTTTAGTACTGTAAAAAAATTAAATATATACATATAT 
               
               
                   
                   
                 ATATATATATATATATATATATATATGAG 
               
               
                   
                   
                 TTTGAAGCAGAATTCACATCATGATGGTGCTACTCAGCCTG 
               
               
                   
                   
                 CTACAAATATATCATAATGTGAGCTAAGA 
               
               
                   
                   
                 ATTCATTAAATGTTTGAGTGATGTTCCTACTTGTCATATACC 
               
               
                   
                   
                 TCAACACTAGTTTGGCAATAGGATATTG 
               
               
                   
                   
                 AACTGAGAGTGAAAGCATTGTGTACCATCATTTTTTTCCAA 
               
               
                   
                   
                 GTCCTTTTTTTTATTGTTAAAAAAAAAAG 
               
               
                   
                   
                 CATACCTTTTTTCAATACTTGATTTCTTAGCAAGTATAACTT 
               
               
                   
                   
                 GAACTTCAACCTTTTTGTTCTAAAAATT 
               
               
                   
                   
                 CAGGGATATTTCAGCTCATGCTCTCCCTATGCCAACATGTCA 
               
               
                   
                   
                 CCTGTGTTTATGTAAAATTGTTGTAGGT 
               
               
                   
                   
                 TAATAAATATATTCTTTGTCAGGGATTTAACCCTTTTATTTT 
               
               
                   
                   
                 GAATCCCTTCTATTTTACTTGTACATGT 
               
               
                   
                   
                 GCTGATGTAACTAAAACTAATTTTGTAAATCTGTTGGCTCTT 
               
               
                   
                   
                 TTTATTGTAAAGAAAAGCATTTTAAAAG 
               
               
                   
                   
                 TTTGAGGAATCTTTTGACTGTTTCAAGCAGGAAAAAAAAAT 
               
               
                   
                   
                 TACATGAAAATAGAATGCACTGAGTTGAT 
               
               
                   
                   
                 AAAGGGAAAAATTGTAAGGCAGGAGTTTGGCAAGTGGCTG 
               
               
                   
                   
                 TTGGCCAGAGACTTACTTGTAACTCTCTAA 
               
               
                   
                   
                 ATGAAGTTTTTTTGATCCTGTAATCACTGAAGGTACATACTC 
               
               
                   
                   
                 CATGTGGACTTCCCTTAAACAGGCAAAC 
               
               
                   
                   
                 ACCTACAGGTATGGTGTGCAACAGATTGTACAATTACATTT 
               
               
                   
                   
                 TGGCCTAAATACATTTTTGCTTACTAGTA 
               
               
                   
                   
                 TTTAAAATAAATTCTTAATCAGAGGAGGCCTTTGGGTTTTAT 
               
               
                   
                   
                 TGGTCAAATCTTTGTAAGCTGGCTTTTG 
               
               
                   
                   
                 TCTTTTTAAAAAATTTCTTGAATTTGTGGTTGTGTCCAATTT 
               
               
                   
                   
                 GCAAACATTTCCAAAAATGTTTGCTTTG 
               
               
                   
                   
                 CTTACAAACCACATGATTTTAATGTTTTTTGTATACCATAAT 
               
               
                   
                   
                 ATCTAGCCCCAAACATTTGATTACTACA 
               
               
                   
                   
                 TGTGCATTGGTGATTTTGATCATCCATTCTTAATATTTGATT 
               
               
                   
                   
                 TCTGTGTCACCTACTGTCATTTGTTAAA 
               
               
                   
                   
                 CTGCTGGCCAACAAGAACAGGAAGTATAGTTTGGGGGGTTG 
               
               
                   
                   
                 GGGAGAGTTTACATAAGGAAGAGAAGAAA 
               
               
                   
                   
                 TTGAGTGGCATATTGTAAATATCAGATCTATAATTGTAAAT 
               
               
                   
                   
                 ATAAAACCTGCCTCAGTTAGAATGAATGG 
               
               
                   
                   
                 AAAGCAGATCTACAATTTGCTAATATAGGAATATCAGGTTG 
               
               
                   
                   
                 ACTATATAGCCATACTTGAAAATGCTTCT 
               
               
                   
                   
                 GAGTGGTGTCAACTTTACTTGAATGAATTTTTCATCTTGATT 
               
               
                   
                   
                 GACGCACAGTGATGTACAGTTCACTTCT 
               
               
                   
                   
                 GAAGCTAGTGGTTAACTTGTGTAGGAAACTTTTGCAGTTTG 
               
               
                   
                   
                 ACACTAAGATAACTTCTGTGTGCATTTTT 
               
               
                   
                   
                 CTATGCTTTTTTAAAAACTAGTTTCATTTCATTTTCATGAGA 
               
               
                   
                   
                 TGTTTGGTTTATAAGATCTGAGGATGGT 
               
               
                   
                   
                 TATAAATACTGTAAGTATTGTAATGTTATGAATGCAGGTTA 
               
               
                   
                   
                 TTTGAAAGCTGTTTATTATTATATCATTC 
               
               
                   
                   
                 CTGATAATGCTATGTGAGTGTTTTTAATAAAATTTATATTTA 
               
               
                   
                   
                 TTTAATGCACTCTAA 
               
               
                   
               
               
                 PPREDICTED: 
                 XXM_006714242.2 
                 GTAGAGAAGCAGAAGGAAGCAAGATGGCTGCCCTTTAGGA 
               
               
                 
                   Homo sapiens 
                 
                   
                 TTTGTTAGAAAGGAGACCCGACTGCAACTG 
               
               
                 tet 
                   
                 CTGGATTGCTGCAAGGCTGAGGGACGAGAACGAGGCTGGC 
               
               
                 methylcytosine 
                   
                 AAACATTCAGCAGCACACCCTCTCAAGATT 
               
               
                 dioxygenase 2 
                   
                 GTTTACTTGCCTTTGCTCCTGTTGAGTTACAACGCTTGGAAG 
               
               
                 (TET2), 
                   
                 CAGGAGATGGGCTCAGCAGCAGCCAATA 
               
               
                 transcript 
                   
                 GGACATGATCCAGGAAGAGCAGTAAGGGACTGAGCTGCTG 
               
               
                 variant X2, 
                   
                 AATTCAACTAGAGGGCAGCCTTGTGGATGG 
               
               
                 mRNA 
                   
                 CCCCGAAGCAAGCCTGATGGAACAGGATAGAACCAACCAT 
               
               
                 [SEQ ID NO: 
                   
                 GTTGAGGGCAACAGACTAAGTCCATTCCTG 
               
               
                 1360] 
                   
                 ATACCATCACCTCCCATTTGCCAGACAGAACCTCTGGCTAC 
               
               
                   
                   
                 AAAGCTCCAGAATGGAAGCCCACTGCCTG 
               
               
                   
                   
                 AGAGAGCTCATCCAGAAGTAAATGGAGACACCAAGTGGCA 
               
               
                   
                   
                 CTCTTTCAAAAGTTATTATGGAATACCCTG 
               
               
                   
                   
                 TATGAAGGGAAGCCAGAATAGTCGTGTGAGTCCTGACTTTA 
               
               
                   
                   
                 CACAAGAAAGTAGAGGGTATTCCAAGTGT 
               
               
                   
                   
                 TTGCAAAATGGAGGAATAAAACGCACAGTTAGTGAACCTTC 
               
               
                   
                   
                 TCTCTCTGGGCTCCTTCAGATCAAGAAAT 
               
               
                   
                   
                 TGAAACAAGACCAAAAGGCTAATGGAGAAAGACGTAACTT 
               
               
                   
                   
                 CGGGGTAAGCCAAGAAAGAAATCCAGGTGA 
               
               
                   
                   
                 AAGCAGTCAACCAAATGTCTCCGATTTGAGTGATAAGAAAG 
               
               
                   
                   
                 AATCTGTGAGTTCTGTAGCCCAAGAAAAT 
               
               
                   
                   
                 GCAGTTAAAGATTTCACCAGTTTTTCAACACATAACTGCAG 
               
               
                   
                   
                 TGGGCCTGAAAATCCAGAGCTTCAGATTC 
               
               
                   
                   
                 TGAATGAGCAGGAGGGGAAAAGTGCTAATTACCATGACAA 
               
               
                   
                   
                 GAACATTGTATTACTTAAAAACAAGGCAGT 
               
               
                   
                   
                 GCTAATGCCTAATGGTGCTACAGTTTCTGCCTCTTCCGTGGA 
               
               
                   
                   
                 ACACACACATGGTGAACTCCTGGAAAAA 
               
               
                   
                   
                 ACACTGTCTCAATATTATCCAGATTGTGTTTCCATTGCGGTG 
               
               
                   
                   
                 CAGAAAACCACATCTCACATAAATGCCA 
               
               
                   
                   
                 TTAACAGTCAGGCTACTAATGAGTTGTCCTGTGAGATCACT 
               
               
                   
                   
                 CACCCATCGCATACCTCAGGGCAGATCAA 
               
               
                   
                   
                 TTCCGCACAGACCTCTAACTCTGAGCTGCCTCCAAAGCCAG 
               
               
                   
                   
                 CTGCAGTGGTGAGTGAGGCCTGTGATGCT 
               
               
                   
                   
                 GATGATGCTGATAATGCCAGTAAACTAGCTGCAATGCTAAA 
               
               
                   
                   
                 TACCTGTTCCTTTCAGAAACCAGAACAAC 
               
               
                   
                   
                 TACAACAACAAAAATCAGTTTTTGAGATATGCCCATCTCCT 
               
               
                   
                   
                 GCAGAAAATAACATCCAGGGAACCACAAA 
               
               
                   
                   
                 GCTAGCGTCTGGTGAAGAATTCTGTTCAGGTTCCAGCAGCA 
               
               
                   
                   
                 ATTTGCAAGCTCCTGGTGGCAGCTCTGAA 
               
               
                   
                   
                 CGGTATTTAAAACAAAATGAAATGAATGGTGCTTACTTCAA 
               
               
                   
                   
                 GCAAAGCTCAGTGTTCACTAAGGATTCCT 
               
               
                   
                   
                 TTTCTGCCACTACCACACCACCACCACCATCACAATTGCTTC 
               
               
                   
                   
                 TTTCTCCCCCTCCTCCTCTTCCACAGGT 
               
               
                   
                   
                 TCCTCAGCTTCCTTCAGAAGGAAAAAGCACTCTGAATGGTG 
               
               
                   
                   
                 GAGTTTTAGAAGAACACCACCACTACCCC 
               
               
                   
                   
                 AACCAAAGTAACACAACACTTTTAAGGGAAGTGAAAATAG 
               
               
                   
                   
                 AGGGTAAACCTGAGGCACCACCTTCCCAGA 
               
               
                   
                   
                 GTCCTAATCCATCTACACATGTATGCAGCCCTTCTCCGATGC 
               
               
                   
                   
                 TTTCTGAAAGGCCTCAGAATAATTGTGT 
               
               
                   
                   
                 GAACAGGAATGACATACAGACTGCAGGGACAATGACTGTT 
               
               
                   
                   
                 CCATTGTGTTCTGAGAAAACAAGACCAATG 
               
               
                   
                   
                 TCAGAACACCTCAAGCATAACCCACCAATTTTTGGTAGCAG 
               
               
                   
                   
                 TGGAGAGCTACAGGACAACTGCCAGCAGT 
               
               
                   
                   
                 TGATGAGAAACAAAGAGCAAGAGATTCTGAAGGGTCGAGA 
               
               
                   
                   
                 CAAGGAGCAAACACGAGATCTTGTGCCCCC 
               
               
                   
                   
                 AACACAGCACTATCTGAAACCAGGATGGATTGAATTGAAG 
               
               
                   
                   
                 GCCCCTCGTTTTCACCAAGCGGAATCCCAT 
               
               
                   
                   
                 CTAAAACGTAATGAGGCATCACTGCCATCAATTCTTCAGTA 
               
               
                   
                   
                 TCAACCCAATCTCTCCAATCAAATGACCT 
               
               
                   
                   
                 CCAAACAATACACTGGAAATTCCAACATGCCTGGGGGGCTC 
               
               
                   
                   
                 CCAAGGCAAGCTTACACCCAGAAAACAAC 
               
               
                   
                   
                 ACAGCTGGAGCACAAGTCACAAATGTACCAAGTTGAAATG 
               
               
                   
                   
                 AATCAAGGGCAGTCCCAAGGTACAGTGGAC 
               
               
                   
                   
                 CAACATCTCCAGTTCCAAAAACCCTCACACCAGGTGCACTT 
               
               
                   
                   
                 CTCCAAAACAGACCATTTACCAAAAGCTC 
               
               
                   
                   
                 ATGTGCAGTCACTGTGTGGCACTAGATTTCATTTTCAACAA 
               
               
                   
                   
                 AGAGCAGATTCCCAAACTGAAAAACTTAT 
               
               
                   
                   
                 GTCCCCAGTGTTGAAACAGCACTTGAATCAACAGGCTTCAG 
               
               
                   
                   
                 AGACTGAGCCATTTTCAAACTCACACCTT 
               
               
                   
                   
                 TTGCAACATAAGCCTCATAAACAGGCAGCACAAACACAAC 
               
               
                   
                   
                 CATCCCAGAGTTCACATCTCCCTCAAAACC 
               
               
                   
                   
                 AGCAACAGCAGCAAAAATTACAAATAAAGAATAAAGAGGA 
               
               
                   
                   
                 AATACTCCAGACTTTTCCTCACCCCCAAAG 
               
               
                   
                   
                 CAACAATGATCAGCAAAGAGAAGGATCATTCTTTGGCCAGA 
               
               
                   
                   
                 CTAAAGTGGAAGAATGTTTTCATGGTGAA 
               
               
                   
                   
                 AATCAGTATTCAAAATCAAGCGAGTTCGAGACTCATAATGT 
               
               
                   
                   
                 CCAAATGGGACTGGAGGAAGTACAGAATA 
               
               
                   
                   
                 TAAATCGTAGAAATTCCCCTTATAGTCAGACCATGAAATCA 
               
               
                   
                   
                 AGTGCATGCAAAATACAGGTTTCTTGTTC 
               
               
                   
                   
                 AAACAATACACACCTAGTTTCAGAGAATAAAGAACAGACT 
               
               
                   
                   
                 ACACATCCTGAACTTTTTGCAGGAAACAAG 
               
               
                   
                   
                 ACCCAAAACTTGCATCACATGCAATATTTTCCAAATAATGT 
               
               
                   
                   
                 GATCCCAAAGCAAGATCTTCTTCACAGGT 
               
               
                   
                   
                 GCTTTCAAGAACAGGAGCAGAAGTCACAACAAGCTTCAGTT 
               
               
                   
                   
                 CTACAGGGATATAAAAATAGAAACCAAGA 
               
               
                   
                   
                 TATGTCTGGTCAACAAGCTGCGCAACTTGCTCAGCAAAGGT 
               
               
                   
                   
                 ACTTGATACATAACCATGCAAATGTTTTT 
               
               
                   
                   
                 CCTGTGCCTGACCAGGGAGGAAGTCACACTCAGACCCCTCC 
               
               
                   
                   
                 CCAGAAGGACACTCAAAAGCATGCTGCTC 
               
               
                   
                   
                 TAAGGTGGCATCTCTTACAGAAGCAAGAACAGCAGCAAAC 
               
               
                   
                   
                 ACAGCAACCCCAAACTGAGTCTTGCCATAG 
               
               
                   
                   
                 TCAGATGCACAGGCCAATTAAGGTGGAACCTGGATGCAAG 
               
               
                   
                   
                 CCACATGCCTGTATGCACACAGCACCACCA 
               
               
                   
                   
                 GAAAACAAAACATGGAAAAAGGTAACTAAGCAAGAGAATC 
               
               
                   
                   
                 CACCTGCAAGCTGTGATAATGTGCAGCAAA 
               
               
                   
                   
                 AGAGCATCATTGAGACCATGGAGCAGCATCTGAAGCAGTTT 
               
               
                   
                   
                 CACGCCAAGTCGTTATTTGACCATAAGGC 
               
               
                   
                   
                 TCTTACTCTCAAATCACAGAAGCAAGTAAAAGTTGAAATGT 
               
               
                   
                   
                 CAGGGCCAGTCACAGTTTTGACTAGACAA 
               
               
                   
                   
                 ACCACTGCTGCAGAACTTGATAGCCACACCCCAGCTTTAGA 
               
               
                   
                   
                 GCAGCAAACAACTTCTTCAGAAAAGACAC 
               
               
                   
                   
                 CAACCAAAAGAACAGCTGCTTCTGTTCTCAATAATTTTATA 
               
               
                   
                   
                 GAGTCACCTTCCAAATTACTAGATACTCC 
               
               
                   
                   
                 TATAAAAAATTTATTGGATACACCTGTCAAGACTCAATATG 
               
               
                   
                   
                 ATTTCCCATCTTGCAGATGTGTAGGTTTG 
               
               
                   
                   
                 GACAGAAGGGTAAAGCTATTAGGATTGAAAGAGTCATCTAT 
               
               
                   
                   
                 ACTGGTAAAGAAGGCAAAAGTTCTCAGGG 
               
               
                   
                   
                 ATGTCCTATTGCTAAGTGGGAGAACTTGCGCCTGTCAGGGG 
               
               
                   
                   
                 CTGGATCCAGAAACCTGTGGTGCCTCCTT 
               
               
                   
                   
                 CTCTTTTGGTTGTTCATGGAGCATGTACTACAATGGATGTAA 
               
               
                   
                   
                 GTTTGCCAGAAGCAAGATCCCAAGGAAG 
               
               
                   
                   
                 TTTAAGCTGCTTGGGGATGACCCAAAAGAGGAAGAGAAAC 
               
               
                   
                   
                 TGGAGTCTCATTTGCAAAACCTGTCCACTC 
               
               
                   
                   
                 TTATGGCACCAACATATAAGAAACTTGCACCTGATGCATAT 
               
               
                   
                   
                 AATAATCAGATTGAATATGAACACAGAGC 
               
               
                   
                   
                 ACCAGAGTGCCGTCTGGGTCTGAAGGAAGGCCGTCCATTCT 
               
               
                   
                   
                 CAGGGGTCACTGCATGTTTGGACTTCTGT 
               
               
                   
                   
                 GCTCATGCCCACAGAGACTTGCACAACATGCAGAATGGCAG 
               
               
                   
                   
                 CACATTGGTATGCACTCTCACTAGAGAAG 
               
               
                   
                   
                 ACAATCGAGAATTTGGAGGAAAACCTGAGGATGAGCAGCT 
               
               
                   
                   
                 TCACGTTCTGCCTTTATACAAAGTCTCTGA 
               
               
                   
                   
                 CGTGGATGAGTTTGGGAGTGTGGAAGCTCAGGAGGAGAAA 
               
               
                   
                   
                 AAACGGAGTGGTGCCATTCAGGTACTGAGT 
               
               
                   
                   
                 TCTTTTCGGCGAAAAGTCAGGATGTTAGCAGAGCCAGTCAA 
               
               
                   
                   
                 GACTTGCCGACAAAGGAAACTAGAAGCCA 
               
               
                   
                   
                 AGAAAGCTGCAGCTGAAAAGCTTTCCTCCCTGGAGAACAGC 
               
               
                   
                   
                 TCAAATAAAAATGAAAAGGAAAAGTCAGC 
               
               
                   
                   
                 CCCATCACGTACAAAACAAACTGAAAACGCAAGCCAGGCT 
               
               
                   
                   
                 AAACAGTTGGCAGAACTTTTGCGACTTTCA 
               
               
                   
                   
                 GGACCAGTCATGCAGCAGTCCCAGCAGCCCCAGCCTCTACA 
               
               
                   
                   
                 GAAGCAGCCACCACAGCCCCAGCAGCAGC 
               
               
                   
                   
                 AGAGACCCCAGCAGCAGCAGCCACATCACCCTCAGACAGA 
               
               
                   
                   
                 GTCTGTCAACTCTTATTCTGCTTCTGGATC 
               
               
                   
                   
                 CACCAATCCATACATGAGACGGCCCAATCCAGTTAGTCCTT 
               
               
                   
                   
                 ATCCAAACTCTTCACACACTTCAGATATC 
               
               
                   
                   
                 TATGGAAGCACCAGCCCTATGAACTTCTATTCCACCTCATCT 
               
               
                   
                   
                 CAAGCTGCAGGTTCATATTTGAATTCTT 
               
               
                   
                   
                 CTAATCCCATGAACCCTTACCCTGGGCTTTTGAATCAGAAT 
               
               
                   
                   
                 ACCCAATATCCATCATATCAATGCAATGG 
               
               
                   
                   
                 AAACCTATCAGTGGACAACTGCTCCCCATATCTGGGTTCCT 
               
               
                   
                   
                 ATTCTCCCCAGTCTCAGCCGATGGATCTG 
               
               
                   
                   
                 TATAGGTATCCAAGCCAAGACCCTCTGTCTAAGCTCAGTCT 
               
               
                   
                   
                 ACCACCCATCCATACACTTTACCAGCCAA 
               
               
                   
                   
                 GGTTTGGAAATAGCCAGAGTTTTACATCTAAATACTTAGGT 
               
               
                   
                   
                 TATGGAAACCAAAATATGCAGGGAGATGG 
               
               
                   
                   
                 TTTCAGCAGTTGTACCATTAGACCAAATGTACATCATGTAG 
               
               
                   
                   
                 GGAAATTGCCTCCTTATCCCACTCATGAG 
               
               
                   
                   
                 ATGGATGGCCACTTCATGGGAGCCACCTCTAGATTACCACC 
               
               
                   
                   
                 CAATCTGAGCAATCCAAACATGGACTATA 
               
               
                   
                   
                 AAAATGGTGAACATCATTCACCTTCTCACATAATCCATAAC 
               
               
                   
                   
                 TACAGTGCAGCTCCGGGCATGTTCAACAG 
               
               
                   
                   
                 CTCTCTTCATGCCCTGCATCTCCAAAACAAGGAGAATGACA 
               
               
                   
                   
                 TGCTTTCCCACACAGCTAATGGGTTATCA 
               
               
                   
                   
                 AAGATGCTTCCAGCTCTTAACCATGATAGAACTGCTTGTGT 
               
               
                   
                   
                 CCAAGGAGGCTTACACAAATTAAGTGATG 
               
               
                   
                   
                 CTAATGGTCAGGAAAAGCAGCCATTGGCACTAGTCCAGGGT 
               
               
                   
                   
                 GTGGCTTCTGGTGCAGAGGACAACGATGA 
               
               
                   
                   
                 GGTCTGGTCAGACAGCGAGCAGAGCTTTCTGGATCCTGACA 
               
               
                   
                   
                 TTGGGGGAGTGGCCGTGGCTCCAACTCAT 
               
               
                   
                   
                 GGGTCAATTCTCATTGAGTGTGCAAAGCGTGAGCTGCATGC 
               
               
                   
                   
                 CACAACCCCTTTAAAGAATCCCAATAGGA 
               
               
                   
                   
                 ATCACCCCACCAGGATCTCCCTCGTCTTTTACCAGCATAAG 
               
               
                   
                   
                 AGCATGAATGAGCCAAAACATGGCTTGGC 
               
               
                   
                   
                 TCTTTGGGAAGCCAAAATGGCTGAAAAAGCCCGTGAGAAA 
               
               
                   
                   
                 GAGGAAGAGTGTGAAAAGTATGGCCCAGAC 
               
               
                   
                   
                 TATGTGCCTCAGAAATCCCATGGCAAAAAAGTGAAACGGG 
               
               
                   
                   
                 AGCCTGCTGAGCCACATGAAACTTCAGAGC 
               
               
                   
                   
                 CCACTTACCTGCGTTTCATCAAGTCTCTTGCCGAAAGGACC 
               
               
                   
                   
                 ATGTCCGTGACCACAGACTCCACAGTAAC 
               
               
                   
                   
                 TACATCTCCATATGCCTTCACTCGGGTCACAGGGCCTTACA 
               
               
                   
                   
                 ACAGATATATATGATATCACCCCCTTTTG 
               
               
                   
                   
                 TTGGTTACCTCACTTGAAAAGACCACAACCAACCTGTCAGT 
               
               
                   
                   
                 AGTATAGTTCTCATGACGTGGGCAGTGGG 
               
               
                   
                   
                 GAAAGGTCACAGTATTCATGACAAATGTGGTGGGAAAAAC 
               
               
                   
                   
                 CTCAGCTCACCAGCAACAAAAGAGGTTATC 
               
               
                   
                   
                 TTACCATAGCACTTAATTTTCACTGGCTCCCAAGTGGTCACA 
               
               
                   
                   
                 GATGGCATCTAGGAAAAGACCAAAGCAT 
               
               
                   
                   
                 TCTATGCAAAAAGAAGGTGGGGAAGAAAGTGTTCCGCAAT 
               
               
                   
                   
                 TTACATTTTTAAACACTGGTTCTATTATTG 
               
               
                   
                   
                 GACGAGATGATATGTAAATGTGATCCCCCCCCCCCGCTTAC 
               
               
                   
                   
                 AACTCTACACATCTGTGACCACTTTTAAT 
               
               
                   
                   
                 AATATCAAGTTTGCATAGTCATGGAACACAAATCAAACAAG 
               
               
                   
                   
                 TACTGTAGTATTACAGTGACAGGAATCTT 
               
               
                   
                   
                 AAAATACCATCTGGTGCTGAATATATGATGTACTGAAATAC 
               
               
                   
                   
                 TGGAATTATGGCTTTTTGAAATGCAGTTT 
               
               
                   
                   
                 TTACTGTAATCTTAACTTTTATTTATCAAAATAGCTACAGGA 
               
               
                   
                   
                 AACATGAATAGCAGGAAAACACTGAATT 
               
               
                   
                   
                 TGTTTGGATGTTCTAAGAAATGGTGCTAAGAAAATGGTGTC 
               
               
                   
                   
                 TTTAATAGCTAAAAATTTAATGCCTTTAT 
               
               
                   
                   
                 ATCATCAAGATGCTATCAGTGTACTCCAGTGCCCTTGAATA 
               
               
                   
                   
                 ATAGGGGTACCTTTTCATTCAAGTTTTTA 
               
               
                   
                   
                 TCATAATTACCTATTCTTACACAAGCTTAGTTTTTAAAATGT 
               
               
                   
                   
                 GGACATTTTAAAGGCCTCTGGATTTTGC 
               
               
                   
                   
                 TCATCCAGTGAAGTCCTTGTAGGACAATAAACGTATATATG 
               
               
                   
                   
                 TACATATATACACAAACATGTATATGTGC 
               
               
                   
                   
                 ACACACATGTATATGTATAAATATTTTAAATGGTGTTTTAGA 
               
               
                   
                   
                 AGCACTTTGTCTACCTAAGCTTTGACAA 
               
               
                   
                   
                 CTTGAACAATGCTAAGGTACTGAGATGTTTAAAAAACAAGT 
               
               
                   
                   
                 TTACTTTCATTTTAGAATGCAAAGTTGAT 
               
               
                   
                   
                 TTTTTTAAGGAAACAAAGAAAGCTTTTAAAATATTTTTGCTT 
               
               
                   
                   
                 TTAGCCATGCATCTGCTGATGAGCAATT 
               
               
                   
                   
                 GTGTCCATTTTTAACACAGCCAGTTAAATCCACCATGGGGC 
               
               
                   
                   
                 TTACTGGATTCAAGGGAATACGTTAGTCC 
               
               
                   
                   
                 ACAAAACATGTTTTCTGGTGCTCATCTCACATGCTATACTGT 
               
               
                   
                   
                 AAAACAGTTTTATACAAAATTGTATGAC 
               
               
                   
                   
                 AAGTTCATTGCTCAAAAATGTACAGTTTTAAGAATTTTCTAT 
               
               
                   
                   
                 TAACTGCAGGTAATAATTAGCTGCATGC 
               
               
                   
                   
                 TGCAGACTCAACAAAGCTAGTTCACTGAAGCCTATGCTATT 
               
               
                   
                   
                 TTATGGATCATAGGCTCTTCAGAGAACTG 
               
               
                   
                   
                 AATGGCAGTCTGCCTTTGTGTTGATAATTATGTACATTGTGA 
               
               
                   
                   
                 CGTTGTCATTTCTTAGCTTAAGTGTCCT 
               
               
                   
                   
                 CTTTAACAAGAGGATTGAGCAGACTGATGCCTGCATAAGAT 
               
               
                   
                   
                 GAATAAACAGGGTTAGTTCCATGTGAATC 
               
               
                   
                   
                 TGTCAGTTAAAAAGAAACAAAAACAGGCAGCTGGTTTGCTG 
               
               
                   
                   
                 TGGTGGTTTTAAATCATTAATTTGTATAA 
               
               
                   
                   
                 AGAAGTGAAAGAGTTGTATAGTAAATTAAATTGTAAACAA 
               
               
                   
                   
                 AACTTTTTTAATGCAATGCTTTAGTATTTT 
               
               
                   
                   
                 AGTACTGTAAAAAAATTAAATATATACATATATATATATAT 
               
               
                   
                   
                 ATATATATATATATATATGAGTTTGAAGC 
               
               
                   
                   
                 AGAATTCACATCATGATGGTGCTACTCAGCCTGCTACAAAT 
               
               
                   
                   
                 ATATCATAATGTGAGCTAAGAATTCATTA 
               
               
                   
                   
                 AATGTTTGAGTGATGTTCCTACTTGTCATATACCTCAACACT 
               
               
                   
                   
                 AGTTTGGCAATAGGATATTGAACTGAGA 
               
               
                   
                   
                 GTGAAAGCATTGTGTACCATCATTTTTTTCCAAGTCCTTTTT 
               
               
                   
                   
                 TTTATTGTTAAAAAAAAAAGCATACCTT 
               
               
                   
                   
                 TTTTCAATACTTGATTTCTTAGCAAGTATAACTTGAACTTCA 
               
               
                   
                   
                 ACCTTTTTGTTCTAAAAATTCAGGGATA 
               
               
                   
                   
                 TTTCAGCTCATGCTCTCCCTATGCCAACATGTCACCTGTGTT 
               
               
                   
                   
                 TATGTAAAATTGTTGTAGGTTAATAAAT 
               
               
                   
                   
                 ATATTCTTTGTCAGGGATTTAACCCTTTTATTTTGAATCCCTT 
               
               
                   
                   
                 CTATTTTACTTGTACATGTGCTGATGT 
               
               
                   
                   
                 AACTAAAACTAATTTTGTAAATCTGTTGGCTCTTTTTATTGT 
               
               
                   
                   
                 AAAGAAAAGCATTTTAAAAGTTTGAGGA 
               
               
                   
                   
                 ATCTTTTGACTGTTTCAAGCAGGAAAAAAAAATTACATGAA 
               
               
                   
                   
                 AATAGAATGCACTGAGTTGATAAAGGGAA 
               
               
                   
                   
                 AAATTGTAAGGCAGGAGTTTGGCAAGTGGCTGTTGGCCAGA 
               
               
                   
                   
                 GACTTACTTGTAACTCTCTAAATGAAGTT 
               
               
                   
                   
                 TTTTTGATCCTGTAATCACTGAAGGTACATACTCCATGTGGA 
               
               
                   
                   
                 CTTCCCTTAAACAGGCAAACACCTACAG 
               
               
                   
                   
                 GTATGGTGTGCAACAGATTGTACAATTACATTTTGGCCTAA 
               
               
                   
                   
                 ATACATTTTTGCTTACTAGTATTTAAAAT 
               
               
                   
                   
                 AAATTCTTAATCAGAGGAGGCCTTTGGGTTTTATTGGTCAA 
               
               
                   
                   
                 ATCTTTGTAAGCTGGCTTTTGTCTTTTTA 
               
               
                   
                   
                 AAAAATTTCTTGAATTTGTGGTTGTGTCCAATTTGCAAACAT 
               
               
                   
                   
                 TTCCAAAAATGTTTGCTTTGCTTACAAA 
               
               
                   
                   
                 CCACATGATTTTAATGTTTTTTGTATACCATAATATCTAGCC 
               
               
                   
                   
                 CCAAACATTTGATTACTACATGTGCATT 
               
               
                   
                   
                 GGTGATTTTGATCATCCATTCTTAATATTTGATTTCTGTGTC 
               
               
                   
                   
                 ACCTACTGTCATTTGTTAAACTGCTGGC 
               
               
                   
                   
                 CAACAAGAACAGGAAGTATAGTTTGGGGGGTTGGGGAGAG 
               
               
                   
                   
                 TTTACATAAGGAAGAGAAGAAATTGAGTGG 
               
               
                   
                   
                 CATATTGTAAATATCAGATCTATAATTGTAAATATAAAACC 
               
               
                   
                   
                 TGCCTCAGTTAGAATGAATGGAAAGCAGA 
               
               
                   
                   
                 TCTACAATTTGCTAATATAGGAATATCAGGTTGACTATATA 
               
               
                   
                   
                 GCCATACTTGAAAATGCTTCTGAGTGGTG 
               
               
                   
                   
                 TCAACTTTACTTGAATGAATTTTTCATCTTGATTGACGCACA 
               
               
                   
                   
                 GTGATGTACAGTTCACTTCTGAAGCTAG 
               
               
                   
                   
                 TGGTTAACTTGTGTAGGAAACTTTTGCAGTTTGACACTAAG 
               
               
                   
                   
                 ATAACTTCTGTGTGCATTTTTCTATGCTT 
               
               
                   
                   
                 TTTTAAAAACTAGTTTCATTTCATTTTCATGAGATGTTTGGT 
               
               
                   
                   
                 TTATAAGATCTGAGGATGGTTATAAATA 
               
               
                   
                   
                 CTGTAAGTATTGTAATGTTATGAATGCAGGTTATTTGAAAG 
               
               
                   
                   
                 CTGTTTATTATTATATCATTCCTGATAAT 
               
               
                   
                   
                 GCTATGTGAGTGTTTTTAATAAAATTTATATTTATTTAATGC 
               
               
                   
                   
                 ACTCTAA 
               
               
                   
               
               
                 H Homo sapiens   
                 NM_017628.4 
                 AAACAGAAGGTGGGCCGGGGCGGGGAGAAACAGAACTCGG 
               
               
                 tet 
                   
                 TCAATTTCCCAGTTTGTCGGGTCTTTAAAA 
               
               
                 methylcytosine 
                   
                 ATACAGGCCCCTAAAGCACTAAGGGCATGCCCTCGGTGAAA 
               
               
                 dioxygenase 2 
                   
                 CAGGGGAGCGCTTCTGCTGAATGAGATTA 
               
               
                 (TET2), 
                   
                 AAGCGACAGAAAAGGGAAAGGAGAGCGCGGGCAACGGGA 
               
               
                 transcript 
                   
                 TCTAAAGGGAGATAGAGACGCGGGCCTCTGA 
               
               
                 variant 2, 
                   
                 GGGCTGGCAAACATTCAGCAGCACACCCTCTCAAGATTGTT 
               
               
                 mRNA 
                   
                 TACTTGCCTTTGCTCCTGTTGAGTTACAA 
               
               
                 [SEQ ID NO: 
                   
                 CGCTTGGAAGCAGGAGATGGGCTCAGCAGCAGCCAATAGG 
               
               
                 1361] 
                   
                 ACATGATCCAGGAAGAGCAGTAAGGGACTG 
               
               
                   
                   
                 AGCTGCTGAATTCAACTAGAGGGCAGCCTTGTGGATGGCCC 
               
               
                   
                   
                 CGAAGCAAGCCTGATGGAACAGGATAGAA 
               
               
                   
                   
                 CCAACCATGTTGAGGGCAACAGACTAAGTCCATTCCTGATA 
               
               
                   
                   
                 CCATCACCTCCCATTTGCCAGACAGAACC 
               
               
                   
                   
                 TCTGGCTACAAAGCTCCAGAATGGAAGCCCACTGCCTGAGA 
               
               
                   
                   
                 GAGCTCATCCAGAAGTAAATGGAGACACC 
               
               
                   
                   
                 AAGTGGCACTCTTTCAAAAGTTATTATGGAATACCCTGTAT 
               
               
                   
                   
                 GAAGGGAAGCCAGAATAGTCGTGTGAGTC 
               
               
                   
                   
                 CTGACTTTACACAAGAAAGTAGAGGGTATTCCAAGTGTTTG 
               
               
                   
                   
                 CAAAATGGAGGAATAAAACGCACAGTTAG 
               
               
                   
                   
                 TGAACCTTCTCTCTCTGGGCTCCTTCAGATCAAGAAATTGAA 
               
               
                   
                   
                 ACAAGACCAAAAGGCTAATGGAGAAAGA 
               
               
                   
                   
                 CGTAACTTCGGGGTAAGCCAAGAAAGAAATCCAGGTGAAA 
               
               
                   
                   
                 GCAGTCAACCAAATGTCTCCGATTTGAGTG 
               
               
                   
                   
                 ATAAGAAAGAATCTGTGAGTTCTGTAGCCCAAGAAAATGCA 
               
               
                   
                   
                 GTTAAAGATTTCACCAGTTTTTCAACACA 
               
               
                   
                   
                 TAACTGCAGTGGGCCTGAAAATCCAGAGCTTCAGATTCTGA 
               
               
                   
                   
                 ATGAGCAGGAGGGGAAAAGTGCTAATTAC 
               
               
                   
                   
                 CATGACAAGAACATTGTATTACTTAAAAACAAGGCAGTGCT 
               
               
                   
                   
                 AATGCCTAATGGTGCTACAGTTTCTGCCT 
               
               
                   
                   
                 CTTCCGTGGAACACACACATGGTGAACTCCTGGAAAAAACA 
               
               
                   
                   
                 CTGTCTCAATATTATCCAGATTGTGTTTC 
               
               
                   
                   
                 CATTGCGGTGCAGAAAACCACATCTCACATAAATGCCATTA 
               
               
                   
                   
                 ACAGTCAGGCTACTAATGAGTTGTCCTGT 
               
               
                   
                   
                 GAGATCACTCACCCATCGCATACCTCAGGGCAGATCAATTC 
               
               
                   
                   
                 CGCACAGACCTCTAACTCTGAGCTGCCTC 
               
               
                   
                   
                 CAAAGCCAGCTGCAGTGGTGAGTGAGGCCTGTGATGCTGAT 
               
               
                   
                   
                 GATGCTGATAATGCCAGTAAACTAGCTGC 
               
               
                   
                   
                 AATGCTAAATACCTGTTCCTTTCAGAAACCAGAACAACTAC 
               
               
                   
                   
                 AACAACAAAAATCAGTTTTTGAGATATGC 
               
               
                   
                   
                 CCATCTCCTGCAGAAAATAACATCCAGGGAACCACAAAGCT 
               
               
                   
                   
                 AGCGTCTGGTGAAGAATTCTGTTCAGGTT 
               
               
                   
                   
                 CCAGCAGCAATTTGCAAGCTCCTGGTGGCAGCTCTGAACGG 
               
               
                   
                   
                 TATTTAAAACAAAATGAAATGAATGGTGC 
               
               
                   
                   
                 TTACTTCAAGCAAAGCTCAGTGTTCACTAAGGATTCCTTTTC 
               
               
                   
                   
                 TGCCACTACCACACCACCACCACCATCA 
               
               
                   
                   
                 CAATTGCTTCTTTCTCCCCCTCCTCCTCTTCCACAGGTTCCTC 
               
               
                   
                   
                 AGCTTCCTTCAGAAGGAAAAAGCACTC 
               
               
                   
                   
                 TGAATGGTGGAGTTTTAGAAGAACACCACCACTACCCCAAC 
               
               
                   
                   
                 CAAAGTAACACAACACTTTTAAGGGAAGT 
               
               
                   
                   
                 GAAAATAGAGGGTAAACCTGAGGCACCACCTTCCCAGAGT 
               
               
                   
                   
                 CCTAATCCATCTACACATGTATGCAGCCCT 
               
               
                   
                   
                 TCTCCGATGCTTTCTGAAAGGCCTCAGAATAATTGTGTGAA 
               
               
                   
                   
                 CAGGAATGACATACAGACTGCAGGGACAA 
               
               
                   
                   
                 TGACTGTTCCATTGTGTTCTGAGAAAACAAGACCAATGTCA 
               
               
                   
                   
                 GAACACCTCAAGCATAACCCACCAATTTT 
               
               
                   
                   
                 TGGTAGCAGTGGAGAGCTACAGGACAACTGCCAGCAGTTG 
               
               
                   
                   
                 ATGAGAAACAAAGAGCAAGAGATTCTGAAG 
               
               
                   
                   
                 GGTCGAGACAAGGAGCAAACACGAGATCTTGTGCCCCCAA 
               
               
                   
                   
                 CACAGCACTATCTGAAACCAGGATGGATTG 
               
               
                   
                   
                 AATTGAAGGCCCCTCGTTTTCACCAAGCGGAATCCCATCTA 
               
               
                   
                   
                 AAACGTAATGAGGCATCACTGCCATCAAT 
               
               
                   
                   
                 TCTTCAGTATCAACCCAATCTCTCCAATCAAATGACCTCCAA 
               
               
                   
                   
                 ACAATACACTGGAAATTCCAACATGCCT 
               
               
                   
                   
                 GGGGGGCTCCCAAGGCAAGCTTACACCCAGAAAACAACAC 
               
               
                   
                   
                 AGCTGGAGCACAAGTCACAAATGTACCAAG 
               
               
                   
                   
                 TTGAAATGAATCAAGGGCAGTCCCAAGGTACAGTGGACCA 
               
               
                   
                   
                 ACATCTCCAGTTCCAAAAACCCTCACACCA 
               
               
                   
                   
                 GGTGCACTTCTCCAAAACAGACCATTTACCAAAAGCTCATG 
               
               
                   
                   
                 TGCAGTCACTGTGTGGCACTAGATTTCAT 
               
               
                   
                   
                 TTTCAACAAAGAGCAGATTCCCAAACTGAAAAACTTATGTC 
               
               
                   
                   
                 CCCAGTGTTGAAACAGCACTTGAATCAAC 
               
               
                   
                   
                 AGGCTTCAGAGACTGAGCCATTTTCAAACTCACACCTTTTG 
               
               
                   
                   
                 CAACATAAGCCTCATAAACAGGCAGCACA 
               
               
                   
                   
                 AACACAACCATCCCAGAGTTCACATCTCCCTCAAAACCAGC 
               
               
                   
                   
                 AACAGCAGCAAAAATTACAAATAAAGAAT 
               
               
                   
                   
                 AAAGAGGAAATACTCCAGACTTTTCCTCACCCCCAAAGCAA 
               
               
                   
                   
                 CAATGATCAGCAAAGAGAAGGATCATTCT 
               
               
                   
                   
                 TTGGCCAGACTAAAGTGGAAGAATGTTTTCATGGTGAAAAT 
               
               
                   
                   
                 CAGTATTCAAAATCAAGCGAGTTCGAGAC 
               
               
                   
                   
                 TCATAATGTCCAAATGGGACTGGAGGAAGTACAGAATATA 
               
               
                   
                   
                 AATCGTAGAAATTCCCCTTATAGTCAGACC 
               
               
                   
                   
                 ATGAAATCAAGTGCATGCAAAATACAGGTTTCTTGTTCAAA 
               
               
                   
                   
                 CAATACACACCTAGTTTCAGAGAATAAAG 
               
               
                   
                   
                 AACAGACTACACATCCTGAACTTTTTGCAGGAAACAAGACC 
               
               
                   
                   
                 CAAAACTTGCATCACATGCAATATTTTCC 
               
               
                   
                   
                 AAATAATGTGATCCCAAAGCAAGATCTTCTTCACAGGTGCT 
               
               
                   
                   
                 TTCAAGAACAGGAGCAGAAGTCACAACAA 
               
               
                   
                   
                 GCTTCAGTTCTACAGGGATATAAAAATAGAAACCAAGATAT 
               
               
                   
                   
                 GTCTGGTCAACAAGCTGCGCAACTTGCTC 
               
               
                   
                   
                 AGCAAAGGTACTTGATACATAACCATGCAAATGTTTTTCCT 
               
               
                   
                   
                 GTGCCTGACCAGGGAGGAAGTCACACTCA 
               
               
                   
                   
                 GACCCCTCCCCAGAAGGACACTCAAAAGCATGCTGCTCTAA 
               
               
                   
                   
                 GGTGGCATCTCTTACAGAAGCAAGAACAG 
               
               
                   
                   
                 CAGCAAACACAGCAACCCCAAACTGAGTCTTGCCATAGTCA 
               
               
                   
                   
                 GATGCACAGGCCAATTAAGGTGGAACCTG 
               
               
                   
                   
                 GATGCAAGCCACATGCCTGTATGCACACAGCACCACCAGAA 
               
               
                   
                   
                 AACAAAACATGGAAAAAGGTAACTAAGCA 
               
               
                   
                   
                 AGAGAATCCACCTGCAAGCTGTGATAATGTGCAGCAAAAG 
               
               
                   
                   
                 AGCATCATTGAGACCATGGAGCAGCATCTG 
               
               
                   
                   
                 AAGCAGTTTCACGCCAAGTCGTTATTTGACCATAAGGCTCT 
               
               
                   
                   
                 TACTCTCAAATCACAGAAGCAAGTAAAAG 
               
               
                   
                   
                 TTGAAATGTCAGGGCCAGTCACAGTTTTGACTAGACAAACC 
               
               
                   
                   
                 ACTGCTGCAGAACTTGATAGCCACACCCC 
               
               
                   
                   
                 AGCTTTAGAGCAGCAAACAACTTCTTCAGAAAAGACACCAA 
               
               
                   
                   
                 CCAAAAGAACAGCTGCTTCTGTTCTCAAT 
               
               
                   
                   
                 AATTTTATAGAGTCACCTTCCAAATTACTAGATACTCCTATA 
               
               
                   
                   
                 AAAAATTTATTGGATACACCTGTCAAGA 
               
               
                   
                   
                 CTCAATATGATTTCCCATCTTGCAGATGTGTAGGTAAGTGCC 
               
               
                   
                   
                 AGAAATGTACTGAGACACATGGCGTTTA 
               
               
                   
                   
                 TCCAGAATTAGCAAATTTATCTTCAGATATGGGATTTTCCTT 
               
               
                   
                   
                 CTTTTTTTAAATCTTGAGTCTGGCAGCA 
               
               
                   
                   
                 ATTTGTAAAGGCTCATAAAAATCTGAAGCTTACATTTTTTGT 
               
               
                   
                   
                 CAAGTTACCGATGCTTGTGTCTTGTGAA 
               
               
                   
                   
                 AGAGAACTTCACTTACATGCAGTTTTTCCAAAAGAATTAAA 
               
               
                   
                   
                 TAATCGTGCATGTTTATTTTTCCCTCTCT 
               
               
                   
                   
                 TCAGATCCTGTAAAATTTGAATGTATCTGTTTTAGATCAATT 
               
               
                   
                   
                 CGCCTATTTAGCTCTTTGTATATTATCT 
               
               
                   
                   
                 CCTGGAGAGACAGCTAGGCAGCAAAAAAACAATCTATTAA 
               
               
                   
                   
                 AATGAGAAAATAACGACCATAGGCAGTCTA 
               
               
                   
                   
                 ATGTACGAACTTTAAATATTTTTTAATTCAAGGTAAAATATA 
               
               
                   
                   
                 TTAGTTTCACAAGATTTCTGGCTAATAG 
               
               
                   
                   
                 GGAAATTATTATCTTCAGTCTTCATGAGTTGGGGGAAATGA 
               
               
                   
                   
                 TAATGCTGACACTCTTAGTGCTCCTAAAG 
               
               
                   
                   
                 TTTCCTTTTCTCCATTTATACATTTGGAATGTTGTGATTTATA 
               
               
                   
                   
                 TTCATTTTGATTCCCTTTTCTCTAAAA 
               
               
                   
                   
                 TTTCATCTTTTTGATTAAAAAATATGATACAGGCATACCTCA 
               
               
                   
                   
                 GAGATATTGTGGGTTTGGCTCCATACCA 
               
               
                   
                   
                 CAATAAAATGAATATTACAATAAAGCAAGTTGTAAGGACTT 
               
               
                   
                   
                 TTTGGTTTCTCACTGTATGTAAAAGTTAT 
               
               
                   
                   
                 TTATATACTATACTGTAACATACTAAGTGTGCAATAGCATT 
               
               
                   
                   
                 GTGTCTAAAAAATATATACTTTAAAAATA 
               
               
                   
                   
                 ATTTATTGTTAAAAAAATGCCAACAATTATCTGGGCCTTTA 
               
               
                   
                   
                 GTGAGTGCTAATCTTTTTGCTGGTGGAGG 
               
               
                   
                   
                 GTCGTGCTTCAGTATTGATCGCTGTGGACTGATCATGGTGGT 
               
               
                   
                   
                 AGTTGCTGAAGGTTGCTGGGATGGCTGT 
               
               
                   
                   
                 GTGTGTGGCAATTTCTTAAAATAAGACAACAGTGAAGTGCT 
               
               
                   
                   
                 GTATCAATTGATTTTTCCATTCACAAAAG 
               
               
                   
                   
                 ATTTCTCTGTAGCATGCAATGCTGTTTGATAGCATTTAACCC 
               
               
                   
                   
                 ACAGCAGAATTTCTTTGAAAATTGGACT 
               
               
                   
                   
                 CAGTCCTCTCAAACTGTGCTGCTGCTTTATCAACTAAGTTTT 
               
               
                   
                   
                 TGTAATTTTCTGAATCCTTTGTTGTCAT 
               
               
                   
                   
                 TTCAGCAGTTTACAGCATCTTCATTGGAAGTATATTCCATCT 
               
               
                   
                   
                 CAAACATTCTTTGTTCATCCATAAGAAG 
               
               
                   
                   
                 CAACTTCTTATCAAGTTTTTTCATGACATTGCAGTAACTCAG 
               
               
                   
                   
                 CCCCATCTTCAGGCTCTACTTCTAATTC 
               
               
                   
                   
                 TGGTTCTCTTGCTACATCTCCCTCATCTGCAGTGACCTCTCC 
               
               
                   
                   
                 ACGGAAGTCTTGAACTCCTCAAAGTAAT 
               
               
                   
                   
                 CCATGAGGGTTGGAATCAACTTCTAAACTCCTGTTAATGTT 
               
               
                   
                   
                 GATATATTGACCCCCTCCCATGAATTATG 
               
               
                   
                   
                 AATGTTCTTAATAACTTCTAAATGGTGATACCTTTCCAGAAG 
               
               
                   
                   
                 GCTTTCAATGTACTTTGCCCGGATCCAT 
               
               
                   
                   
                 CAGAAGACTATCTTGGCAGCTGTAGACTAACAATATATTTC 
               
               
                   
                   
                 TTAAATGATAAGACTTGAAAGTCAAAAGT 
               
               
                   
                   
                 ACTCCTTAATCCATAGGCTGCAGAATCAATGTTGTATTAAC 
               
               
                   
                   
                 AGGCACGAAAACAGCATTAATCTTGTGCA 
               
               
                   
                   
                 TCTCCATCGGAGCTCTTGGGTGACTAGGTGCCTTGAGCAGT 
               
               
                   
                   
                 AATATTTTGAAAGGAGGTTTTGGTTTTGT 
               
               
                   
                   
                 TTTTTGTTTTTTTTTTTTGTTTTTTAGCAGTAAGTCTCAACAC 
               
               
                   
                   
                 TGGGCTTAAAATATTCAGTAAACTATG 
               
               
                   
                   
                 TTGTAAAAAGATGTGTTATCATCCAGACTTTGTTGTTCCATT 
               
               
                   
                   
                 ACTCTACACAAGCAGGGTACACTTAGCA 
               
               
                   
                   
                 TAATTCTTAAGGGCCTTGGAATTTTCAGAATGGTAAATGAG 
               
               
                   
                   
                 TATGGGCTTCAACTTAAAATCATCAACTG 
               
               
                   
                   
                 CATTAGCCTGTAACAAGAGAGTCAGCCTGTCCTTTGAAGCA 
               
               
                   
                   
                 AGGCATTGACTTCTATCTATGAAAGTCTT 
               
               
                   
                   
                 AGATGGCACCTTGTTTCAATAGTAGGCTGTTTAGTACAGCC 
               
               
                   
                   
                 ACCTTCATCAGTGATCTTAGCTAGATCTT 
               
               
                   
                   
                 CTGCATAACTTGCTGCAGCTTCTACATCAGCACTTGCTGCCT 
               
               
                   
                   
                 CACCTTGTCCTTTTATGTTATAGAGACA 
               
               
                   
                   
                 GCTGCGCTTCTTAAACTTTATAAACCAACTTCTGCTAGCTTC 
               
               
                   
                   
                 CAACTTCTCTTCTGCAGCTTCCTCATTC 
               
               
                   
                   
                 TCTTCATAGAACTGAAGGGAGTCAAGGCCTTGCTCTGGATT 
               
               
                   
                   
                 AAGCTTTGGCTTAAGGAATGTTGTGGCTG 
               
               
                   
                   
                 ACGTGATCTTCTATCCAGACCACTAAAGCGCTCTCCATATC 
               
               
                   
                   
                 AGCAATAAGGCCGTTTTGCTTTCTTACCT 
               
               
                   
                   
                 TTCATGTGTTCACTGGAGTAATTTCCTTCAAGAATTTTTCCT 
               
               
                   
                   
                 TTACATTCACAACTTGGCTAACTGGCAT 
               
               
                   
                   
                 GCAAGGCCTAGCTTTCAGCCTGTCTTGGCTTTTGACATGCCT 
               
               
                   
                   
                 TCCTCACTTAGCTCGTCATATCTAGCTT 
               
               
                   
                   
                 TTGATTTAAAGTGGCAGGCATACAACTCTTCCTTTCACTTGA 
               
               
                   
                   
                 ACACTTAGAGGCCACTGTAGGGTTATTA 
               
               
                   
                   
                 ATTGGCCTAATTTCAATATTGTTGTGTTTTAGGGAATAGAGA 
               
               
                   
                   
                 GGCCCAGGGAGAGGGAGAGAGCCCAAAC 
               
               
                   
                   
                 GGCTGGTTGATAGAGCAGGCAGAATGCACACAACATTTATC 
               
               
                   
                   
                 AGATTATGTTTGCACCATTTACCAGATTA 
               
               
                   
                   
                 TGGGTACGGTTTGTGGCACCCCCCAAAAATTAGAATAGTAA 
               
               
                   
                   
                 CATCAAAGATCACTGATCACAGATCGCCA 
               
               
                   
                   
                 TAACATAAATAATAATAAACTTTAAAATACTGTGAGAATTA 
               
               
                   
                   
                 CCAAAATGTGATACAGAGACATGAAGTGA 
               
               
                   
                   
                 GCACATGCTGTTGAAAAAAATGACACTGATAGACATACTTA 
               
               
                   
                   
                 ACACGTGGGATTGCCACAAACCTTCAGTT 
               
               
                   
                   
                 TGTAAAAGTCACAGTAACTGTGACTCACAAAAGAACAAAG 
               
               
                   
                   
                 CACAATAAAACGAGGTATGCCTGTATTTTT 
               
               
                   
                   
                 AAAAAAAGCTTTTTGTTAAAATTCAGGATATGTAATAGGTC 
               
               
                   
                   
                 TGTAGGAATAGTGAAATATTTTTGCTGAT 
               
               
                   
                   
                 GGATGTAGATATATACGTGGATAGAGATGAAGATCTTAATT 
               
               
                   
                   
                 ATAGCTATGCAGCATAGATTTAGTCAAAG 
               
               
                   
                   
                 ACATTTGAAAAGACAAATGTTAAATTAGTGTGGCTAATGAC 
               
               
                   
                   
                 CTACCCGTGCCATGTTTTCCCTCTTGCAA 
               
               
                   
                   
                 TGAGATACCCCACACTGTGTAGAAGGATGGAGGGAGGACT 
               
               
                   
                   
                 CCTACTGTCCCTCTTTGCGTGTGGTTATTA 
               
               
                   
                   
                 AGTTGCCTCACTGGGCTAAAACACCACACATCTCATAGATA 
               
               
                   
                   
                 ATATTTGGTAAGTTGTAATCGTCTTCACT 
               
               
                   
                   
                 CTTCTCTTATCACCCACCCCTATCTTCCCACTTTTCCATCTTT 
               
               
                   
                   
                 GTTGGTTTGCAACAGCCCCTTCTTTTT 
               
               
                   
                   
                 GCCTGACTCTCCAGGATTTTCTCTCATCATAAATTGTTCTAA 
               
               
                   
                   
                 AGTACATACTAATATGGGTCTGGATTGA 
               
               
                   
                   
                 CTATTCTTATTTGCAAAACAGCAATTAAATGTTATAGGGAA 
               
               
                   
                   
                 GTAGGAAGAAAAAGGGGTATCCTTGACAA 
               
               
                   
                   
                 TAAACCAAGCAATATTCTGGGGGTGGGATAGAGCAGGAAA 
               
               
                   
                   
                 TTTTATTTTTAATCTTTTAAAATCCAAGTA 
               
               
                   
                   
                 ATAGGTAGGCTTCCAGTTAGCTTTAAATGTTTTTTTTTTCCA 
               
               
                   
                   
                 GCTCAAAAAATTGGATTGTAGTTGATAC 
               
               
                   
                   
                 TACATATAATACATTCTAATTCCCTCACTGTATTCTTTGTTT 
               
               
                   
                   
                 AGTTTCATTTATTTGGTTTAAAATAATT 
               
               
                   
                   
                 TTTTATCCCATATCTGAAATGTAATATATTTTTATCCAACAA 
               
               
                   
                   
                 CCAGCATGTACATATACTTAATTATGTG 
               
               
                   
                   
                 GCACATTTTCTAATAGATCAGTCCATCAATCTACTCATTTTA 
               
               
                   
                   
                 AAGAAAAAAAAATTTTAAAGTCACTTTT 
               
               
                   
                   
                 AGAGCCCTTAATGTGTAGTTGGGGGTTAAGCTTTGTGGATG 
               
               
                   
                   
                 TAGCCTTTATATTTAGTATAATTGAGGTC 
               
               
                   
                   
                 TAAAATAATAATCTTCTATTATCTCAACAGAGCAAATTATT 
               
               
                   
                   
                 GAAAAAGATGAAGGTCCTTTTTATACCCA 
               
               
                   
                   
                 TCTAGGAGCAGGTCCTAATGTGGCAGCTATTAGAGAAATCA 
               
               
                   
                   
                 TGGAAGAAAGGTAATTAACGCAAAGGCAC 
               
               
                   
                   
                 AGGGCAGATTAACGTTTATCCTTTTGTATATGTCAGAATTTT 
               
               
                   
                   
                 TCCAGCCTTCACACACAAAGCAGTAAAC 
               
               
                   
                   
                 AATTGTAAATTGAGTAATTATTAGTAGGCTTAGCTATTCTAG 
               
               
                   
                   
                 GGTTGCCAACACTACACACTGTGCTATT 
               
               
                   
                   
                 CACCAGAGAGTCACAATATTTGACAGGACTAATAGTCTGCT 
               
               
                   
                   
                 AGCTGGCACAGGCTGCCCACTTTGCGATG 
               
               
                   
                   
                 GATGCCAGAAAACCCAGGCATGAACAGGAATCGGCCAGCC 
               
               
                   
                   
                 AGGCTGCCAGCCACAAGGTACTGGCACAGG 
               
               
                   
                   
                 CTCCAACGAGAGGTCCCACTCTGGCTTTCCCACCTGATAAT 
               
               
                   
                   
                 AAAGTGTCAAAGCAGAAAGACTGGTAAAG 
               
               
                   
                   
                 TGTGGTATAAGAAAAGAACCACTGAATTAAATTCACCTAGT 
               
               
                   
                   
                 GTTGCAAATGAGTACTTATCTCTAAGTTT 
               
               
                   
                   
                 TCTTTTACCATAAAAAGAGAGCAAGTGTGATATGTTGAATA 
               
               
                   
                   
                 GAAAGAGAAACATACTATTTACAGCTGCC 
               
               
                   
                   
                 TTTTTTTTTTTTTTTCGCTATCAATCACAGGTATACAAGTACT 
               
               
                   
                   
                 TGCCTTTACTCCTGCATGTAGAAGACT 
               
               
                   
                   
                 CTTATGAGCGAGATAATGCAGAGAAGGCCTTTCATATAAAT 
               
               
                   
                   
                 TTATACAGCTCTGAGCTGTTCTTCTTCTA 
               
               
                   
                   
                 GGGTGCCTTTTCATTAAGAGGTAGGCAGTATTATTATTAAA 
               
               
                   
                   
                 GTACTTAGGATACATTGGGGCAGCTAGGA 
               
               
                   
                   
                 CATATTCAGTATCATTCTTGCTCCATTTCCAAATTATTCATTT 
               
               
                   
                   
                 CTAAATTAGCATGTAGAAGTTCACTAA 
               
               
                   
                   
                 ATAATCATCTAGTGGCCTGGCAGAAATAGTGAATTTCCCTA 
               
               
                   
                   
                 AGTGCCTTTTTTTTGTTGTTTTTTTGTTT 
               
               
                   
                   
                 TGTTTTTTAAACAAGCAGTAGGTGGTGCTTTGGTCATAAGG 
               
               
                   
                   
                 GAAGATATAGTCTATTTCTAGGACTATTC 
               
               
                   
                   
                 CATATTTTCCATGTGGCTGGATACTAACTATTTGCCAGCCTC 
               
               
                   
                   
                 CTTTTCTAAATTGTGAGACATTCTTGGA 
               
               
                   
                   
                 GGAACAGTTCTAACTAAAATCTATTATGACTCCCCAAGTTTT 
               
               
                   
                   
                 AAAATAGCTAAATTTAGTAAGGGAAAAA 
               
               
                   
                   
                 ATAGTTTATGTTTTAGAAGACTGAACTTAGCAAACTAACCT 
               
               
                   
                   
                 GAATTTTGTGCTTTGTGAAATTTTATATC 
               
               
                   
                   
                 GAAATGAGCTTTCCCATTTTCACCCACATGTAATTTACAAA 
               
               
                   
                   
                 ATAGTTCATTACAATTATCTGTACATTTT 
               
               
                   
                   
                 GATATTGAGGAAAAACAAGGCTTAAAAACCATTATCCAGTT 
               
               
                   
                   
                 TGCTTGGCGTAGACCTGTTTAAAAAATAA 
               
               
                   
                   
                 TAAACCGTTCATTTCTCAGGATGTGGTCATAGAATAAAGTT 
               
               
                   
                   
                 ATGCTCAAATGTTCAAATATTTAAA 
               
               
                   
               
               
                 PPREDICTED: 
                 XXM_011532044.1 
                 TCAGGCTCTACTTCTAATTCTGGTTCTCTTGCTACATCTCCCT 
               
               
                 
                   Homo sapiens 
                 
                   
                 CATCTGCAGTGACCTCTCCACGGAAGT 
               
               
                 tet 
                   
                 CTTGAACTCCTCAAAAGCAAATTATTGAAAAAGATGAAGGT 
               
               
                 methylcytosine 
                   
                 CCTTTTTATACCCATCTAGGAGCAGGTCC 
               
               
                 dioxygenase 2 
                   
                 TAATGTGGCAGCTATTAGAGAAATCATGGAAGAAAGGTTTG 
               
               
                 (TET2), 
                   
                 GACAGAAGGGTAAAGCTATTAGGATTGAA 
               
               
                 transcript 
                   
                 AGAGTCATCTATACTGGTAAAGAAGGCAAAAGTTCTCAGGG 
               
               
                 variant X9, 
                   
                 ATGTCCTATTGCTAAGTGGGTGGTTCGCA 
               
               
                 mRNA 
                   
                 GAAGCAGCAGTGAAGAGAAGCTACTGTGTTTGGTGCGGGA 
               
               
                 [SEQ ID NO: 
                   
                 GCGAGCTGGCCACACCTGTGAGGCTGCAGT 
               
               
                 1362] 
                   
                 GATTGTGATTCTCATCCTGGTGTGGGAAGGAATCCCGCTGT 
               
               
                   
                   
                 CTCTGGCTGACAAACTCTACTCGGAGCTT 
               
               
                   
                   
                 ACCGAGACGCTGAGGAAATACGGCACGCTCACCAATCGCC 
               
               
                   
                   
                 GGTGTGCCTTGAATGAAGAGAGAACTTGCG 
               
               
                   
                   
                 CCTGTCAGGGGCTGGATCCAGAAACCTGTGGTGCCTCCTTC 
               
               
                   
                   
                 TCTTTTGGTTGTTCATGGAGCATGTACTA 
               
               
                   
                   
                 CAATGGATGTAAGTTTGCCAGAAGCAAGATCCCAAGGAAG 
               
               
                   
                   
                 TTTAAGCTGCTTGGGGATGACCCAAAAGAG 
               
               
                   
                   
                 GAAGAGAAACTGGAGTCTCATTTGCAAAACCTGTCCACTCT 
               
               
                   
                   
                 TATGGCACCAACATATAAGAAACTTGCAC 
               
               
                   
                   
                 CTGATGCATATAATAATCAGATTGAATATGAACACAGAGCA 
               
               
                   
                   
                 CCAGAGTGCCGTCTGGGTCTGAAGGAAGG 
               
               
                   
                   
                 CCGTCCATTCTCAGGGGTCACTGCATGTTTGGACTTCTGTGC 
               
               
                   
                   
                 TCATGCCCACAGAGACTTGCACAACATG 
               
               
                   
                   
                 CAGAATGGCAGCACATTGGTATGCACTCTCACTAGAGAAGA 
               
               
                   
                   
                 CAATCGAGAATTTGGAGGAAAACCTGAGG 
               
               
                   
                   
                 ATGAGCAGCTTCACGTTCTGCCTTTATACAAAGTCTCTGACG 
               
               
                   
                   
                 TGGATGAGTTTGGGAGTGTGGAAGCTCA 
               
               
                   
                   
                 GGAGGAGAAAAAACGGAGTGGTGCCATTCAGGTACTGAGT 
               
               
                   
                   
                 TCTTTTCGGCGAAAAGTCAGGATGTTAGCA 
               
               
                   
                   
                 GAGCCAGTCAAGACTTGCCGACAAAGGAAACTAGAAGCCA 
               
               
                   
                   
                 AGAAAGCTGCAGCTGAAAAGCTTTCCTCCC 
               
               
                   
                   
                 TGGAGAACAGCTCAAATAAAAATGAAAAGGAAAAGTCAGC 
               
               
                   
                   
                 CCCATCACGTACAAAACAAACTGAAAACGC 
               
               
                   
                   
                 AAGCCAGGCTAAACAGTTGGCAGAACTTTTGCGACTTTCAG 
               
               
                   
                   
                 GACCAGTCATGCAGCAGTCCCAGCAGCCC 
               
               
                   
                   
                 CAGCCTCTACAGAAGCAGCCACCACAGCCCCAGCAGCAGC 
               
               
                   
                   
                 AGAGACCCCAGCAGCAGCAGCCACATCACC 
               
               
                   
                   
                 CTCAGACAGAGTCTGTCAACTCTTATTCTGCTTCTGGATCCA 
               
               
                   
                   
                 CCAATCCATACATGAGACGGCCCAATCC 
               
               
                   
                   
                 AGTTAGTCCTTATCCAAACTCTTCACACACTTCAGATATCTA 
               
               
                   
                   
                 TGGAAGCACCAGCCCTATGAACTTCTAT 
               
               
                   
                   
                 TCCACCTCATCTCAAGCTGCAGGTTCATATTTGAATTCTTCT 
               
               
                   
                   
                 AATCCCATGAACCCTTACCCTGGGCTTT 
               
               
                   
                   
                 TGAATCAGAATACCCAATATCCATCATATCAATGCAATGGA 
               
               
                   
                   
                 AACCTATCAGTGGACAACTGCTCCCCATA 
               
               
                   
                   
                 TCTGGGTTCCTATTCTCCCCAGTCTCAGCCGATGGATCTGTA 
               
               
                   
                   
                 TAGGTATCCAAGCCAAGACCCTCTGTCT 
               
               
                   
                   
                 AAGCTCAGTCTACCACCCATCCATACACTTTACCAGCCAAG 
               
               
                   
                   
                 GTTTGGAAATAGCCAGAGTTTTACATCTA 
               
               
                   
                   
                 AATACTTAGGTTATGGAAACCAAAATATGCAGGGAGATGGT 
               
               
                   
                   
                 TTCAGCAGTTGTACCATTAGACCAAATGT 
               
               
                   
                   
                 ACATCATGTAGGGAAATTGCCTCCTTATCCCACTCATGAGA 
               
               
                   
                   
                 TGGATGGCCACTTCATGGGAGCCACCTCT 
               
               
                   
                   
                 AGATTACCACCCAATCTGAGCAATCCAAACATGGACTATAA 
               
               
                   
                   
                 AAATGGTGAACATCATTCACCTTCTCACA 
               
               
                   
                   
                 TAATCCATAACTACAGTGCAGCTCCGGGCATGTTCAACAGC 
               
               
                   
                   
                 TCTCTTCATGCCCTGCATCTCCAAAACAA 
               
               
                   
                   
                 GGAGAATGACATGCTTTCCCACACAGCTAATGGGTTATCAA 
               
               
                   
                   
                 AGATGCTTCCAGCTCTTAACCATGATAGA 
               
               
                   
                   
                 ACTGCTTGTGTCCAAGGAGGCTTACACAAATTAAGTGATGC 
               
               
                   
                   
                 TAATGGTCAGGAAAAGCAGCCATTGGCAC 
               
               
                   
                   
                 TAGTCCAGGGTGTGGCTTCTGGTGCAGAGGACAACGATGAG 
               
               
                   
                   
                 GTCTGGTCAGACAGCGAGCAGAGCTTTCT 
               
               
                   
                   
                 GGATCCTGACATTGGGGGAGTGGCCGTGGCTCCAACTCATG 
               
               
                   
                   
                 GGTCAATTCTCATTGAGTGTGCAAAGCGT 
               
               
                   
                   
                 GAGCTGCATGCCACAACCCCTTTAAAGAATCCCAATAGGAA 
               
               
                   
                   
                 TCACCCCACCAGGATCTCCCTCGTCTTTT 
               
               
                   
                   
                 ACCAGCATAAGAGCATGAATGAGCCAAAACATGGCTTGGC 
               
               
                   
                   
                 TCTTTGGGAAGCCAAAATGGCTGAAAAAGC 
               
               
                   
                   
                 CCGTGAGAAAGAGGAAGAGTGTGAAAAGTATGGCCCAGAC 
               
               
                   
                   
                 TATGTGCCTCAGAAATCCCATGGCAAAAAA 
               
               
                   
                   
                 GTGAAACGGGAGCCTGCTGAGCCACATGAAACTTCAGAGC 
               
               
                   
                   
                 CCACTTACCTGCGTTTCATCAAGTCTCTTG 
               
               
                   
                   
                 CCGAAAGGACCATGTCCGTGACCACAGACTCCACAGTAACT 
               
               
                   
                   
                 ACATCTCCATATGCCTTCACTCGGGTCAC 
               
               
                   
                   
                 AGGGCCTTACAACAGATATATATGATATCACCCCCTTTTGTT 
               
               
                   
                   
                 GGTTACCTCACTTGAAAAGACCACAACC 
               
               
                   
                   
                 AACCTGTCAGTAGTATAGTTCTCATGACGTGGGCAGTGGGG 
               
               
                   
                   
                 AAAGGTCACAGTATTCATGACAAATGTGG 
               
               
                   
                   
                 TGGGAAAAACCTCAGCTCACCAGCAACAAAAGAGGTTATCT 
               
               
                   
                   
                 TACCATAGCACTTAATTTTCACTGGCTCC 
               
               
                   
                   
                 CAAGTGGTCACAGATGGCATCTAGGAAAAGACCAAAGCAT 
               
               
                   
                   
                 TCTATGCAAAAAGAAGGTGGGGAAGAAAGT 
               
               
                   
                   
                 GTTCCGCAATTTACATTTTTAAACACTGGTTCTATTATTGGA 
               
               
                   
                   
                 CGAGATGATATGTAAATGTGATCCCCCC 
               
               
                   
                   
                 CCCCCGCTTACAACTCTACACATCTGTGACCACTTTTAATAA 
               
               
                   
                   
                 TATCAAGTTTGCATAGTCATGGAACACA 
               
               
                   
                   
                 AATCAAACAAGTACTGTAGTATTACAGTGACAGGAATCTTA 
               
               
                   
                   
                 AAATACCATCTGGTGCTGAATATATGATG 
               
               
                   
                   
                 TACTGAAATACTGGAATTATGGCTTTTTGAAATGCAGTTTTT 
               
               
                   
                   
                 ACTGTAATCTTAACTTTTATTTATCAAA 
               
               
                   
                   
                 ATAGCTACAGGAAACATGAATAGCAGGAAAACACTGAATT 
               
               
                   
                   
                 TGTTTGGATGTTCTAAGAAATGGTGCTAAG 
               
               
                   
                   
                 AAAATGGTGTCTTTAATAGCTAAAAATTTAATGCCTTTATAT 
               
               
                   
                   
                 CATCAAGATGCTATCAGTGTACTCCAGT 
               
               
                   
                   
                 GCCCTTGAATAATAGGGGTACCTTTTCATTCAAGTTTTTATC 
               
               
                   
                   
                 ATAATTACCTATTCTTACACAAGCTTAG 
               
               
                   
                   
                 TTTTTAAAATGTGGACATTTTAAAGGCCTCTGGATTTTGCTC 
               
               
                   
                   
                 ATCCAGTGAAGTCCTTGTAGGACAATAA 
               
               
                   
                   
                 ACGTATATATGTACATATATACACAAACATGTATATGTGCA 
               
               
                   
                   
                 CACACATGTATATGTATAAATATTTTAAA 
               
               
                   
                   
                 TGGTGTTTTAGAAGCACTTTGTCTACCTAAGCTTTGACAACT 
               
               
                   
                   
                 TGAACAATGCTAAGGTACTGAGATGTTT 
               
               
                   
                   
                 AAAAAACAAGTTTACTTTCATTTTAGAATGCAAAGTTGATT 
               
               
                   
                   
                 TTTTTAAGGAAACAAAGAAAGCTTTTAAA 
               
               
                   
                   
                 ATATTTTTGCTTTTAGCCATGCATCTGCTGATGAGCAATTGT 
               
               
                   
                   
                 GTCCATTTTTAACACAGCCAGTTAAATC 
               
               
                   
                   
                 CACCATGGGGCTTACTGGATTCAAGGGAATACGTTAGTCCA 
               
               
                   
                   
                 CAAAACATGTTTTCTGGTGCTCATCTCAC 
               
               
                   
                   
                 ATGCTATACTGTAAAACAGTTTTATACAAAATTGTATGACA 
               
               
                   
                   
                 AGTTCATTGCTCAAAAATGTACAGTTTTA 
               
               
                   
                   
                 AGAATTTTCTATTAACTGCAGGTAATAATTAGCTGCATGCT 
               
               
                   
                   
                 GCAGACTCAACAAAGCTAGTTCACTGAAG 
               
               
                   
                   
                 CCTATGCTATTTTATGGATCATAGGCTCTTCAGAGAACTGA 
               
               
                   
                   
                 ATGGCAGTCTGCCTTTGTGTTGATAATTA 
               
               
                   
                   
                 TGTACATTGTGACGTTGTCATTTCTTAGCTTAAGTGTCCTCT 
               
               
                   
                   
                 TTAACAAGAGGATTGAGCAGACTGATGC 
               
               
                   
                   
                 CTGCATAAGATGAATAAACAGGGTTAGTTCCATGTGAATCT 
               
               
                   
                   
                 GTCAGTTAAAAAGAAACAAAAACAGGCAG 
               
               
                   
                   
                 CTGGTTTGCTGTGGTGGTTTTAAATCATTAATTTGTATAAAG 
               
               
                   
                   
                 AAGTGAAAGAGTTGTATAGTAAATTAAA 
               
               
                   
                   
                 TTGTAAACAAAACTTTTTTAATGCAATGCTTTAGTATTTTAG 
               
               
                   
                   
                 TACTGTAAAAAAATTAAATATATACATA 
               
               
                   
                   
                 TATATATATATATATATATATATATATATGAGTTTGAAGCAG 
               
               
                   
                   
                 AATTCACATCATGATGGTGCTACTCAGC 
               
               
                   
                   
                 CTGCTACAAATATATCATAATGTGAGCTAAGAATTCATTAA 
               
               
                   
                   
                 ATGTTTGAGTGATGTTCCTACTTGTCATA 
               
               
                   
                   
                 TACCTCAACACTAGTTTGGCAATAGGATATTGAACTGAGAG 
               
               
                   
                   
                 TGAAAGCATTGTGTACCATCATTTTTTTC 
               
               
                   
                   
                 CAAGTCCTTTTTTTTATTGTTAAAAAAAAAAGCATACCTTTT 
               
               
                   
                   
                 TTCAATACTTGATTTCTTAGCAAGTATA 
               
               
                   
                   
                 ACTTGAACTTCAACCTTTTTGTTCTAAAAATTCAGGGATATT 
               
               
                   
                   
                 TCAGCTCATGCTCTCCCTATGCCAACAT 
               
               
                   
                   
                 GTCACCTGTGTTTATGTAAAATTGTTGTAGGTTAATAAATAT 
               
               
                   
                   
                 ATTCTTTGTCAGGGATTTAACCCTTTTA 
               
               
                   
                   
                 TTTTGAATCCCTTCTATTTTACTTGTACATGTGCTGATGTAA 
               
               
                   
                   
                 CTAAAACTAATTTTGTAAATCTGTTGGC 
               
               
                   
                   
                 TCTTTTTATTGTAAAGAAAAGCATTTTAAAAGTTTGAGGAA 
               
               
                   
                   
                 TCTTTTGACTGTTTCAAGCAGGAAAAAAA 
               
               
                   
                   
                 AATTACATGAAAATAGAATGCACTGAGTTGATAAAGGGAA 
               
               
                   
                   
                 AAATTGTAAGGCAGGAGTTTGGCAAGTGGC 
               
               
                   
                   
                 TGTTGGCCAGAGACTTACTTGTAACTCTCTAAATGAAGTTTT 
               
               
                   
                   
                 TTTGATCCTGTAATCACTGAAGGTACAT 
               
               
                   
                   
                 ACTCCATGTGGACTTCCCTTAAACAGGCAAACACCTACAGG 
               
               
                   
                   
                 TATGGTGTGCAACAGATTGTACAATTACA 
               
               
                   
                   
                 TTTTGGCCTAAATACATTTTTGCTTACTAGTATTTAAAATAA 
               
               
                   
                   
                 ATTCTTAATCAGAGGAGGCCTTTGGGTT 
               
               
                   
                   
                 TTATTGGTCAAATCTTTGTAAGCTGGCTTTTGTCTTTTTAAA 
               
               
                   
                   
                 AAATTTCTTGAATTTGTGGTTGTGTCCA 
               
               
                   
                   
                 ATTTGCAAACATTTCCAAAAATGTTTGCTTTGCTTACAAACC 
               
               
                   
                   
                 ACATGATTTTAATGTTTTTTGTATACCA 
               
               
                   
                   
                 TAATATCTAGCCCCAAACATTTGATTACTACATGTGCATTGG 
               
               
                   
                   
                 TGATTTTGATCATCCATTCTTAATATTT 
               
               
                   
                   
                 GATTTCTGTGTCACCTACTGTCATTTGTTAAACTGCTGGCCA 
               
               
                   
                   
                 ACAAGAACAGGAAGTATAGTTTGGGGGG 
               
               
                   
                   
                 TTGGGGAGAGTTTACATAAGGAAGAGAAGAAATTGAGTGG 
               
               
                   
                   
                 CATATTGTAAATATCAGATCTATAATTGTA 
               
               
                   
                   
                 AATATAAAACCTGCCTCAGTTAGAATGAATGGAAAGCAGAT 
               
               
                   
                   
                 CTACAATTTGCTAATATAGGAATATCAGG 
               
               
                   
                   
                 TTGACTATATAGCCATACTTGAAAATGCTTCTGAGTGGTGTC 
               
               
                   
                   
                 AACTTTACTTGAATGAATTTTTCATCTT 
               
               
                   
                   
                 GATTGACGCACAGTGATGTACAGTTCACTTCTGAAGCTAGT 
               
               
                   
                   
                 GGTTAACTTGTGTAGGAAACTTTTGCAGT 
               
               
                   
                   
                 TTGACACTAAGATAACTTCTGTGTGCATTTTTCTATGCTTTT 
               
               
                   
                   
                 TTAAAAACTAGTTTCATTTCATTTTCAT 
               
               
                   
                   
                 GAGATGTTTGGTTTATAAGATCTGAGGATGGTTATAAATAC 
               
               
                   
                   
                 TGTAAGTATTGTAATGTTATGAATGCAGG 
               
               
                   
                   
                 TTATTTGAAAGCTGTTTATTATTATATCATTCCTGATAATGC 
               
               
                   
                   
                 TATGTGAGTGTTTTTAATAAAATTTATA 
               
               
                   
                   
                 TTTATTTAATGCACTCTAA 
               
               
                   
               
               
                 PPREDICTED: 
                 XXM_011532043.1 
                 GTAGAGAAGCAGAAGGAAGCAAGATGGCTGCCCTTTAGGA 
               
               
                 
                   Homo sapiens 
                 
                   
                 TTTGTTAGAAAGGAGACCCGACTGCAACTG 
               
               
                 tet 
                   
                 CTGGATTGCTGCAAGGCTGAGGGACGAGAACGAGGCTGGC 
               
               
                 methylcytosine 
                   
                 AAACATTCAGCAGCACACCCTCTCAAGATT 
               
               
                 dioxygenase 2 
                   
                 GTTTACTTGCCTTTGCTCCTGTTGAGTTACAACGCTTGGAAG 
               
               
                 (TET2), 
                   
                 CAGGAGATGGGCTCAGCAGCAGCCAATA 
               
               
                 transcript 
                   
                 GGACATGATCCAGGAAGAGCAGTAAGGGACTGAGCTGCTG 
               
               
                 variant X7, 
                   
                 AATTCAACTAGAGGGCAGCCTTGTGGATGG 
               
               
                 mRNA 
                   
                 CCCCGAAGCAAGCCTGATGGAACAGGATAGAACCAACCAT 
               
               
                 [SEQ ID NO: 
                   
                 GTTGAGGGCAACAGACTAAGTCCATTCCTG 
               
               
                 1363] 
                   
                 ATACCATCACCTCCCATTTGCCAGACAGAACCTCTGGCTAC 
               
               
                   
                   
                 AAAGCTCCAGAATGGAAGCCCACTGCCTG 
               
               
                   
                   
                 AGAGAGCTCATCCAGAAGTAAATGGAGACACCAAGTGGCA 
               
               
                   
                   
                 CTCTTTCAAAAGTTATTATGGAATACCCTG 
               
               
                   
                   
                 TATGAAGGGAAGCCAGAATAGTCGTGTGAGTCCTGACTTTA 
               
               
                   
                   
                 CACAAGAAAGTAGAGGGTATTCCAAGTGT 
               
               
                   
                   
                 TTGCAAAATGGAGGAATAAAACGCACAGTTAGTGAACCTTC 
               
               
                   
                   
                 TCTCTCTGGGCTCCTTCAGATCAAGAAAT 
               
               
                   
                   
                 TGAAACAAGACCAAAAGGCTAATGGAGAAAGACGTAACTT 
               
               
                   
                   
                 CGGGGTAAGCCAAGAAAGAAATCCAGGTGA 
               
               
                   
                   
                 AAGCAGTCAACCAAATGTCTCCGATTTGAGTGATAAGAAAG 
               
               
                   
                   
                 AATCTGTGAGTTCTGTAGCCCAAGAAAAT 
               
               
                   
                   
                 GCAGTTAAAGATTTCACCAGTTTTTCAACACATAACTGCAG 
               
               
                   
                   
                 TGGGCCTGAAAATCCAGAGCTTCAGATTC 
               
               
                   
                   
                 TGAATGAGCAGGAGGGGAAAAGTGCTAATTACCATGACAA 
               
               
                   
                   
                 GAACATTGTATTACTTAAAAACAAGGCAGT 
               
               
                   
                   
                 GCTAATGCCTAATGGTGCTACAGTTTCTGCCTCTTCCGTGGA 
               
               
                   
                   
                 ACACACACATGGTGAACTCCTGGAAAAA 
               
               
                   
                   
                 ACACTGTCTCAATATTATCCAGATTGTGTTTCCATTGCGGTG 
               
               
                   
                   
                 CAGAAAACCACATCTCACATAAATGCCA 
               
               
                   
                   
                 TTAACAGTCAGGCTACTAATGAGTTGTCCTGTGAGATCACT 
               
               
                   
                   
                 CACCCATCGCATACCTCAGGGCAGATCAA 
               
               
                   
                   
                 TTCCGCACAGACCTCTAACTCTGAGCTGCCTCCAAAGCCAG 
               
               
                   
                   
                 CTGCAGTGGTGAGTGAGGCCTGTGATGCT 
               
               
                   
                   
                 GATGATGCTGATAATGCCAGTAAACTAGCTGCAATGCTAAA 
               
               
                   
                   
                 TACCTGTTCCTTTCAGAAACCAGAACAAC 
               
               
                   
                   
                 TACAACAACAAAAATCAGTTTTTGAGATATGCCCATCTCCT 
               
               
                   
                   
                 GCAGAAAATAACATCCAGGGAACCACAAA 
               
               
                   
                   
                 GCTAGCGTCTGGTGAAGAATTCTGTTCAGGTTCCAGCAGCA 
               
               
                   
                   
                 ATTTGCAAGCTCCTGGTGGCAGCTCTGAA 
               
               
                   
                   
                 CGGTATTTAAAACAAAATGAAATGAATGGTGCTTACTTCAA 
               
               
                   
                   
                 GCAAAGCTCAGTGTTCACTAAGGATTCCT 
               
               
                   
                   
                 TTTCTGCCACTACCACACCACCACCACCATCACAATTGCTTC 
               
               
                   
                   
                 TTTCTCCCCCTCCTCCTCTTCCACAGGT 
               
               
                   
                   
                 TCCTCAGCTTCCTTCAGAAGGAAAAAGCACTCTGAATGGTG 
               
               
                   
                   
                 GAGTTTTAGAAGAACACCACCACTACCCC 
               
               
                   
                   
                 AACCAAAGTAACACAACACTTTTAAGGGAAGTGAAAATAG 
               
               
                   
                   
                 AGGGTAAACCTGAGGCACCACCTTCCCAGA 
               
               
                   
                   
                 GTCCTAATCCATCTACACATGTATGCAGCCCTTCTCCGATGC 
               
               
                   
                   
                 TTTCTGAAAGGCCTCAGAATAATTGTGT 
               
               
                   
                   
                 GAACAGGAATGACATACAGACTGCAGGGACAATGACTGTT 
               
               
                   
                   
                 CCATTGTGTTCTGAGAAAACAAGACCAATG 
               
               
                   
                   
                 TCAGAACACCTCAAGCATAACCCACCAATTTTTGGTAGCAG 
               
               
                   
                   
                 TGGAGAGCTACAGGACAACTGCCAGCAGT 
               
               
                   
                   
                 TGATGAGAAACAAAGAGCAAGAGATTCTGAAGGGTCGAGA 
               
               
                   
                   
                 CAAGGAGCAAACACGAGATCTTGTGCCCCC 
               
               
                   
                   
                 AACACAGCACTATCTGAAACCAGGATGGATTGAATTGAAG 
               
               
                   
                   
                 GCCCCTCGTTTTCACCAAGCGGAATCCCAT 
               
               
                   
                   
                 CTAAAACGTAATGAGGCATCACTGCCATCAATTCTTCAGTA 
               
               
                   
                   
                 TCAACCCAATCTCTCCAATCAAATGACCT 
               
               
                   
                   
                 CCAAACAATACACTGGAAATTCCAACATGCCTGGGGGGCTC 
               
               
                   
                   
                 CCAAGGCAAGCTTACACCCAGAAAACAAC 
               
               
                   
                   
                 ACAGCTGGAGCACAAGTCACAAATGTACCAAGTTGAAATG 
               
               
                   
                   
                 AATCAAGGGCAGTCCCAAGGTACAGTGGAC 
               
               
                   
                   
                 CAACATCTCCAGTTCCAAAAACCCTCACACCAGGTGCACTT 
               
               
                   
                   
                 CTCCAAAACAGACCATTTACCAAAAGCTC 
               
               
                   
                   
                 ATGTGCAGTCACTGTGTGGCACTAGATTTCATTTTCAACAA 
               
               
                   
                   
                 AGAGCAGATTCCCAAACTGAAAAACTTAT 
               
               
                   
                   
                 GTCCCCAGTGTTGAAACAGCACTTGAATCAACAGGCTTCAG 
               
               
                   
                   
                 AGACTGAGCCATTTTCAAACTCACACCTT 
               
               
                   
                   
                 TTGCAACATAAGCCTCATAAACAGGCAGCACAAACACAAC 
               
               
                   
                   
                 CATCCCAGAGTTCACATCTCCCTCAAAACC 
               
               
                   
                   
                 AGCAACAGCAGCAAAAATTACAAATAAAGAATAAAGAGGA 
               
               
                   
                   
                 AATACTCCAGACTTTTCCTCACCCCCAAAG 
               
               
                   
                   
                 CAACAATGATCAGCAAAGAGAAGGATCATTCTTTGGCCAGA 
               
               
                   
                   
                 CTAAAGTGGAAGAATGTTTTCATGGTGAA 
               
               
                   
                   
                 AATCAGTATTCAAAATCAAGCGAGTTCGAGACTCATAATGT 
               
               
                   
                   
                 CCAAATGGGACTGGAGGAAGTACAGAATA 
               
               
                   
                   
                 TAAATCGTAGAAATTCCCCTTATAGTCAGACCATGAAATCA 
               
               
                   
                   
                 AGTGCATGCAAAATACAGGTTTCTTGTTC 
               
               
                   
                   
                 AAACAATACACACCTAGTTTCAGAGAATAAAGAACAGACT 
               
               
                   
                   
                 ACACATCCTGAACTTTTTGCAGGAAACAAG 
               
               
                   
                   
                 ACCCAAAACTTGCATCACATGCAATATTTTCCAAATAATGT 
               
               
                   
                   
                 GATCCCAAAGCAAGATCTTCTTCACAGGT 
               
               
                   
                   
                 GCTTTCAAGAACAGGAGCAGAAGTCACAACAAGCTTCAGTT 
               
               
                   
                   
                 CTACAGGGATATAAAAATAGAAACCAAGA 
               
               
                   
                   
                 TATGTCTGGTCAACAAGCTGCGCAACTTGCTCAGCAAAGGT 
               
               
                   
                   
                 ACTTGATACATAACCATGCAAATGTTTTT 
               
               
                   
                   
                 CCTGTGCCTGACCAGGGAGGAAGTCACACTCAGACCCCTCC 
               
               
                   
                   
                 CCAGAAGGACACTCAAAAGCATGCTGCTC 
               
               
                   
                   
                 TAAGGTGGCATCTCTTACAGAAGCAAGAACAGCAGCAAAC 
               
               
                   
                   
                 ACAGCAACCCCAAACTGAGTCTTGCCATAG 
               
               
                   
                   
                 TCAGATGCACAGGCCAATTAAGGTGGAACCTGGATGCAAG 
               
               
                   
                   
                 CCACATGCCTGTATGCACACAGCACCACCA 
               
               
                   
                   
                 GAAAACAAAACATGGAAAAAGGTAACTAAGCAAGAGAATC 
               
               
                   
                   
                 CACCTGCAAGCTGTGATAATGTGCAGCAAA 
               
               
                   
                   
                 AGAGCATCATTGAGACCATGGAGCAGCATCTGAAGCAGTTT 
               
               
                   
                   
                 CACGCCAAGTCGTTATTTGACCATAAGGC 
               
               
                   
                   
                 TCTTACTCTCAAATCACAGAAGCAAGTAAAAGTTGAAATGT 
               
               
                   
                   
                 CAGGGCCAGTCACAGTTTTGACTAGACAA 
               
               
                   
                   
                 ACCACTGCTGCAGAACTTGATAGCCACACCCCAGCTTTAGA 
               
               
                   
                   
                 GCAGCAAACAACTTCTTCAGAAAAGACAC 
               
               
                   
                   
                 CAACCAAAAGAACAGCTGCTTCTGTTCTCAATAATTTTATA 
               
               
                   
                   
                 GAGTCACCTTCCAAATTACTAGATACTCC 
               
               
                   
                   
                 TATAAAAAATTTATTGGATACACCTGTCAAGACTCAATATG 
               
               
                   
                   
                 ATTTCCCATCTTGCAGATGTGTAGAGCAA 
               
               
                   
                   
                 ATTATTGAAAAAGATGAAGGTCCTTTTTATACCCATCTAGG 
               
               
                   
                   
                 AGCAGGTCCTAATGTGGCAGCTATTAGAG 
               
               
                   
                   
                 AAATCATGGAAGAAAGGTATACAAGTACTTGCCTTTACTCC 
               
               
                   
                   
                 TGCATGTAGAAGACTCTTATGAGCGAGAT 
               
               
                   
                   
                 AATGCAGAGAAGGCCTTTCATATAAATTTATACAGCTCTGA 
               
               
                   
                   
                 GCTGTTCTTCTTCTAGGGTGCCTTTTCAT 
               
               
                   
                   
                 TAAGAGGTAGGCAGTATTATTATTAAAGTACTTAGGATACA 
               
               
                   
                   
                 TTGGGGCAGCTAGGACATATTCAGTATCA 
               
               
                   
                   
                 TTCTTGCTCCATTTCCAAATTATTCATTTCTAAATTAGCATG 
               
               
                   
                   
                 TAGAAGTTCACTAAATAATCATCTAGTG 
               
               
                   
                   
                 GCCTGGCAGAAATAGTGAATTTCCCTAAGTGCCTTTTTTTTG 
               
               
                   
                   
                 TTGTTTTTTTGTTTTGTTTTTTAAACAA 
               
               
                   
                   
                 GCAGTAGGTGGTGCTTTGGTCATAAGGGAAGATATAGTCTA 
               
               
                   
                   
                 TTTCTAGGACTATTCCATATTTTCCATGT 
               
               
                   
                   
                 GGCTGGATACTAACTATTTGCCAGCCTCCTTTTCTAAATTGT 
               
               
                   
                   
                 GAGACATTCTTGGAGGAACAGTTCTAAC 
               
               
                   
                   
                 TAAAATCTATTATGACTCCCCAAGTTTTAAAATAGCTAAATT 
               
               
                   
                   
                 TAGTAAGGGAAAAAATAGTTTATGTTTT 
               
               
                   
                   
                 AGAAGACTGAACTTAGCAAACTAACCTGAATTTTGTGCTTT 
               
               
                   
                   
                 GTGAAATTTTATATCGAAATGAGCTTTCC 
               
               
                   
                   
                 CATTTTCACCCACATGTAATTTACAAAATAGTTCATTACAAT 
               
               
                   
                   
                 TATCTGTACATTTTGATATTGAGGAAAA 
               
               
                   
                   
                 ACAAGGCTTAAAAACCATTATCCAGTTTGCTTGGCGTAGAC 
               
               
                   
                   
                 CTGTTTAAAAAATAATAAACCGTTCATTT 
               
               
                   
                   
                 CTCAGGATGTGGTCATAGAATAAAGTTATGCTCAAATGTTC 
               
               
                   
                   
                 AAA 
               
               
                   
               
            
           
         
       
     
     “Tet inhibitor” or “Tet[x] inhibitor” (e.g., “Tet1 inhibitor,” “Tet2 inhibitor”, or “Tet3 inhibitor”) as the terms are used herein, refers to a molecule, or group of molecules (e.g., a system) that reduces or eliminates the function and/or expression of the corresponding Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2. In embodiments, a Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2 inhibitor is a molecule that inhibits the expression of Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, e.g., reduces or eliminates expression of Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2. In embodiments, the Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2 inhibitor is a molecule that inhibits the function of Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2. An example of Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2 inhibitor that inhibits the expression of Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2 is a gene editing system, e.g., as described herein, that is targeted to nucleic acid within the Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2 gene, or its regulatory elements, such that modification of the nucleic acid at or near the gene editing system binding site(s) is modified to reduce or eliminate expression of Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2. Another example of a Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2 inhibitor that inhibits the expression of Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2 is a nucleic acid molecule, e.g., RNA molecule, e.g., a short hairpin RNA (shRNA) or short interfering RNA (siRNA), capable of hybridizing with Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2 mRNA and causing a reduction or elimination of Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2 translation. Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2 inhibitors also include nucleic acids encoding molecules which inhibit Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2 expression (e.g., nucleic acid encoding an anti-Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2 shRNA or siRNA, or nucleic acid encoding one or more, e.g., all, components of an anti-Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2 gene editing system). An example of a molecule that inhibits the function of Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2 is a molecule, e.g., a protein or small molecule which inhibits one or more activities of Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2. An example is a small molecule inhibitor of Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2. Another example is a dominant negative Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2 protein. Another example is a dominant negative version of a Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2 binding partner, e.g., an associated histone deacetylase (HDAC). Another example is a molecule, e.g., a small molecule, which inhibits a Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2 binding partner, e.g., a Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2-associated HDAC inhibitor. Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2 inhibitors also include nucleic acids encoding inhibitors of Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2 function. 
     A “system” as the term is used herein in connection with gene editing or Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2 inhibition, refers to a group of molecules, e.g., one or more molecules, which together act to effect a desired function. 
     A “gene editing system” as the term is used herein, refers to a system, e.g., one or more molecules, that direct and effect an alteration, e.g., a deletion, of one or more nucleic acids at or near a site of genomic DNA targeted by said system. Gene editing systems are known in the art, and are described more fully below. 
     “binding partner” as the term is used herein in the context of a Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2 binding partner, refers to a molecule, e.g., a protein, which interacts, e.g., binds to, Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2 protein. Without being bound by theory, it is believed that Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2 binds to one or more HDAC proteins. Such HDAC proteins are considered examples of Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2 binding partners. 
     A “dominant negative” gene product or protein is one that interferes with the function of another gene product or protein. The other gene product affected can be the same or different from the dominant negative protein. Dominant negative gene products can be of many forms, including truncations, full length proteins with point mutations or fragments thereof, or fusions of full length wild type or mutant proteins or fragments thereof with other proteins. The level of inhibition observed can be very low. For example, it may require a large excess of the dominant negative protein compared to the functional protein or proteins involved in a process in order to see an effect. It may be difficult to see effects under normal biological assay conditions. In one embodiment, a dominant negative Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2 is a catalytically inactive Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2. In another embodiment, a dominant negative Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2 binding partner is a catalytically inactive Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2-binding HDAC inhibitor. 
     Description 
     The present invention provides Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, inhibitors and methods of use therefore. In particular, the invention provides CAR-expressing T cells comprising Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, inhibitors, and use of Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, in connection with CAR T cells. Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, inhibitor of the present invention, together with their methods of use, are described in more detail below. CARs, CAR T cells, and methods of use are further described below. 
     Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2 Inhibitors 
     The present invention provides compositions, e.g., Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2 inhibitors, and methods for enhancing immune effector cell functions, e.g., CAR-expressing cell functions, by using such compositions and/or other means as described herein. Any Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, inhibitors known in the art can be used as a Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, inhibitor according to the present invention. Examples of Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, inhibitors are described below. 
     Gene Editing Systems 
     According to the present invention, gene editing systems can be used as Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, inhibitors. Also contemplated by the present invention are the uses of nucleic acid encoding one or more components of a Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, gene editing system. 
     CRISPR/Cas9 Gene Editing Systems 
     Naturally-occurring CRISPR/Cas systems are found in approximately 40% of sequenced eubacteria genomes and 90% of sequenced archaea. Grissa et al. (2007) BMC Bioinformatics 8: 172. This system is a type of prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages and provides a form of acquired immunity. Barrangou et al. (2007) Science 315: 1709-1712; Marragini et al. (2008) Science 322: 1843-1845. 
     The CRISPR/Cas system has been modified for use in gene editing (silencing, enhancing or changing specific genes) in eukaryotes such as mice or primates. Wiedenheft et al. (2012) Nature 482: 331-8. This is accomplished by, for example, introducing into the eukaryotic cell a plasmid containing a specifically designed CRISPR and one or more appropriate Cas. 
     The CRISPR sequence, sometimes called a CRISPR locus, comprises alternating repeats and spacers. In a naturally-occurring CRISPR, the spacers usually comprise sequences foreign to the bacterium such as a plasmid or phage sequence; in an exemplary Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, CRISPR/Cas system, the spacers are derived from the Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, gene sequence, or a sequence of its regulatory elements. 
     RNA from the CRISPR locus is constitutively expressed and processed into small RNAs. These comprise a spacer flanked by a repeat sequence. The RNAs guide other Cas proteins to silence exogenous genetic elements at the RNA or DNA level. Horvath et al. (2010) Science 327: 167-170; Makarova et al. (2006) Biology Direct 1: 7. The spacers thus serve as templates for RNA molecules, analogously to siRNAs. Pennisi (2013) Science 341: 833-836. 
     As these naturally occur in many different types of bacteria, the exact arrangements of the CRISPR and structure, function and number of Cas genes and their product differ somewhat from species to species. Haft et al. (2005) PLoS Comput. Biol. 1: e60; Kunin et al. (2007) Genome Biol. 8: R61; Mojica et al. (2005) J. Mol. Evol. 60: 174-182; Bolotin et al. (2005) Microbiol. 151: 2551-2561; Pourcel et al. (2005) Microbiol. 151: 653-663; and Stern et al. (2010) Trends. Genet. 28: 335-340. For example, the Cse (Cas subtype,  E. coli ) proteins (e.g., CasA) form a functional complex, Cascade, that processes CRISPR RNA transcripts into spacer-repeat units that Cascade retains. Brouns et al. (2008) Science 321: 960-964. In other prokaryotes, Cas6 processes the CRISPR transcript. The CRISPR-based phage inactivation in  E. coli  requires Cascade and Cas3, but not Cas1 or Cas2. The Cmr (Cas RAMP module) proteins in  Pyrococcus furiosus  and other prokaryotes form a functional complex with small CRISPR RNAs that recognizes and cleaves complementary target RNAs. A simpler CRISPR system relies on the protein Cas9, which is a nuclease with two active cutting sites, one for each strand of the double helix. Combining Cas9 and modified CRISPR locus RNA can be used in a system for gene editing. Pennisi (2013) Science 341: 833-836. 
     The CRISPR/Cas system can thus be used to modify, e.g., delete one or more nucleic acids, the Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, gene, or a Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, gene regulatory element, or introduce a premature stop which thus decreases expression of a functional Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2. The CRISPR/Cas system can alternatively be used like RNA interference, turning off the Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, gene in a reversible fashion. In a mammalian cell, for example, the RNA can guide the Cas protein to a Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, promoter, sterically blocking RNA polymerases. 
     CRISPR/Cas systems for gene editing in eukaryotic cells typically involve (1) a guide RNA molecule (gRNA) comprising a targeting sequence (which is capable of hybridizing to the genomic DNA target sequence), and sequence which is capable of binding to a Cas, e.g., Cas9 enzyme, and (2) a Cas, e.g., Cas9, protein. The targeting sequence and the sequence which is capable of binding to a Cas, e.g., Cas9 enzyme, may be disposed on the same or different molecules. If disposed on different molecules, each includes a hybridization domain which allows the molecules to associate, e.g., through hybridization. 
     An exemplary gRNA molecule of the present invention comprises, e.g., consists of a first nucleic acid having the sequence (where the “n” &#39;s refer to the residues of the targeting sequence (e.g., as described herein, e.g., in Table 3), and may consist of 15-25 nucelotides, e.g., consist of 20 nucleotides): 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 40) 
               
               
                   
                 nnnnnnnnnnnnnnnnnnnnGUUUUAGAGCUAUGCUGUUUUG; 
               
            
           
         
       
     
     and a second nucleic acid sequence having the sequence: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 41) 
               
               
                 AACUUACCAAGGAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC 
               
               
                   
               
               
                 AACUUGAAAAAGUGGCACCGAGUCGGUGC, 
               
               
                 optionally with 1, 2, 3, 4, 5, 6, or 7 (e.g., 4 
               
               
                 or 7, e.g., 7) additional U nucleotides at the 3′ 
               
               
                 end. 
               
            
           
         
       
     
     The second nucleic acid molecule may alternatively consist of a fragment of the sequence above, wherein such fragment is capable of hybridizing to the first nucleic acid. An example of such second nucleic acid molecule is: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 42) 
               
               
                 AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAG 
               
               
                   
               
               
                 UGGCACCGAGUCGGUGC, 
               
               
                 optionally with 1, 2, 3, 4, 5, 6, or 7 (e.g., 4 
               
               
                 or 7, e.g., 7) additional U nucleotides at the 3′ 
               
               
                 end. 
               
            
           
         
       
     
     Another exemplary gRNA molecule of the present invention comprises, e.g., consists of a first nucleic acid having the sequence (where the “n” &#39;s refer to the residues of the targeting sequence (e.g., as described herein, e.g., in Table 3), and may consist of 15-25 nucelotides, e.g., consist of 20 nucleotides): 
                    (SEQ ID NO: 43)       nnnnnnnnnnnnnnnnnnn GUUUUAGAGCUA GAAA UAGCAAGUUAAAAUA                   AGGCUAGUCCG UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC,            
optionally with 1, 2, 3, 4, 5, 6, or 7 (e.g., 4 or 7, e.g., 4) additional U nucleotides at the 3′ end. Artificial CRISPR/Cas systems can be generated which inhibit Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, using technology known in the art, e.g., that are described in U.S. Publication No. 20140068797, WO2015/048577, and Cong (2013) Science 339: 819-823. Other artificial CRISPR/Cas systems that are known in the art may also be generated which inhibit Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, e.g., that described in Tsai (2014) Nature Biotechnol., 32:6 569-576, U.S. Pat. Nos. 8,871,445; 8,865,406; 8,795,965; 8,771,945; and 8,697,359, the contents of which are hereby incorporated by reference in their entirety. Such systems can be generated which inhibit Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, by, for example, engineering a CRISPR/Cas system to include a gRNA molecule comprising a targeting sequence that hybridizes to a sequence of a tet gene, e.g., a Tet1, Tet2 and/or Tet3, e.g., Tet2 gene. In embodiments, the gRNA comprises a targeting sequence which is fully complementarity to 15-25 nucleotides, e.g., 20 nucleotides, of a tet gene, e.g., a Tet1, Tet2 and/or Tet3, e.g., Tet2 gene. In embodiments, the 15-25 nucleotides, e.g., 20 nucleotides, of a tet gene, e.g., a Tet1, Tet2 and/or Tet3, e.g., Tet2 gene, are disposed immediately 5′ to a protospacer adjacent motif (PAM) sequence recognized by the Cas protein of the CRISPR/Cas system (e.g., where the system comprises a  S. pyogenes  Cas9 protein, the PAM sequence comprises NGG, where N can be any of A, T, G or C). In embodiments, the targeting sequence of the gRNA comprises, e.g., consists of, a RNA sequence complementary to a sequence listed in Table 2. In embodiments, the gRNA comprises a targeting sequence listed in Table 3.
 
     In one embodiment, foreign DNA can be introduced into the cell along with the CRISPR/Cas system, e.g., DNA encoding a CAR, e.g., as described herein; depending on the sequences of the foreign DNA and chromosomal sequence, this process can be used to integrate the DNA encoding the CAR, e.g., as described herein, at or near the site targeted by the CRISPR/Cas system. As shown herein, in the examples, but without being bound by theory, such integration may lead to the expression of the CAR as well as disruption of the Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, gene. Such foreign DNA molecule is referred to herein as “template DNA.” In embodiments, the template DNA further comprises homology arms 5′ to, 3′ to, or both 5′ and 3′ to the nucleic acid of the template DNA which encodes the molecule or molecules of interest (e.g., which encodes a CAR described herein), wherein said homology arms are complementary to genomic DNA sequence flanking the target sequence. 
     In an embodiment, the CRISPR/Cas system of the present invention comprises Cas9, e.g.,  S. pyogenes  Cas9, and a gRNA comprising a targeting sequence which hybridizes to a sequence of the Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, gene. In an embodiment, the CRISPR/Cas system comprises nucleic acid encoding a Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, gRNA and nucleic acid encoding a Cas protein, e.g., Cas9, e.g.,  S. pyogenes  Cas9. In an embodiment, the CRISPR/Cas system comprises a Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, gRNA and nucleic acid encoding a Cas protein, e.g., Cas9, e.g.,  S. pyogenes  Cas9. 
     Examples of genomic target sequences for Tet2, for which gRNAs comprising complementary targeting sequences can be generated for use in the present invention are listed in the table 2 below. In embodiments, the gRNA comprises an RNA complement of a Target Sequence of the table below (e.g., for sgTET2_1, the gRNA would comprise CCUUGGACACCUUCUCCUCC (SEQ ID NO: 44)). In embodiments, the gRNA comprises the RNA analog of a Target sequence of the table 2 below (e.g., for sgTET2_1, the gRNA would comprise GGAACCUGUGGAAGAGGAGG (SEQ ID NO: 45). In embodiments, the Tet2 inhibitor is nucleic acid encoding a gRNA molecule specific for Tet2, wherein the nucleic acid comprises the sequence of a Target Sequence from the 2 table below, e.g., under the control of a U6- or H1-promoter: 
     
       
         
           
               
               
               
               
               
               
             
               
                 TABLE 2 
               
               
                   
               
               
                   
                 Gene 
                   
                   
                   
                 Target Sequence within 
               
               
                 gRNA ID 
                 Symbol 
                 Chromosome 
                 Position 
                 Strand 
                 the Tet2 gene sequence 
               
               
                   
               
             
            
               
                 sgTET2_1 
                 TET2 
                 chr4 
                 106156327 
                 − 
                 GGAACCTGTGGAAGAGGAGG 
               
               
                   
                   
                   
                   
                   
                 (SEQ ID NO: 46) 
               
               
                   
               
               
                 sgTET2_2 
                 TET2 
                 chr4 
                 106156339 
                 − 
                 GAAGGAAGCTGAGGAACCTG 
               
               
                   
                   
                   
                   
                   
                 (SEQ ID NO: 47) 
               
               
                   
               
               
                 sgTET2_3 
                 TET2 
                 chr4 
                 106156897 
                 + 
                 ATGACCTCCAAACAATACAC 
               
               
                   
                   
                   
                   
                   
                 (SEQ ID NO: 48) 
               
               
                   
               
               
                 sgTET2_4 
                 TET2 
                 chr4 
                 106157189 
                 − 
                 CAAGTGCTGTTTCAACACTG 
               
               
                   
                   
                   
                   
                   
                 (SEQ ID NO: 49) 
               
               
                   
               
               
                 sgTET2_5 
                 TET2 
                 chr4 
                 106157296 
                 − 
                 GGGAGATGTGAACTCTGGGA 
               
               
                   
                   
                   
                   
                   
                 (SEQ ID NO: 50) 
               
               
                   
               
               
                 sgTET2_6 
                 TET2 
                 chr4 
                 106155148 
                 − 
                 GGAGGTGATGGTATCAGGAA 
               
               
                   
                   
                   
                   
                   
                 (SEQ ID NO: 51) 
               
               
                   
               
               
                 sgTET2_7 
                 TET2 
                 chr4 
                 106155166 
                 − 
                 GGTTCTGTCTGGCAAATGGG 
               
               
                   
                   
                   
                   
                   
                 (SEQ ID NO: 52) 
               
               
                   
               
               
                 sgTET2_8 
                 TET2 
                 chr4 
                 106155217 
                 − 
                 GGATGAGCTCTCTCAGGCAG 
               
               
                   
                   
                   
                   
                   
                 (SEQ ID NO: 53) 
               
               
                   
               
               
                 sgTET2_9 
                 TET2 
                 chr4 
                 106155403 
                 − 
                 TGAAGGAGCCCAGAGAGAGA 
               
               
                   
                   
                   
                   
                   
                 (SEQ ID NO: 65) 
               
               
                   
               
               
                 sgTET2_10 
                 TET2 
                 chr4 
                 106155478 
                 + 
                 GTAAGCCAAGAAAGAAATCC 
               
               
                   
                   
                   
                   
                   
                 (SEQ ID NO: 66) 
               
               
                   
               
            
           
         
       
     
     Examples of gRNA targeting sequences which are useful in the various embodiments of the present invention to inhibit a Tet, e.g., Tet2, are provided below in Table 3. In embodiments a CRISPR/Cas system of the present invention comprises a gRNA molecule comprising a targeting sequence comprising a sequence listed in Table 3. In embodiments, a CRISPR/Cas system of the present invention comprises a gRNA molecule comprising a targeting sequence that is a sequence listed in Table 3. 
     
       
         
           
               
               
               
               
               
               
               
             
               
                 TABLE 3 
               
               
                   
               
               
                   
                   
                   
                   
                 Location of 
                   
                 SEQ 
               
               
                   
                   
                 TARGET 
                   
                 Genomic Target 
                   
                 ID 
               
               
                 ID 
                 TARGET 
                 REGION 
                 STRAND 
                 Sequence (hg38) 
                 gRNA Targeting sequence 
                 NO: 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 54790_1_1 
                 TET2 
                 EXON 
                 + 
                 chr4: 105145928-105145948 
                 UGUCGGGUCUUUAAAAAUAC 
                 73 
               
               
                   
               
               
                 54790_1_3 
                 TET2 
                 EXON 
                 + 
                 chr4: 105145945-105145965 
                 UACAGGCCCCUAAAGCACUA 
                 74 
               
               
                   
               
               
                 54790_1_4 
                 TET2 
                 EXON 
                 + 
                 chr4: 105145946-105145966 
                 ACAGGCCCCUAAAGCACUAA 
                 75 
               
               
                   
               
               
                 54790_1_5 
                 TET2 
                 EXON 
                 + 
                 chr4: 105145957-105145977 
                 AAGCACUAAGGGCAUGCCCU 
                 76 
               
               
                   
               
               
                 54790_1_8 
                 TET2 
                 EXON 
                 + 
                 chr4: 105145966-105145986 
                 GGGCAUGCCCUCGGUGAAAC 
                 77 
               
               
                   
               
               
                 54790_1_10 
                 TET2 
                 EXON 
                 + 
                 chr4: 105145967-105145987 
                 GGCAUGCCCUCGGUGAAACA 
                 78 
               
               
                   
               
               
                 54790_1_12 
                 TET2 
                 EXON 
                 + 
                 chr4: 105145968-105145988 
                 GCAUGCCCUCGGUGAAACAG 
                 79 
               
               
                   
               
               
                 54790_1_20 
                 TET2 
                 EXON 
                 + 
                 chr4: 105146006-105146026 
                 UGAGAUUAAAGCGACAGAAA 
                 80 
               
               
                   
               
               
                 54790_1_23 
                 TET2 
                 EXON 
                 + 
                 chr4: 105146007-105146027 
                 GAGAUUAAAGCGACAGAAAA 
                 81 
               
               
                   
               
               
                 54790_1_25 
                 TET2 
                 EXON 
                 + 
                 chr4: 105146012-105146032 
                 UAAAGCGACAGAAAAGGGAA 
                 82 
               
               
                   
               
               
                 54790_1_30 
                 TET2 
                 EXON 
                 + 
                 chr4: 105146021-105146041 
                 AGAAAAGGGAAAGGAGAGCG 
                 83 
               
               
                   
               
               
                 54790_1_31 
                 TET2 
                 EXON 
                 + 
                 chr4: 105146022-105146042 
                 GAAAAGGGAAAGGAGAGCGC 
                 84 
               
               
                   
               
               
                 54790_1_33 
                 TET2 
                 EXON 
                 + 
                 chr4: 105146028-105146048 
                 GGAAAGGAGAGCGCGGGCAA 
                 85 
               
               
                   
               
               
                 54790_1_35 
                 TET2 
                 EXON 
                 + 
                 chr4: 105146029-105146049 
                 GAAAGGAGAGCGCGGGCAAC 
                 86 
               
               
                   
               
               
                 54790_1_38 
                 TET2 
                 EXON 
                 + 
                 chr4: 105146038-105146058 
                 GCGCGGGCAACGGGAUCUAA 
                 87 
               
               
                   
               
               
                 54790_1_39 
                 TET2 
                 EXON 
                 + 
                 chr4: 105146039-105146059 
                 CGCGGGCAACGGGAUCUAAA 
                 88 
               
               
                   
               
               
                 54790_1_43 
                 TET2 
                 EXON 
                 + 
                 chr4: 105146053-105146073 
                 UCUAAAGGGAGAUAGAGACG 
                 89 
               
               
                   
               
               
                 54790_1_44 
                 TET2 
                 EXON 
                 + 
                 chr4: 105146054-105146074 
                 CUAAAGGGAGAUAGAGACGC 
                 90 
               
               
                   
               
               
                 54790_1_47 
                 TET2 
                 EXON 
                 + 
                 chr4: 105146063-105146083 
                 GAUAGAGACGCGGGCCUCUG 
                 91 
               
               
                   
               
               
                 54790_1_48 
                 TET2 
                 EXON 
                 + 
                 chr4: 105146064-105146084 
                 AUAGAGACGCGGGCCUCUGA 
                 92 
               
               
                   
               
               
                 54790_1_49 
                 TET2 
                 EXON 
                 + 
                 chr4: 105146069-105146089 
                 GACGCGGGCCUCUGAGGGUA 
                 93 
               
               
                   
               
               
                 54790_1_51 
                 TET2 
                 EXON 
                 + 
                 chr4: 105146072-105146092 
                 GCGGGCCUCUGAGGGUAAGG 
                 94 
               
               
                   
               
               
                 54790_1_52 
                 TET2 
                 EXON 
                 + 
                 chr4: 105146073-105146093 
                 CGGGCCUCUGAGGGUAAGGU 
                 95 
               
               
                   
               
               
                 54790_1_54 
                 TET2 
                 EXON 
                 + 
                 chr4: 105146082-105146102 
                 GAGGGUAAGGUGGGCGCAAG 
                 96 
               
               
                   
               
               
                 54790_1_61 
                 TET2 
                 EXON 
                 − 
                 chr4: 105145954-105145974 
                 GCAUGCCCUUAGUGCUUUAG 
                 97 
               
               
                   
               
               
                 54790_1_62 
                 TET2 
                 EXON 
                 − 
                 chr4: 105145955-105145975 
                 GGCAUGCCCUUAGUGCUUUA 
                 98 
               
               
                   
               
               
                 54790_1_64 
                 TET2 
                 EXON 
                 − 
                 chr4: 105145956-105145976 
                 GGGCAUGCCCUUAGUGCUUU 
                 99 
               
               
                   
               
               
                 54790_1_68 
                 TET2 
                 EXON 
                 − 
                 chr4: 105145976-105145996 
                 GCGCUCCCCUGUUUCACCGA 
                 100 
               
               
                   
               
               
                 54790_1_69 
                 TET2 
                 EXON 
                 − 
                 chr4: 105145977-105145997 
                 AGCGCUCCCCUGUUUCACCG 
                 101 
               
               
                   
               
               
                 54790_1_87 
                 TET2 
                 EXON 
                 − 
                 chr4: 105146080-105146100 
                 UGCGCCCACCUUACCCUCAG 
                 102 
               
               
                   
               
               
                 54790_2_1 
                 TET2 
                 EXON 
                 + 
                 chr4: 105146669-105146689 
                 AGAGCCGGCGGUAGCGGCAG 
                 103 
               
               
                   
               
               
                 54790_2_2 
                 TET2 
                 EXON 
                 + 
                 chr4: 105146675-105146695 
                 GGCGGUAGCGGCAGUGGCAG 
                 104 
               
               
                   
               
               
                 54790_2_6 
                 TET2 
                 EXON 
                 + 
                 chr4: 105146686-105146706 
                 CAGUGGCAGCGGCGAGAGCU 
                 105 
               
               
                   
               
               
                 54790_2_7 
                 TET2 
                 EXON 
                 + 
                 chr4: 105146687-105146707 
                 AGUGGCAGCGGCGAGAGCUU 
                 106 
               
               
                   
               
               
                 54790_2_8 
                 TET2 
                 EXON 
                 + 
                 chr4: 105146690-105146710 
                 GGCAGCGGCGAGAGCUUGGG 
                 107 
               
               
                   
               
               
                 54790_2_12 
                 TET2 
                 EXON 
                 + 
                 chr4: 105146725-105146745 
                 CCUCGCGAGCGCCGCGCGCC 
                 108 
               
               
                   
               
               
                 54790_2_13 
                 TET2 
                 EXON 
                 + 
                 chr4: 105146726-105146746 
                 CUCGCGAGCGCCGCGCGCCC 
                 109 
               
               
                   
               
               
                 54790_2_14 
                 TET2 
                 EXON 
                 + 
                 chr4: 105146761-105146781 
                 GCAAGUCACGUCCGCCCCCU 
                 110 
               
               
                   
               
               
                 54790_2_15 
                 TET2 
                 EXON 
                 + 
                 chr4: 105146766-105146786 
                 UCACGUCCGCCCCCUCGGCG 
                 111 
               
               
                   
               
               
                 54790_2_17 
                 TET2 
                 EXON 
                 + 
                 chr4: 105146783-105146803 
                 GCGCGGCCGCCCCGAGACGC 
                 112 
               
               
                   
               
               
                 54790_2_24 
                 TET2 
                 EXON 
                 + 
                 chr4: 105146836-105146856 
                 CUGCCUUAUGAAUAUUGAUG 
                 113 
               
               
                   
               
               
                 54790_2_25 
                 TET2 
                 EXON 
                 + 
                 chr4: 105146839-105146859 
                 CCUUAUGAAUAUUGAUGCGG 
                 114 
               
               
                   
               
               
                 54790_2_27 
                 TET2 
                 EXON 
                 + 
                 chr4: 105146844-105146864 
                 UGAAUAUUGAUGCGGAGGCU 
                 115 
               
               
                   
               
               
                 54790_2_34 
                 TET2 
                 EXON 
                 + 
                 chr4: 105146868-105146888 
                 UGCUUUCGUAGAGAAGCAGA 
                 116 
               
               
                   
               
               
                 54790_2_37 
                 TET2 
                 EXON 
                 + 
                 chr4: 105146879-105146899 
                 AGAAGCAGAAGGAAGCAAGA 
                 117 
               
               
                   
               
               
                 54790_2_39 
                 TET2 
                 EXON 
                 + 
                 chr4: 105146891-105146911 
                 AAGCAAGAUGGCUGCCCUUU 
                 118 
               
               
                   
               
               
                 54790_2_44 
                 TET2 
                 EXON 
                 + 
                 chr4: 105146905-105146925 
                 CCCUUUAGGAUUUGUUAGAA 
                 119 
               
               
                   
               
               
                 54790_2_51 
                 TET2 
                 EXON 
                 + 
                 chr4: 105146926-105146946 
                 GGAGACCCGACUGCAACUGC 
                 120 
               
               
                   
               
               
                 54790_2_52 
                 TET2 
                 EXON 
                 + 
                 chr4: 105146938-105146958 
                 GCAACUGCUGGAUUGCUGCA 
                 121 
               
               
                   
               
               
                 54790_2_56 
                 TET2 
                 EXON 
                 + 
                 chr4: 105146944-105146964 
                 GCUGGAUUGCUGCAAGGCUG 
                 122 
               
               
                   
               
               
                 54790_2_57 
                 TET2 
                 EXON 
                 + 
                 chr4: 105146945-105146965 
                 CUGGAUUGCUGCAAGGCUGA 
                 123 
               
               
                   
               
               
                 54790_2_62 
                 TET2 
                 EXON 
                 + 
                 chr4: 105146957-105146977 
                 AAGGCUGAGGGACGAGAACG 
                 124 
               
               
                   
               
               
                 54790_2_64 
                 TET2 
                 EXON 
                 − 
                 chr4: 105146676-105146696 
                 GCUGCCACUGCCGCUACCGC 
                 125 
               
               
                   
               
               
                 54790_2_65 
                 TET2 
                 EXON 
                 − 
                 chr4: 105146716-105146736 
                 CGCUCGCGAGGAGGCGGCGG 
                 126 
               
               
                   
               
               
                 54790_2_66 
                 TET2 
                 EXON 
                 − 
                 chr4: 105146719-105146739 
                 CGGCGCUCGCGAGGAGGCGG 
                 127 
               
               
                   
               
               
                 54790_2_67 
                 TET2 
                 EXON 
                 − 
                 chr4: 105146722-105146742 
                 GCGCGGCGCUCGCGAGGAGG 
                 128 
               
               
                   
               
               
                 54790_2_68 
                 TET2 
                 EXON 
                 − 
                 chr4: 105146725-105146745 
                 GGCGCGCGGCGCUCGCGAGG 
                 129 
               
               
                   
               
               
                 54790_2_69 
                 TET2 
                 EXON 
                 − 
                 chr4: 105146728-105146748 
                 CCGGGCGCGCGGCGCUCGCG 
                 130 
               
               
                   
               
               
                 54790_2_74 
                 TET2 
                 EXON 
                 − 
                 chr4: 105146739-105146759 
                 GCGAGCGGGACCCGGGCGCG 
                 131 
               
               
                   
               
               
                 54790_2_75 
                 TET2 
                 EXON 
                 − 
                 chr4: 105146746-105146766 
                 CUUGCAUGCGAGCGGGACCC 
                 132 
               
               
                   
               
               
                 54790_2_76 
                 TET2 
                 EXON 
                 − 
                 chr4: 105146747-105146767 
                 ACUUGCAUGCGAGCGGGACC 
                 133 
               
               
                   
               
               
                 54790_2_78 
                 TET2 
                 EXON 
                 − 
                 chr4: 105146753-105146773 
                 GACGUGACUUGCAUGCGAGC 
                 134 
               
               
                   
               
               
                 54790_2_79 
                 TET2 
                 EXON 
                 − 
                 chr4: 105146754-105146774 
                 GGACGUGACUUGCAUGCGAG 
                 135 
               
               
                   
               
               
                 54790_2_83 
                 TET2 
                 EXON 
                 − 
                 chr4: 105146775-105146795 
                 GGGCGGCCGCGCCGAGGGGG 
                 136 
               
               
                   
               
               
                 54790_2_85 
                 TET2 
                 EXON 
                 − 
                 chr4: 105146778-105146798 
                 UCGGGGCGGCCGCGCCGAGG 
                 137 
               
               
                   
               
               
                 54790_2_86 
                 TET2 
                 EXON 
                 − 
                 chr4: 105146779-105146799 
                 CUCGGGGCGGCCGCGCCGAG 
                 138 
               
               
                   
               
               
                 54790_2_88 
                 TET2 
                 EXON 
                 − 
                 chr4: 105146780-105146800 
                 UCUCGGGGCGGCCGCGCCGA 
                 139 
               
               
                   
               
               
                 54790_2_89 
                 TET2 
                 EXON 
                 − 
                 chr4: 105146781-105146801 
                 GUCUCGGGGCGGCCGCGCCG 
                 140 
               
               
                   
               
               
                 54790_2_93 
                 TET2 
                 EXON 
                 − 
                 chr4: 105146792-105146812 
                 GCGGGGCCGGCGUCUCGGGG 
                 141 
               
               
                   
               
               
                 54790_2_94 
                 TET2 
                 EXON 
                 − 
                 chr4: 105146795-105146815 
                 UCAGCGGGGCCGGCGUCUCG 
                 142 
               
               
                   
               
               
                 54790_2_95 
                 TET2 
                 EXON 
                 − 
                 chr4: 105146796-105146816 
                 CUCAGCGGGGCCGGCGUCUC 
                 143 
               
               
                   
               
               
                 54790_2_97 
                 TET2 
                 EXON 
                 − 
                 chr4: 105146797-105146817 
                 ACUCAGCGGGGCCGGCGUCU 
                 144 
               
               
                   
               
               
                 54790_2_100 
                 TET2 
                 EXON 
                 − 
                 chr4: 105146805-105146825 
                 UUCUCAUCACUCAGCGGGGC 
                 145 
               
               
                   
               
               
                 54790_2_101 
                 TET2 
                 EXON 
                 − 
                 chr4: 105146809-105146829 
                 UCUGUUCUCAUCACUCAGCG 
                 146 
               
               
                   
               
               
                 54790_2_103 
                 TET2 
                 EXON 
                 − 
                 chr4: 105146810-105146830 
                 GUCUGUUCUCAUCACUCAGC 
                 147 
               
               
                   
               
               
                 54790_2_106 
                 TET2 
                 EXON 
                 − 
                 chr4: 105146811-105146831 
                 CGUCUGUUCUCAUCACUCAG 
                 148 
               
               
                   
               
               
                 54790_2_109 
                 TET2 
                 EXON 
                 − 
                 chr4: 105146842-105146862 
                 CCUCCGCAUCAAUAUUCAUA 
                 149 
               
               
                   
               
               
                 54790_2_117 
                 TET2 
                 EXON 
                 − 
                 chr4: 105146908-105146928 
                 CCUUUCUAACAAAUCCUAAA 
                 150 
               
               
                   
               
               
                 54790_2_118 
                 TET2 
                 EXON 
                 − 
                 chr4: 105146909-105146929 
                 UCCUUUCUAACAAAUCCUAA 
                 151 
               
               
                   
               
               
                 54790_2_122 
                 TET2 
                 EXON 
                 − 
                 chr4: 105146934-105146954 
                 GCAAUCCAGCAGUUGCAGUC 
                 152 
               
               
                   
               
               
                 54790_2_123 
                 TET2 
                 EXON 
                 − 
                 chr4: 105146935-105146955 
                 AGCAAUCCAGCAGUUGCAGU 
                 153 
               
               
                   
               
               
                 54790_3_1 
                 TET2 
                 EXON 
                 + 
                 chr4: 105190341-105190361 
                 AAACUCUGUCUUCUCUAGGC 
                 154 
               
               
                   
               
               
                 54790_3_13 
                 TET2 
                 EXON 
                 + 
                 chr4: 105190411-105190431 
                 UCCUGUUGAGUUACAACGCU 
                 155 
               
               
                   
               
               
                 54790_3_16 
                 TET2 
                 EXON 
                 + 
                 chr4: 105190418-105190438 
                 GAGUUACAACGCUUGGAAGC 
                 156 
               
               
                   
               
               
                 54790_3_19 
                 TET2 
                 EXON 
                 + 
                 chr4: 105190424-105190444 
                 CAACGCUUGGAAGCAGGAGA 
                 157 
               
               
                   
               
               
                 54790_3_21 
                 TET2 
                 EXON 
                 + 
                 chr4: 105190425-105190445 
                 AACGCUUGGAAGCAGGAGAU 
                 158 
               
               
                   
               
               
                 54790_3_24 
                 TET2 
                 EXON 
                 + 
                 chr4: 105190444-105190464 
                 UGGGCUCAGCAGCAGCCAAU 
                 159 
               
               
                   
               
               
                 54790_3_26 
                 TET2 
                 EXON 
                 + 
                 chr4: 105190456-105190476 
                 CAGCCAAUAGGACAUGAUCC 
                 160 
               
               
                   
               
               
                 54790_3_30 
                 TET2 
                 EXON 
                 + 
                 chr4: 105190469-105190489 
                 AUGAUCCAGGAAGAGCAGUA 
                 161 
               
               
                   
               
               
                 54790_3_32 
                 TET2 
                 EXON 
                 + 
                 chr4: 105190470-105190490 
                 UGAUCCAGGAAGAGCAGUAA 
                 162 
               
               
                   
               
               
                 54790_3_34 
                 TET2 
                 EXON 
                 + 
                 chr4: 105190483-105190503 
                 GCAGUAAGGGACUGAGCUGC 
                 163 
               
               
                   
               
               
                 54790_3_37 
                 TET2 
                 EXON 
                 + 
                 chr4: 105190494-105190514 
                 CUGAGCUGCUGGUAAGACAG 
                 164 
               
               
                   
               
               
                 54790_3_46 
                 TET2 
                 EXON 
                 − 
                 chr4: 105190385-105190405 
                 GCAAGUAAACAAUCUUGAGA 
                 165 
               
               
                   
               
               
                 54790_3_47 
                 TET2 
                 EXON 
                 − 
                 chr4: 105190386-105190406 
                 GGCAAGUAAACAAUCUUGAG 
                 166 
               
               
                   
               
               
                 54790_3_52 
                 TET2 
                 EXON 
                 − 
                 chr4: 105190407-105190427 
                 UUGUAACUCAACAGGAGCAA 
                 167 
               
               
                   
               
               
                 54790_3_55 
                 TET2 
                 EXON 
                 − 
                 chr4: 105190415-105190435 
                 UCCAAGCGUUGUAACUCAAC 
                 168 
               
               
                   
               
               
                 54790_3_60 
                 TET2 
                 EXON 
                 − 
                 chr4: 105190462-105190482 
                 CUUCCUGGAUCAUGUCCUAU 
                 169 
               
               
                   
               
               
                 54790_3_62 
                 TET2 
                 EXON 
                 − 
                 chr4: 105190477-105190497 
                 CAGUCCCUUACUGCUCUUCC 
                 170 
               
               
                   
               
               
                 54790_4_7 
                 TET2 
                 EXON 
                 + 
                 chr4: 105233887-105233907 
                 GCUCUUUAGAAUUCAACUAG 
                 171 
               
               
                   
               
               
                 54790_4_8 
                 TET2 
                 EXON 
                 + 
                 chr4: 105233888-105233908 
                 CUCUUUAGAAUUCAACUAGA 
                 172 
               
               
                   
               
               
                 54790_4_12 
                 TET2 
                 EXON 
                 + 
                 chr4: 105233899-105233919 
                 UCAACUAGAGGGCAGCCUUG 
                 173 
               
               
                   
               
               
                 54790_4_14 
                 TET2 
                 EXON 
                 + 
                 chr4: 105233903-105233923 
                 CUAGAGGGCAGCCUUGUGGA 
                 174 
               
               
                   
               
               
                 54790_4_19 
                 TET2 
                 EXON 
                 + 
                 chr4: 105233923-105233943 
                 UGGCCCCGAAGCAAGCCUGA 
                 175 
               
               
                   
               
               
                 54790_4_21 
                 TET2 
                 EXON 
                 + 
                 chr4: 105233929-105233949 
                 CGAAGCAAGCCUGAUGGAAC 
                 176 
               
               
                   
               
               
                 54790_4_25 
                 TET2 
                 EXON 
                 + 
                 chr4: 105233950-105233970 
                 GGAUAGAACCAACCAUGUUG 
                 177 
               
               
                   
               
               
                 54790_4_26 
                 TET2 
                 EXON 
                 + 
                 chr4: 105233951-105233971 
                 GAUAGAACCAACCAUGUUGA 
                 178 
               
               
                   
               
               
                 54790_4_30 
                 TET2 
                 EXON 
                 + 
                 chr4: 105234010-105234030 
                 CAUUUGCCAGACAGAACCUC 
                 179 
               
               
                   
               
               
                 54790_4_37 
                 TET2 
                 EXON 
                 + 
                 chr4: 105234029-105234049 
                 CUGGCUACAAAGCUCCAGAA 
                 180 
               
               
                   
               
               
                 54790_4_44 
                 TET2 
                 EXON 
                 + 
                 chr4: 105234068-105234088 
                 AGAGCUCAUCCAGAAGUAAA 
                 181 
               
               
                   
               
               
                 54790_4_45 
                 TET2 
                 EXON 
                 + 
                 chr4: 105234081-105234101 
                 AAGUAAAUGGAGACACCAAG 
                 182 
               
               
                   
               
               
                 54790_4_47 
                 TET2 
                 EXON 
                 + 
                 chr4: 105234104-105234124 
                 CACUCUUUCAAAAGUUAUUA 
                 183 
               
               
                   
               
               
                 54790_4_54 
                 TET2 
                 EXON 
                 + 
                 chr4: 105234121-105234141 
                 UUAUGGAAUACCCUGUAUGA 
                 184 
               
               
                   
               
               
                 54790_4_57 
                 TET2 
                 EXON 
                 + 
                 chr4: 105234122-105234142 
                 UAUGGAAUACCCUGUAUGAA 
                 185 
               
               
                   
               
               
                 54790_4_66 
                 TET2 
                 EXON 
                 + 
                 chr4: 105234170-105234190 
                 GACUUUACACAAGAAAGUAG 
                 186 
               
               
                   
               
               
                 54790_4_67 
                 TET2 
                 EXON 
                 + 
                 chr4: 105234171-105234191 
                 ACUUUACACAAGAAAGUAGA 
                 187 
               
               
                   
               
               
                 54790_4_72 
                 TET2 
                 EXON 
                 + 
                 chr4: 105234194-105234214 
                 UAUUCCAAGUGUUUGCAAAA 
                 188 
               
               
                   
               
               
                 54790_4_74 
                 TET2 
                 EXON 
                 + 
                 chr4: 105234197-105234217 
                 UCCAAGUGUUUGCAAAAUGG 
                 189 
               
               
                   
               
               
                 54790_4_81 
                 TET2 
                 EXON 
                 + 
                 chr4: 105234233-105234253 
                 GUUAGUGAACCUUCUCUCUC 
                 190 
               
               
                   
               
               
                 54790_4_82 
                 TET2 
                 EXON 
                 + 
                 chr4: 105234234-105234254 
                 UUAGUGAACCUUCUCUCUCU 
                 191 
               
               
                   
               
               
                 54790_4_89 
                 TET2 
                 EXON 
                 + 
                 chr4: 105234271-105234291 
                 GAAAUUGAAACAAGACCAAA 
                 192 
               
               
                   
               
               
                 54790_4_93 
                 TET2 
                 EXON 
                 + 
                 chr4: 105234278-105234298 
                 AAACAAGACCAAAAGGCUAA 
                 193 
               
               
                   
               
               
                 54790_4_97 
                 TET2 
                 EXON 
                 + 
                 chr4: 105234296-105234316 
                 AAUGGAGAAAGACGUAACUU 
                 194 
               
               
                   
               
               
                 54790_4_99 
                 TET2 
                 EXON 
                 + 
                 chr4: 105234297-105234317 
                 AUGGAGAAAGACGUAACUUC 
                 195 
               
               
                   
               
               
                 54790_4_100 
                 TET2 
                 EXON 
                 + 
                 chr4: 105234298-105234318 
                 UGGAGAAAGACGUAACUUCG 
                 196 
               
               
                   
               
               
                 54790_4_106 
                 TET2 
                 EXON 
                 + 
                 chr4: 105234320-105234340 
                 GUAAGCCAAGAAAGAAAUCC 
                 197 
               
               
                   
               
               
                 54790_4_123 
                 TET2 
                 EXON 
                 + 
                 chr4: 105234437-105234457 
                 UUUUCAACACAUAACUGCAG 
                 198 
               
               
                   
               
               
                 54790_4_124 
                 TET2 
                 EXON 
                 + 
                 chr4: 105234438-105234458 
                 UUUCAACACAUAACUGCAGU 
                 199 
               
               
                   
               
               
                 54790_4_134 
                 TET2 
                 EXON 
                 + 
                 chr4: 105234475-105234495 
                 GCUUCAGAUUCUGAAUGAGC 
                 200 
               
               
                   
               
               
                 54790_4_138 
                 TET2 
                 EXON 
                 + 
                 chr4: 105234478-105234498 
                 UCAGAUUCUGAAUGAGCAGG 
                 201 
               
               
                   
               
               
                 54790_4_140 
                 TET2 
                 EXON 
                 + 
                 chr4: 105234479-105234499 
                 CAGAUUCUGAAUGAGCAGGA 
                 202 
               
               
                   
               
               
                 54790_4_141 
                 TET2 
                 EXON 
                 + 
                 chr4: 105234480-105234500 
                 AGAUUCUGAAUGAGCAGGAG 
                 203 
               
               
                   
               
               
                 54790_4_147 
                 TET2 
                 EXON 
                 + 
                 chr4: 105234529-105234549 
                 CAUUGUAUUACUUAAAAACA 
                 204 
               
               
                   
               
               
                 54790_4_151 
                 TET2 
                 EXON 
                 + 
                 chr4: 105234548-105234568 
                 AAGGCAGUGCUAAUGCCUAA 
                 205 
               
               
                   
               
               
                 54790_4_153 
                 TET2 
                 EXON 
                 + 
                 chr4: 105234574-105234594 
                 UACAGUUUCUGCCUCUUCCG 
                 206 
               
               
                   
               
               
                 54790_4_157 
                 TET2 
                 EXON 
                 + 
                 chr4: 105234587-105234607 
                 UCUUCCGUGGAACACACACA 
                 207 
               
               
                   
               
               
                 54790_4_161 
                 TET2 
                 EXON 
                 + 
                 chr4: 105234598-105234618 
                 ACACACACAUGGUGAACUCC 
                 208 
               
               
                   
               
               
                 54790_4_163 
                 TET2 
                 EXON 
                 + 
                 chr4: 105234643-105234663 
                 UCCAGAUUGUGUUUCCAUUG 
                 209 
               
               
                   
               
               
                 54790_4_171 
                 TET2 
                 EXON 
                 + 
                 chr4: 105234685-105234705 
                 CAUAAAUGCCAUUAACAGUC 
                 210 
               
               
                   
               
               
                 54790_4_177 
                 TET2 
                 EXON 
                 + 
                 chr4: 105234734-105234754 
                 ACUCACCCAUCGCAUACCUC 
                 211 
               
               
                   
               
               
                 54790_4_178 
                 TET2 
                 EXON 
                 + 
                 chr4: 105234735-105234755 
                 CUCACCCAUCGCAUACCUCA 
                 212 
               
               
                   
               
               
                 54790_4_181 
                 TET2 
                 EXON 
                 + 
                 chr4: 105234793-105234813 
                 GCCUCCAAAGCCAGCUGCAG 
                 213 
               
               
                   
               
               
                 54790_4_184 
                 TET2 
                 EXON 
                 + 
                 chr4: 105234802-105234822 
                 GCCAGCUGCAGUGGUGAGUG 
                 214 
               
               
                   
               
               
                 54790_4_200 
                 TET2 
                 EXON 
                 + 
                 chr4: 105234943-105234963 
                 UCCUGCAGAAAAUAACAUCC 
                 215 
               
               
                   
               
               
                 54790_4_201 
                 TET2 
                 EXON 
                 + 
                 chr4: 105234944-105234964 
                 CCUGCAGAAAAUAACAUCCA 
                 216 
               
               
                   
               
               
                 54790_4_203 
                 TET2 
                 EXON 
                 + 
                 chr4: 105234965-105234985 
                 GGAACCACAAAGCUAGCGUC 
                 217 
               
               
                   
               
               
                 54790_4_207 
                 TET2 
                 EXON 
                 + 
                 chr4: 105234983-105235003 
                 UCUGGUGAAGAAUUCUGUUC 
                 218 
               
               
                   
               
               
                 54790_4_211 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235010-105235030 
                 AGCAGCAAUUUGCAAGCUCC 
                 219 
               
               
                   
               
               
                 54790_4_212 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235013-105235033 
                 AGCAAUUUGCAAGCUCCUGG 
                 220 
               
               
                   
               
               
                 54790_4_216 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235026-105235046 
                 CUCCUGGUGGCAGCUCUGAA 
                 221 
               
               
                   
               
               
                 54790_4_219 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235052-105235072 
                 UUAAAACAAAAUGAAAUGAA 
                 222 
               
               
                   
               
               
                 54790_4_225 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235087-105235107 
                 GCAAAGCUCAGUGUUCACUA 
                 223 
               
               
                   
               
               
                 54790_4_235 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235162-105235182 
                 UCCCCCUCCUCCUCUUCCAC 
                 224 
               
               
                   
               
               
                 54790_4_240 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235184-105235204 
                 GUUCCUCAGCUUCCUUCAGA 
                 225 
               
               
                   
               
               
                 54790_4_245 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235202-105235222 
                 GAAGGAAAAAGCACUCUGAA 
                 226 
               
               
                   
               
               
                 54790_4_247 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235205-105235225 
                 GGAAAAAGCACUCUGAAUGG 
                 227 
               
               
                   
               
               
                 54790_4_256 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235260-105235280 
                 AAAGUAACACAACACUUUUA 
                 228 
               
               
                   
               
               
                 54790_4_258 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235261-105235281 
                 AAGUAACACAACACUUUUAA 
                 229 
               
               
                   
               
               
                 54790_4_262 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235276-105235296 
                 UUUAAGGGAAGUGAAAAUAG 
                 230 
               
               
                   
               
               
                 54790_4_263 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235277-105235297 
                 UUAAGGGAAGUGAAAAUAGA 
                 231 
               
               
                   
               
               
                 54790_4_268 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235288-105235308 
                 GAAAAUAGAGGGUAAACCUG 
                 232 
               
               
                   
               
               
                 54790_4_272 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235356-105235376 
                 CUUCUCCGAUGCUUUCUGAA 
                 233 
               
               
                   
               
               
                 54790_4_280 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235380-105235400 
                 CUCAGAAUAAUUGUGUGAAC 
                 234 
               
               
                   
               
               
                 54790_4_284 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235400-105235420 
                 AGGAAUGACAUACAGACUGC 
                 235 
               
               
                   
               
               
                 54790_4_286 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235401-105235421 
                 GGAAUGACAUACAGACUGCA 
                 236 
               
               
                   
               
               
                 54790_4_294 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235478-105235498 
                 AAGCAUAACCCACCAAUUUU 
                 237 
               
               
                   
               
               
                 54790_4_297 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235487-105235507 
                 CCACCAAUUUUUGGUAGCAG 
                 238 
               
               
                   
               
               
                 54790_4_302 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235498-105235518 
                 UGGUAGCAGUGGAGAGCUAC 
                 239 
               
               
                   
               
               
                 54790_4_313 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235546-105235566 
                 CAAAGAGCAAGAGAUUCUGA 
                 240 
               
               
                   
               
               
                 54790_4_314 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235547-105235567 
                 AAAGAGCAAGAGAUUCUGAA 
                 241 
               
               
                   
               
               
                 54790_4_317 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235558-105235578 
                 GAUUCUGAAGGGUCGAGACA 
                 242 
               
               
                   
               
               
                 54790_4_324 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235607-105235627 
                 ACACAGCACUAUCUGAAACC 
                 243 
               
               
                   
               
               
                 54790_4_326 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235611-105235631 
                 AGCACUAUCUGAAACCAGGA 
                 244 
               
               
                   
               
               
                 54790_4_329 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235624-105235644 
                 ACCAGGAUGGAUUGAAUUGA 
                 245 
               
               
                   
               
               
                 54790_4_333 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235645-105235665 
                 GGCCCCUCGUUUUCACCAAG 
                 246 
               
               
                   
               
               
                 54790_4_339 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235669-105235689 
                 AUCCCAUCUAAAACGUAAUG 
                 247 
               
               
                   
               
               
                 54790_4_343 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235739-105235759 
                 AUGACCUCCAAACAAUACAC 
                 248 
               
               
                   
               
               
                 54790_4_347 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235757-105235777 
                 ACUGGAAAUUCCAACAUGCC 
                 249 
               
               
                   
               
               
                 54790_4_349 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235758-105235778 
                 CUGGAAAUUCCAACAUGCCU 
                 250 
               
               
                   
               
               
                 54790_4_351 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235759-105235779 
                 UGGAAAUUCCAACAUGCCUG 
                 251 
               
               
                   
               
               
                 54790_4_352 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235760-105235780 
                 GGAAAUUCCAACAUGCCUGG 
                 252 
               
               
                   
               
               
                 54790_4_353 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235761-105235781 
                 GAAAUUCCAACAUGCCUGGG 
                 253 
               
               
                   
               
               
                 54790_4_355 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235770-105235790 
                 ACAUGCCUGGGGGGCUCCCA 
                 254 
               
               
                   
               
               
                 54790_4_360 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235801-105235821 
                 CACCCAGAAAACAACACAGC 
                 255 
               
               
                   
               
               
                 54790_4_365 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235841-105235861 
                 UACCAAGUUGAAAUGAAUCA 
                 256 
               
               
                   
               
               
                 54790_4_366 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235842-105235862 
                 ACCAAGUUGAAAUGAAUCAA 
                 257 
               
               
                   
               
               
                 54790_4_368 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235853-105235873 
                 AUGAAUCAAGGGCAGUCCCA 
                 258 
               
               
                   
               
               
                 54790_4_370 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235861-105235881 
                 AGGGCAGUCCCAAGGUACAG 
                 259 
               
               
                   
               
               
                 54790_4_371 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235897-105235917 
                 GUUCCAAAAACCCUCACACC 
                 260 
               
               
                   
               
               
                 54790_4_376 
                 TET2 
                 EXON 
                 + 
                 chr4: 105235952-105235972 
                 GCUCAUGUGCAGUCACUGUG 
                 261 
               
               
                   
               
               
                 54790_4_388 
                 TET2 
                 EXON 
                 + 
                 chr4: 105236038-105236058 
                 GAAACAGCACUUGAAUCAAC 
                 262 
               
               
                   
               
               
                 54790_4_399 
                 TET2 
                 EXON 
                 + 
                 chr4: 105236098-105236118 
                 GCAACAUAAGCCUCAUAAAC 
                 263 
               
               
                   
               
               
                 54790_4_407 
                 TET2 
                 EXON 
                 + 
                 chr4: 105236182-105236202 
                 AUUACAAAUAAAGAAUAAAG 
                 264 
               
               
                   
               
               
                 54790_4_416 
                 TET2 
                 EXON 
                 + 
                 chr4: 105236237-105236257 
                 AACAAUGAUCAGCAAAGAGA 
                 265 
               
               
                   
               
               
                 54790_4_417 
                 TET2 
                 EXON 
                 + 
                 chr4: 105236249-105236269 
                 CAAAGAGAAGGAUCAUUCUU 
                 266 
               
               
                   
               
               
                 54790_4_419 
                 TET2 
                 EXON 
                 + 
                 chr4: 105236263-105236283 
                 AUUCUUUGGCCAGACUAAAG 
                 267 
               
               
                   
               
               
                 54790_4_426 
                 TET2 
                 EXON 
                 + 
                 chr4: 105236279-105236299 
                 AAAGUGGAAGAAUGUUUUCA 
                 268 
               
               
                   
               
               
                 54790_4_435 
                 TET2 
                 EXON 
                 + 
                 chr4: 105236332-105236352 
                 CGAGACUCAUAAUGUCCAAA 
                 269 
               
               
                   
               
               
                 54790_4_438 
                 TET2 
                 EXON 
                 + 
                 chr4: 105236333-105236353 
                 GAGACUCAUAAUGUCCAAAU 
                 270 
               
               
                   
               
               
                 54790_4_440 
                 TET2 
                 EXON 
                 + 
                 chr4: 105236338-105236358 
                 UCAUAAUGUCCAAAUGGGAC 
                 271 
               
               
                   
               
               
                 54790_4_444 
                 TET2 
                 EXON 
                 + 
                 chr4: 105236341-105236361 
                 UAAUGUCCAAAUGGGACUGG 
                 272 
               
               
                   
               
               
                 54790_4_452 
                 TET2 
                 EXON 
                 + 
                 chr4: 105236413-105236433 
                 AUCAAGUGCAUGCAAAAUAC 
                 273 
               
               
                   
               
               
                 54790_4_466 
                 TET2 
                 EXON 
                 + 
                 chr4: 105236486-105236506 
                 ACACAUCCUGAACUUUUUGC 
                 274 
               
               
                   
               
               
                 54790_4_475 
                 TET2 
                 EXON 
                 + 
                 chr4: 105236562-105236582 
                 CAAAGCAAGAUCUUCUUCAC 
                 275 
               
               
                   
               
               
                 54790_4_479 
                 TET2 
                 EXON 
                 + 
                 chr4: 105236578-105236598 
                 UCACAGGUGCUUUCAAGAAC 
                 276 
               
               
                   
               
               
                 54790_4_486 
                 TET2 
                 EXON 
                 + 
                 chr4: 105236611-105236631 
                 ACAACAAGCUUCAGUUCUAC 
                 277 
               
               
                   
               
               
                 54790_4_488 
                 TET2 
                 EXON 
                 + 
                 chr4: 105236612-105236632 
                 CAACAAGCUUCAGUUCUACA 
                 278 
               
               
                   
               
               
                 54790_4_493 
                 TET2 
                 EXON 
                 + 
                 chr4: 105236642-105236662 
                 AAUAGAAACCAAGAUAUGUC 
                 279 
               
               
                   
               
               
                 54790_4_494 
                 TET2 
                 EXON 
                 + 
                 chr4: 105236673-105236693 
                 CUGCGCAACUUGCUCAGCAA 
                 280 
               
               
                   
               
               
                 54790_4_498 
                 TET2 
                 EXON 
                 + 
                 chr4: 105236719-105236739 
                 UGUUUUUCCUGUGCCUGACC 
                 281 
               
               
                   
               
               
                 54790_4_501 
                 TET2 
                 EXON 
                 + 
                 chr4: 105236720-105236740 
                 GUUUUUCCUGUGCCUGACCA 
                 282 
               
               
                   
               
               
                 54790_4_503 
                 TET2 
                 EXON 
                 + 
                 chr4: 105236723-105236743 
                 UUUCCUGUGCCUGACCAGGG 
                 283 
               
               
                   
               
               
                 54790_4_511 
                 TET2 
                 EXON 
                 + 
                 chr4: 105236752-105236772 
                 CACUCAGACCCCUCCCCAGA 
                 284 
               
               
                   
               
               
                 54790_4_512 
                 TET2 
                 EXON 
                 + 
                 chr4: 105236778-105236798 
                 CUCAAAAGCAUGCUGCUCUA 
                 285 
               
               
                   
               
               
                 54790_4_513 
                 TET2 
                 EXON 
                 + 
                 chr4: 105236781-105236801 
                 AAAAGCAUGCUGCUCUAAGG 
                 286 
               
               
                   
               
               
                 54790_4_518 
                 TET2 
                 EXON 
                 + 
                 chr4: 105236856-105236876 
                 CUUGCCAUAGUCAGAUGCAC 
                 287 
               
               
                   
               
               
                 54790_4_520 
                 TET2 
                 EXON 
                 + 
                 chr4: 105236866-105236886 
                 UCAGAUGCACAGGCCAAUUA 
                 288 
               
               
                   
               
               
                 54790_4_522 
                 TET2 
                 EXON 
                 + 
                 chr4: 105236869-105236889 
                 GAUGCACAGGCCAAUUAAGG 
                 289 
               
               
                   
               
               
                 54790_4_525 
                 TET2 
                 EXON 
                 + 
                 chr4: 105236876-105236896 
                 AGGCCAAUUAAGGUGGAACC 
                 290 
               
               
                   
               
               
                 54790_4_531 
                 TET2 
                 EXON 
                 + 
                 chr4: 105236928-105236948 
                 CACCACCAGAAAACAAAACA 
                 291 
               
               
                   
               
               
                 54790_4_532 
                 TET2 
                 EXON 
                 + 
                 chr4: 105236935-105236955 
                 AGAAAACAAAACAUGGAAAA 
                 292 
               
               
                   
               
               
                 54790_4_540 
                 TET2 
                 EXON 
                 + 
                 chr4: 105237004-105237024 
                 AAAGAGCAUCAUUGAGACCA 
                 293 
               
               
                   
               
               
                 54790_4_545 
                 TET2 
                 EXON 
                 + 
                 chr4: 105237052-105237072 
                 CAAGUCGUUAUUUGACCAUA 
                 294 
               
               
                   
               
               
                 54790_4_553 
                 TET2 
                 EXON 
                 + 
                 chr4: 105237098-105237118 
                 CAAGUAAAAGUUGAAAUGUC 
                 295 
               
               
                   
               
               
                 54790_4_554 
                 TET2 
                 EXON 
                 + 
                 chr4: 105237099-105237119 
                 AAGUAAAAGUUGAAAUGUCA 
                 296 
               
               
                   
               
               
                 54790_4_578 
                 TET2 
                 EXON 
                 + 
                 chr4: 105237280-105237300 
                 UACUCCUAUAAAAAAUUUAU 
                 297 
               
               
                   
               
               
                 54790_4_582 
                 TET2 
                 EXON 
                 + 
                 chr4: 105237329-105237349 
                 UUCCCAUCUUGCAGAUGUGU 
                 298 
               
               
                   
               
               
                 54790_4_589 
                 TET2 
                 EXON 
                 + 
                 chr4: 105237359-105237379 
                 CAGAAAUGUACUGAGACACA 
                 299 
               
               
                   
               
               
                 54790_4_596 
                 TET2 
                 EXON 
                 + 
                 chr4: 105237397-105237417 
                 AGCAAAUUUAUCUUCAGAUA 
                 300 
               
               
                   
               
               
                 54790_4_597 
                 TET2 
                 EXON 
                 + 
                 chr4: 105237398-105237418 
                 GCAAAUUUAUCUUCAGAUAU 
                 301 
               
               
                   
               
               
                 54790_4_606 
                 TET2 
                 EXON 
                 + 
                 chr4: 105237430-105237450 
                 CUUUUUUUAAAUCUUGAGUC 
                 302 
               
               
                   
               
               
                 54790_4_614 
                 TET2 
                 EXON 
                 + 
                 chr4: 105237446-105237466 
                 AGUCUGGCAGCAAUUUGUAA 
                 303 
               
               
                   
               
               
                 54790_4_657 
                 TET2 
                 EXON 
                 + 
                 chr4: 105237650-105237670 
                 GCUCUUUGUAUAUUAUCUCC 
                 304 
               
               
                   
               
               
                 54790_4_662 
                 TET2 
                 EXON 
                 + 
                 chr4: 105237663-105237683 
                 UAUCUCCUGGAGAGACAGCU 
                 305 
               
               
                   
               
               
                 54790_4_668 
                 TET2 
                 EXON 
                 + 
                 chr4: 105237708-105237728 
                 AAUGAGAAAAUAACGACCAU 
                 306 
               
               
                   
               
               
                 54790_4_670 
                 TET2 
                 EXON 
                 + 
                 chr4: 105237748-105237768 
                 UUUAAAUAUUUUUUAAUUCA 
                 307 
               
               
                   
               
               
                 54790_4_679 
                 TET2 
                 EXON 
                 + 
                 chr4: 105237778-105237798 
                 UAUUAGUUUCACAAGAUUUC 
                 308 
               
               
                   
               
               
                 54790_4_682 
                 TET2 
                 EXON 
                 + 
                 chr4: 105237786-105237806 
                 UCACAAGAUUUCUGGCUAAU 
                 309 
               
               
                   
               
               
                 54790_4_686 
                 TET2 
                 EXON 
                 + 
                 chr4: 105237787-105237807 
                 CACAAGAUUUCUGGCUAAUA 
                 310 
               
               
                   
               
               
                 54790_4_693 
                 TET2 
                 EXON 
                 + 
                 chr4: 105237817-105237837 
                 UAUCUUCAGUCUUCAUGAGU 
                 311 
               
               
                   
               
               
                 54790_4_695 
                 TET2 
                 EXON 
                 + 
                 chr4: 105237818-105237838 
                 AUCUUCAGUCUUCAUGAGUU 
                 312 
               
               
                   
               
               
                 54790_4_697 
                 TET2 
                 EXON 
                 + 
                 chr4: 105237819-105237839 
                 UCUUCAGUCUUCAUGAGUUG 
                 313 
               
               
                   
               
               
                 54790_4_700 
                 TET2 
                 EXON 
                 + 
                 chr4: 105237820-105237840 
                 CUUCAGUCUUCAUGAGUUGG 
                 314 
               
               
                   
               
               
                 54790_4_709 
                 TET2 
                 EXON 
                 + 
                 chr4: 105237882-105237902 
                 CUUUUCUCCAUUUAUACAUU 
                 315 
               
               
                   
               
               
                 54790_4_741 
                 TET2 
                 EXON 
                 + 
                 chr4: 105240332-105240352 
                 AAAGCUUUUUGUUAAAAUUC 
                 316 
               
               
                   
               
               
                 54790_4_746 
                 TET2 
                 EXON 
                 + 
                 chr4: 105240344-105240364 
                 UAAAAUUCAGGAUAUGUAAU 
                 317 
               
               
                   
               
               
                 54790_4_750 
                 TET2 
                 EXON 
                 + 
                 chr4: 105240352-105240372 
                 AGGAUAUGUAAUAGGUCUGU 
                 318 
               
               
                   
               
               
                 54790_4_754 
                 TET2 
                 EXON 
                 + 
                 chr4: 105240377-105240397 
                 UAGUGAAAUAUUUUUGCUGA 
                 319 
               
               
                   
               
               
                 54790_4_760 
                 TET2 
                 EXON 
                 + 
                 chr4: 105240395-105240415 
                 GAUGGAUGUAGAUAUAUACG 
                 320 
               
               
                   
               
               
                 54790_4_770 
                 TET2 
                 EXON 
                 + 
                 chr4: 105240478-105240498 
                 AGACAAAUGUUAAAUUAGUG 
                 321 
               
               
                   
               
               
                 54790_4_780 
                 TET2 
                 EXON 
                 + 
                 chr4: 105240541-105240561 
                 GAUACCCCACACUGUGUAGA 
                 322 
               
               
                   
               
               
                 54790_4_783 
                 TET2 
                 EXON 
                 + 
                 chr4: 105240545-105240565 
                 CCCCACACUGUGUAGAAGGA 
                 323 
               
               
                   
               
               
                 54790_4_785 
                 TET2 
                 EXON 
                 + 
                 chr4: 105240548-105240568 
                 CACACUGUGUAGAAGGAUGG 
                 324 
               
               
                   
               
               
                 54790_4_787 
                 TET2 
                 EXON 
                 + 
                 chr4: 105240549-105240569 
                 ACACUGUGUAGAAGGAUGGA 
                 325 
               
               
                   
               
               
                 54790_4_790 
                 TET2 
                 EXON 
                 + 
                 chr4: 105240552-105240572 
                 CUGUGUAGAAGGAUGGAGGG 
                 326 
               
               
                   
               
               
                 54790_4_791 
                 TET2 
                 EXON 
                 + 
                 chr4: 105240579-105240599 
                 CUACUGUCCCUCUUUGCGUG 
                 327 
               
               
                   
               
               
                 54790_4_795 
                 TET2 
                 EXON 
                 + 
                 chr4: 105240599-105240619 
                 UGGUUAUUAAGUUGCCUCAC 
                 328 
               
               
                   
               
               
                 54790_4_796 
                 TET2 
                 EXON 
                 + 
                 chr4: 105240600-105240620 
                 GGUUAUUAAGUUGCCUCACU 
                 329 
               
               
                   
               
               
                 54790_4_800 
                 TET2 
                 EXON 
                 + 
                 chr4: 105240634-105240654 
                 CACAUCUCAUAGAUAAUAUU 
                 330 
               
               
                   
               
               
                 54790_4_807 
                 TET2 
                 EXON 
                 + 
                 chr4: 105240703-105240723 
                 UCCCACUUUUCCAUCUUUGU 
                 331 
               
               
                   
               
               
                 54790_4_818 
                 TET2 
                 EXON 
                 + 
                 chr4: 105240740-105240760 
                 UUCUUUUUGCCUGACUCUCC 
                 332 
               
               
                   
               
               
                 54790_4_829 
                 TET2 
                 EXON 
                 + 
                 chr4: 105240784-105240804 
                 UUCUAAAGUACAUACUAAUA 
                 333 
               
               
                   
               
               
                 54790_4_830 
                 TET2 
                 EXON 
                 + 
                 chr4: 105240785-105240805 
                 UCUAAAGUACAUACUAAUAU 
                 334 
               
               
                   
               
               
                 54790_4_833 
                 TET2 
                 EXON 
                 + 
                 chr4: 105240790-105240810 
                 AGUACAUACUAAUAUGGGUC 
                 335 
               
               
                   
               
               
                 54790_4_841 
                 TET2 
                 EXON 
                 + 
                 chr4: 105240833-105240853 
                 AAACAGCAAUUAAAUGUUAU 
                 336 
               
               
                   
               
               
                 54790_4_842 
                 TET2 
                 EXON 
                 + 
                 chr4: 105240834-105240854 
                 AACAGCAAUUAAAUGUUAUA 
                 337 
               
               
                   
               
               
                 54790_4_845 
                 TET2 
                 EXON 
                 + 
                 chr4: 105240841-105240861 
                 AUUAAAUGUUAUAGGGAAGU 
                 338 
               
               
                   
               
               
                 54790_4_851 
                 TET2 
                 EXON 
                 + 
                 chr4: 105240851-105240871 
                 AUAGGGAAGUAGGAAGAAAA 
                 339 
               
               
                   
               
               
                 54790_4_853 
                 TET2 
                 EXON 
                 + 
                 chr4: 105240852-105240872 
                 UAGGGAAGUAGGAAGAAAAA 
                 340 
               
               
                   
               
               
                 54790_4_855 
                 TET2 
                 EXON 
                 + 
                 chr4: 105240853-105240873 
                 AGGGAAGUAGGAAGAAAAAG 
                 341 
               
               
                   
               
               
                 54790_4_858 
                 TET2 
                 EXON 
                 + 
                 chr4: 105240885-105240905 
                 CAAUAAACCAAGCAAUAUUC 
                 342 
               
               
                   
               
               
                 54790_4_861 
                 TET2 
                 EXON 
                 + 
                 chr4: 105240886-105240906 
                 AAUAAACCAAGCAAUAUUCU 
                 343 
               
               
                   
               
               
                 54790_4_862 
                 TET2 
                 EXON 
                 + 
                 chr4: 105240887-105240907 
                 AUAAACCAAGCAAUAUUCUG 
                 344 
               
               
                   
               
               
                 54790_4_863 
                 TET2 
                 EXON 
                 + 
                 chr4: 105240888-105240908 
                 UAAACCAAGCAAUAUUCUGG 
                 345 
               
               
                   
               
               
                 54790_4_865 
                 TET2 
                 EXON 
                 + 
                 chr4: 105240891-105240911 
                 ACCAAGCAAUAUUCUGGGGG 
                 346 
               
               
                   
               
               
                 54790_4_867 
                 TET2 
                 EXON 
                 + 
                 chr4: 105240892-105240912 
                 CCAAGCAAUAUUCUGGGGGU 
                 347 
               
               
                   
               
               
                 54790_4_870 
                 TET2 
                 EXON 
                 + 
                 chr4: 105240902-105240922 
                 UUCUGGGGGUGGGAUAGAGC 
                 348 
               
               
                   
               
               
                 54790_4_880 
                 TET2 
                 EXON 
                 + 
                 chr4: 105240940-105240960 
                 UCUUUUAAAAUCCAAGUAAU 
                 349 
               
               
                   
               
               
                 54790_4_881 
                 TET2 
                 EXON 
                 + 
                 chr4: 105240944-105240964 
                 UUAAAAUCCAAGUAAUAGGU 
                 350 
               
               
                   
               
               
                 54790_4_891 
                 TET2 
                 EXON 
                 + 
                 chr4: 105240991-105241011 
                 UUUUUUCCAGCUCAAAAAAU 
                 351 
               
               
                   
               
               
                 54790_4_905 
                 TET2 
                 EXON 
                 + 
                 chr4: 105241063-105241083 
                 UUUGUUUAGUUUCAUUUAUU 
                 352 
               
               
                   
               
               
                 54790_4_929 
                 TET2 
                 EXON 
                 + 
                 chr4: 105241146-105241166 
                 UGUACAUAUACUUAAUUAUG 
                 353 
               
               
                   
               
               
                 54790_4_945 
                 TET2 
                 EXON 
                 + 
                 chr4: 105241237-105241257 
                 UAGAGCCCUUAAUGUGUAGU 
                 354 
               
               
                   
               
               
                 54790_4_949 
                 TET2 
                 EXON 
                 + 
                 chr4: 105241238-105241258 
                 AGAGCCCUUAAUGUGUAGUU 
                 355 
               
               
                   
               
               
                 54790_4_951 
                 TET2 
                 EXON 
                 + 
                 chr4: 105241239-105241259 
                 GAGCCCUUAAUGUGUAGUUG 
                 356 
               
               
                   
               
               
                 54790_4_953 
                 TET2 
                 EXON 
                 + 
                 chr4: 105241240-105241260 
                 AGCCCUUAAUGUGUAGUUGG 
                 357 
               
               
                   
               
               
                 54790_4_956 
                 TET2 
                 EXON 
                 + 
                 chr4: 105241253-105241273 
                 UAGUUGGGGGUUAAGCUUUG 
                 358 
               
               
                   
               
               
                 54790_4_962 
                 TET2 
                 EXON 
                 + 
                 chr4: 105241283-105241303 
                 CUUUAUAUUUAGUAUAAUUG 
                 359 
               
               
                   
               
               
                 54790_4_973 
                 TET2 
                 EXON 
                 + 
                 chr4: 105241340-105241360 
                 CAAAUUAUUGAAAAAGAUGA 
                 360 
               
               
                   
               
               
                 54790_4_977 
                 TET2 
                 EXON 
                 + 
                 chr4: 105241361-105241381 
                 GGUCCUUUUUAUACCCAUCU 
                 361 
               
               
                   
               
               
                 54790_4_979 
                 TET2 
                 EXON 
                 + 
                 chr4: 105241367-105241387 
                 UUUUAUACCCAUCUAGGAGC 
                 362 
               
               
                   
               
               
                 54790_4_984 
                 TET2 
                 EXON 
                 + 
                 chr4: 105241378-105241398 
                 UCUAGGAGCAGGUCCUAAUG 
                 363 
               
               
                   
               
               
                 54790_4_990 
                 TET2 
                 EXON 
                 + 
                 chr4: 105241399-105241419 
                 GGCAGCUAUUAGAGAAAUCA 
                 364 
               
               
                   
               
               
                 54790_4_993 
                 TET2 
                 EXON 
                 + 
                 chr4: 105241407-105241427 
                 UUAGAGAAAUCAUGGAAGAA 
                 365 
               
               
                   
               
               
                 54790_4_995 
                 TET2 
                 EXON 
                 + 
                 chr4: 105241422-105241442 
                 AAGAAAGGUAAUUAACGCAA 
                 366 
               
               
                   
               
               
                 54790_4_997 
                 TET2 
                 EXON 
                 + 
                 chr4: 105241428-105241448 
                 GGUAAUUAACGCAAAGGCAC 
                 367 
               
               
                   
               
               
                 54790_4_998 
                 TET2 
                 EXON 
                 + 
                 chr4: 105241429-105241449 
                 GUAAUUAACGCAAAGGCACA 
                 368 
               
               
                   
               
               
                 54790_4_1014 
                 TET2 
                 EXON 
                 + 
                 chr4: 105241523-105241543 
                 UAAAUUGAGUAAUUAUUAGU 
                 369 
               
               
                   
               
               
                 54790_4_1019 
                 TET2 
                 EXON 
                 + 
                 chr4: 105241538-105241558 
                 UUAGUAGGCUUAGCUAUUCU 
                 370 
               
               
                   
               
               
                 54790_4_1020 
                 TET2 
                 EXON 
                 + 
                 chr4: 105241539-105241559 
                 UAGUAGGCUUAGCUAUUCUA 
                 371 
               
               
                   
               
               
                 54790_4_1029 
                 TET2 
                 EXON 
                 + 
                 chr4: 105241592-105241612 
                 AGAGAGUCACAAUAUUUGAC 
                 372 
               
               
                   
               
               
                 54790_4_1032 
                 TET2 
                 EXON 
                 + 
                 chr4: 105241612-105241632 
                 AGGACUAAUAGUCUGCUAGC 
                 373 
               
               
                   
               
               
                 54790_4_1033 
                 TET2 
                 EXON 
                 + 
                 chr4: 105241618-105241638 
                 AAUAGUCUGCUAGCUGGCAC 
                 374 
               
               
                   
               
               
                 54790_4_1035 
                 TET2 
                 EXON 
                 + 
                 chr4: 105241636-105241656 
                 ACAGGCUGCCCACUUUGCGA 
                 375 
               
               
                   
               
               
                 54790_4_1040 
                 TET2 
                 EXON 
                 + 
                 chr4: 105241653-105241673 
                 CGAUGGAUGCCAGAAAACCC 
                 376 
               
               
                   
               
               
                 54790_4_1043 
                 TET2 
                 EXON 
                 + 
                 chr4: 105241663-105241683 
                 CAGAAAACCCAGGCAUGAAC 
                 377 
               
               
                   
               
               
                 54790_4_1045 
                 TET2 
                 EXON 
                 + 
                 chr4: 105241669-105241689 
                 ACCCAGGCAUGAACAGGAAU 
                 378 
               
               
                   
               
               
                 54790_4_1046 
                 TET2 
                 EXON 
                 + 
                 chr4: 105241678-105241698 
                 UGAACAGGAAUCGGCCAGCC 
                 379 
               
               
                   
               
               
                 54790_4_1047 
                 TET2 
                 EXON 
                 + 
                 chr4: 105241693-105241713 
                 CAGCCAGGCUGCCAGCCACA 
                 380 
               
               
                   
               
               
                 54790_4_1048 
                 TET2 
                 EXON 
                 + 
                 chr4: 105241699-105241719 
                 GGCUGCCAGCCACAAGGUAC 
                 381 
               
               
                   
               
               
                 54790_4_1049 
                 TET2 
                 EXON 
                 + 
                 chr4: 105241705-105241725 
                 CAGCCACAAGGUACUGGCAC 
                 382 
               
               
                   
               
               
                 54790_4_1052 
                 TET2 
                 EXON 
                 + 
                 chr4: 105241718-105241738 
                 CUGGCACAGGCUCCAACGAG 
                 383 
               
               
                   
               
               
                 54790_4_1053 
                 TET2 
                 EXON 
                 + 
                 chr4: 105241729-105241749 
                 UCCAACGAGAGGUCCCACUC 
                 384 
               
               
                   
               
               
                 54790_4_1058 
                 TET2 
                 EXON 
                 + 
                 chr4: 105241770-105241790 
                 AAGUGUCAAAGCAGAAAGAC 
                 385 
               
               
                   
               
               
                 54790_4_1059 
                 TET2 
                 EXON 
                 + 
                 chr4: 105241780-105241800 
                 GCAGAAAGACUGGUAAAGUG 
                 386 
               
               
                   
               
               
                 54790_4_1092 
                 TET2 
                 EXON 
                 + 
                 chr4: 105241946-105241966 
                 UUUUUUUCGCUAUCAAUCAC 
                 387 
               
               
                   
               
               
                 54790_4_1109 
                 TET2 
                 EXON 
                 + 
                 chr4: 105242012-105242032 
                 UGAGCGAGAUAAUGCAGAGA 
                 388 
               
               
                   
               
               
                 54790_4_1117 
                 TET2 
                 EXON 
                 + 
                 chr4: 105242057-105242077 
                 CUCUGAGCUGUUCUUCUUCU 
                 389 
               
               
                   
               
               
                 54790_4_1118 
                 TET2 
                 EXON 
                 + 
                 chr4: 105242058-105242078 
                 UCUGAGCUGUUCUUCUUCUA 
                 390 
               
               
                   
               
               
                 54790_4_1123 
                 TET2 
                 EXON 
                 + 
                 chr4: 105242076-105242096 
                 UAGGGUGCCUUUUCAUUAAG 
                 391 
               
               
                   
               
               
                 54790_4_1124 
                 TET2 
                 EXON 
                 + 
                 chr4: 105242080-105242100 
                 GUGCCUUUUCAUUAAGAGGU 
                 392 
               
               
                   
               
               
                 54790_4_1130 
                 TET2 
                 EXON 
                 + 
                 chr4: 105242105-105242125 
                 GUAUUAUUAUUAAAGUACUU 
                 393 
               
               
                   
               
               
                 54790_4_1135 
                 TET2 
                 EXON 
                 + 
                 chr4: 105242114-105242134 
                 UUAAAGUACUUAGGAUACAU 
                 394 
               
               
                   
               
               
                 54790_4_1136 
                 TET2 
                 EXON 
                 + 
                 chr4: 105242115-105242135 
                 UAAAGUACUUAGGAUACAUU 
                 395 
               
               
                   
               
               
                 54790_4_1137 
                 TET2 
                 EXON 
                 + 
                 chr4: 105242116-105242136 
                 AAAGUACUUAGGAUACAUUG 
                 396 
               
               
                   
               
               
                 54790_4_1140 
                 TET2 
                 EXON 
                 + 
                 chr4: 105242124-105242144 
                 UAGGAUACAUUGGGGCAGCU 
                 397 
               
               
                   
               
               
                 54790_4_1154 
                 TET2 
                 EXON 
                 + 
                 chr4: 105242210-105242230 
                 UUCACUAAAUAAUCAUCUAG 
                 398 
               
               
                   
               
               
                 54790_4_1156 
                 TET2 
                 EXON 
                 + 
                 chr4: 105242215-105242235 
                 UAAAUAAUCAUCUAGUGGCC 
                 399 
               
               
                   
               
               
                 54790_4_1162 
                 TET2 
                 EXON 
                 + 
                 chr4: 105242287-105242307 
                 UUGUUUUUUAAACAAGCAGU 
                 400 
               
               
                   
               
               
                 54790_4_1163 
                 TET2 
                 EXON 
                 + 
                 chr4: 105242290-105242310 
                 UUUUUUAAACAAGCAGUAGG 
                 401 
               
               
                   
               
               
                 54790_4_1164 
                 TET2 
                 EXON 
                 + 
                 chr4: 105242298-105242318 
                 ACAAGCAGUAGGUGGUGCUU 
                 402 
               
               
                   
               
               
                 54790_4_1167 
                 TET2 
                 EXON 
                 + 
                 chr4: 105242306-105242326 
                 UAGGUGGUGCUUUGGUCAUA 
                 403 
               
               
                   
               
               
                 54790_4_1169 
                 TET2 
                 EXON 
                 + 
                 chr4: 105242307-105242327 
                 AGGUGGUGCUUUGGUCAUAA 
                 404 
               
               
                   
               
               
                 54790_4_1173 
                 TET2 
                 EXON 
                 + 
                 chr4: 105242328-105242348 
                 GGAAGAUAUAGUCUAUUUCU 
                 405 
               
               
                   
               
               
                 54790_4_1176 
                 TET2 
                 EXON 
                 + 
                 chr4: 105242351-105242371 
                 ACUAUUCCAUAUUUUCCAUG 
                 406 
               
               
                   
               
               
                 54790_4_1178 
                 TET2 
                 EXON 
                 + 
                 chr4: 105242355-105242375 
                 UUCCAUAUUUUCCAUGUGGC 
                 407 
               
               
                   
               
               
                 54790_4_1187 
                 TET2 
                 EXON 
                 + 
                 chr4: 105242404-105242424 
                 UCUAAAUUGUGAGACAUUCU 
                 408 
               
               
                   
               
               
                 54790_4_1193 
                 TET2 
                 EXON 
                 + 
                 chr4: 105242407-105242427 
                 AAAUUGUGAGACAUUCUUGG 
                 409 
               
               
                   
               
               
                 54790_4_1201 
                 TET2 
                 EXON 
                 + 
                 chr4: 105242469-105242489 
                 UAAAAUAGCUAAAUUUAGUA 
                 410 
               
               
                   
               
               
                 54790_4_1205 
                 TET2 
                 EXON 
                 + 
                 chr4: 105242470-105242490 
                 AAAAUAGCUAAAUUUAGUAA 
                 411 
               
               
                   
               
               
                 54790_4_1241 
                 TET2 
                 EXON 
                 + 
                 chr4: 105242625-105242645 
                 AUCUGUACAUUUUGAUAUUG 
                 412 
               
               
                   
               
               
                 54790_4_1244 
                 TET2 
                 EXON 
                 + 
                 chr4: 105242635-105242655 
                 UUUGAUAUUGAGGAAAAACA 
                 413 
               
               
                   
               
               
                 54790_4_1250 
                 TET2 
                 EXON 
                 + 
                 chr4: 105242663-105242683 
                 AAACCAUUAUCCAGUUUGCU 
                 414 
               
               
                   
               
               
                 54790_4_1258 
                 TET2 
                 EXON 
                 + 
                 chr4: 105242705-105242725 
                 UAAUAAACCGUUCAUUUCUC 
                 415 
               
               
                   
               
               
                 54790_4_1259 
                 TET2 
                 EXON 
                 + 
                 chr4: 105242711-105242731 
                 ACCGUUCAUUUCUCAGGAUG 
                 416 
               
               
                   
               
               
                 54790_4_1269 
                 TET2 
                 EXON 
                 − 
                 chr4: 105233886-105233906 
                 UAGUUGAAUUCUAAAGAGCA 
                 417 
               
               
                   
               
               
                 54790_4_1276 
                 TET2 
                 EXON 
                 − 
                 chr4: 105233917-105233937 
                 UUGCUUCGGGGCCAUCCACA 
                 418 
               
               
                   
               
               
                 54790_4_1278 
                 TET2 
                 EXON 
                 − 
                 chr4: 105233929-105233949 
                 GUUCCAUCAGGCUUGCUUCG 
                 419 
               
               
                   
               
               
                 54790_4_1279 
                 TET2 
                 EXON 
                 − 
                 chr4: 105233930-105233950 
                 UGUUCCAUCAGGCUUGCUUC 
                 420 
               
               
                   
               
               
                 54790_4_1281 
                 TET2 
                 EXON 
                 − 
                 chr4: 105233931-105233951 
                 CUGUUCCAUCAGGCUUGCUU 
                 421 
               
               
                   
               
               
                 54790_4_1285 
                 TET2 
                 EXON 
                 − 
                 chr4: 105233941-105233961 
                 UGGUUCUAUCCUGUUCCAUC 
                 422 
               
               
                   
               
               
                 54790_4_1288 
                 TET2 
                 EXON 
                 − 
                 chr4: 105233961-105233981 
                 UCUGUUGCCCUCAACAUGGU 
                 423 
               
               
                   
               
               
                 54790_4_1289 
                 TET2 
                 EXON 
                 − 
                 chr4: 105233965-105233985 
                 UUAGUCUGUUGCCCUCAACA 
                 424 
               
               
                   
               
               
                 54790_4_1290 
                 TET2 
                 EXON 
                 − 
                 chr4: 105233990-105234010 
                 GGAGGUGAUGGUAUCAGGAA 
                 425 
               
               
                   
               
               
                 54790_4_1293 
                 TET2 
                 EXON 
                 − 
                 chr4: 105233995-105234015 
                 AAAUGGGAGGUGAUGGUAUC 
                 426 
               
               
                   
               
               
                 54790_4_1296 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234002-105234022 
                 GUCUGGCAAAUGGGAGGUGA 
                 427 
               
               
                   
               
               
                 54790_4_1297 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234008-105234028 
                 GGUUCUGUCUGGCAAAUGGG 
                 428 
               
               
                   
               
               
                 54790_4_1298 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234011-105234031 
                 AGAGGUUCUGUCUGGCAAAU 
                 429 
               
               
                   
               
               
                 54790_4_1300 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234012-105234032 
                 CAGAGGUUCUGUCUGGCAAA 
                 430 
               
               
                   
               
               
                 54790_4_1305 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234019-105234039 
                 UUGUAGCCAGAGGUUCUGUC 
                 431 
               
               
                   
               
               
                 54790_4_1308 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234029-105234049 
                 UUCUGGAGCUUUGUAGCCAG 
                 432 
               
               
                   
               
               
                 54790_4_1310 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234046-105234066 
                 CAGGCAGUGGGCUUCCAUUC 
                 433 
               
               
                   
               
               
                 54790_4_1314 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234058-105234078 
                 GAUGAGCUCUCUCAGGCAGU 
                 434 
               
               
                   
               
               
                 54790_4_1315 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234059-105234079 
                 GGAUGAGCUCUCUCAGGCAG 
                 435 
               
               
                   
               
               
                 54790_4_1319 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234065-105234085 
                 ACUUCUGGAUGAGCUCUCUC 
                 436 
               
               
                   
               
               
                 54790_4_1322 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234080-105234100 
                 UUGGUGUCUCCAUUUACUUC 
                 437 
               
               
                   
               
               
                 54790_4_1327 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234099-105234119 
                 ACUUUUGAAAGAGUGCCACU 
                 438 
               
               
                   
               
               
                 54790_4_1334 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234134-105234154 
                 UUCUGGCUUCCCUUCAUACA 
                 439 
               
               
                   
               
               
                 54790_4_1335 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234135-105234155 
                 AUUCUGGCUUCCCUUCAUAC 
                 440 
               
               
                   
               
               
                 54790_4_1337 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234151-105234171 
                 CAGGACUCACACGACUAUUC 
                 441 
               
               
                   
               
               
                 54790_4_1341 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234170-105234190 
                 CUACUUUCUUGUGUAAAGUC 
                 442 
               
               
                   
               
               
                 54790_4_1351 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234201-105234221 
                 UCCUCCAUUUUGCAAACACU 
                 443 
               
               
                   
               
               
                 54790_4_1355 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234245-105234265 
                 UGAAGGAGCCCAGAGAGAGA 
                 444 
               
               
                   
               
               
                 54790_4_1367 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234262-105234282 
                 GUUUCAAUUUCUUGAUCUGA 
                 445 
               
               
                   
               
               
                 54790_4_1378 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234289-105234309 
                 GUCUUUCUCCAUUAGCCUUU 
                 446 
               
               
                   
               
               
                 54790_4_1388 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234328-105234348 
                 UUUCACCUGGAUUUCUUUCU 
                 447 
               
               
                   
               
               
                 54790_4_1392 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234341-105234361 
                 UUUGGUUGACUGCUUUCACC 
                 448 
               
               
                   
               
               
                 54790_4_1396 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234359-105234379 
                 UCACUCAAAUCGGAGACAUU 
                 449 
               
               
                   
               
               
                 54790_4_1399 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234369-105234389 
                 UUCUUUCUUAUCACUCAAAU 
                 450 
               
               
                   
               
               
                 54790_4_1410 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234408-105234428 
                 AUCUUUAACUGCAUUUUCUU 
                 451 
               
               
                   
               
               
                 54790_4_1411 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234409-105234429 
                 AAUCUUUAACUGCAUUUUCU 
                 452 
               
               
                   
               
               
                 54790_4_1416 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234435-105234455 
                 GCAGUUAUGUGUUGAAAAAC 
                 453 
               
               
                   
               
               
                 54790_4_1422 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234464-105234484 
                 AUCUGAAGCUCUGGAUUUUC 
                 454 
               
               
                   
               
               
                 54790_4_1423 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234473-105234493 
                 UCAUUCAGAAUCUGAAGCUC 
                 455 
               
               
                   
               
               
                 54790_4_1435 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234520-105234540 
                 GUAAUACAAUGUUCUUGUCA 
                 456 
               
               
                   
               
               
                 54790_4_1441 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234566-105234586 
                 GCAGAAACUGUAGCACCAUU 
                 457 
               
               
                   
               
               
                 54790_4_1444 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234588-105234608 
                 AUGUGUGUGUUCCACGGAAG 
                 458 
               
               
                   
               
               
                 54790_4_1448 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234594-105234614 
                 UUCACCAUGUGUGUGUUCCA 
                 459 
               
               
                   
               
               
                 54790_4_1457 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234619-105234639 
                 AUUGAGACAGUGUUUUUUCC 
                 460 
               
               
                   
               
               
                 54790_4_1461 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234647-105234667 
                 ACCGCAAUGGAAACACAAUC 
                 461 
               
               
                   
               
               
                 54790_4_1466 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234660-105234680 
                 UGUGGUUUUCUGCACCGCAA 
                 462 
               
               
                   
               
               
                 54790_4_1471 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234678-105234698 
                 AAUGGCAUUUAUGUGAGAUG 
                 463 
               
               
                   
               
               
                 54790_4_1474 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234696-105234716 
                 AUUAGUAGCCUGACUGUUAA 
                 464 
               
               
                   
               
               
                 54790_4_1475 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234726-105234746 
                 CGAUGGGUGAGUGAUCUCAC 
                 465 
               
               
                   
               
               
                 54790_4_1479 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234742-105234762 
                 UCUGCCCUGAGGUAUGCGAU 
                 466 
               
               
                   
               
               
                 54790_4_1480 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234743-105234763 
                 AUCUGCCCUGAGGUAUGCGA 
                 467 
               
               
                   
               
               
                 54790_4_1482 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234753-105234773 
                 UGCGGAAUUGAUCUGCCCUG 
                 468 
               
               
                   
               
               
                 54790_4_1485 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234771-105234791 
                 CUCAGAGUUAGAGGUCUGUG 
                 469 
               
               
                   
               
               
                 54790_4_1490 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234780-105234800 
                 UGGAGGCAGCUCAGAGUUAG 
                 470 
               
               
                   
               
               
                 54790_4_1493 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234797-105234817 
                 ACCACUGCAGCUGGCUUUGG 
                 471 
               
               
                   
               
               
                 54790_4_1495 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234800-105234820 
                 CUCACCACUGCAGCUGGCUU 
                 472 
               
               
                   
               
               
                 54790_4_1497 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234806-105234826 
                 GCCUCACUCACCACUGCAGC 
                 473 
               
               
                   
               
               
                 54790_4_1499 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234828-105234848 
                 AUCAGCAUCAUCAGCAUCAC 
                 474 
               
               
                   
               
               
                 54790_4_1505 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234855-105234875 
                 UAGCAUUGCAGCUAGUUUAC 
                 475 
               
               
                   
               
               
                 54790_4_1510 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234882-105234902 
                 UUCUGGUUUCUGAAAGGAAC 
                 476 
               
               
                   
               
               
                 54790_4_1514 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234888-105234908 
                 UAGUUGUUCUGGUUUCUGAA 
                 477 
               
               
                   
               
               
                 54790_4_1521 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234899-105234919 
                 UUUUGUUGUUGUAGUUGUUC 
                 478 
               
               
                   
               
               
                 54790_4_1526 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234940-105234960 
                 UGUUAUUUUCUGCAGGAGAU 
                 479 
               
               
                   
               
               
                 54790_4_1527 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234941-105234961 
                 AUGUUAUUUUCUGCAGGAGA 
                 480 
               
               
                   
               
               
                 54790_4_1531 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234947-105234967 
                 CCCUGGAUGUUAUUUUCUGC 
                 481 
               
               
                   
               
               
                 54790_4_1535 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234964-105234984 
                 ACGCUAGCUUUGUGGUUCCC 
                 482 
               
               
                   
               
               
                 54790_4_1539 
                 TET2 
                 EXON 
                 − 
                 chr4: 105234972-105234992 
                 UUCACCAGACGCUAGCUUUG 
                 483 
               
               
                   
               
               
                 54790_4_1545 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235011-105235031 
                 AGGAGCUUGCAAAUUGCUGC 
                 484 
               
               
                   
               
               
                 54790_4_1551 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235031-105235051 
                 UACCGUUCAGAGCUGCCACC 
                 485 
               
               
                   
               
               
                 54790_4_1569 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235116-105235136 
                 ACCACACCAUCACCCAGAAA 
                 486 
               
               
                   
               
               
                 54790_4_1577 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235166-105235186 
                 ACCUGUGGAAGAGGAGGAGG 
                 487 
               
               
                   
               
               
                 54790_4_1579 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235167-105235187 
                 AACCUGUGGAAGAGGAGGAG 
                 488 
               
               
                   
               
               
                 54790_4_1581 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235168-105235188 
                 GAACCUGUGGAAGAGGAGGA 
                 489 
               
               
                   
               
               
                 54790_4_1582 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235169-105235189 
                 GGAACCUGUGGAAGAGGAGG 
                 490 
               
               
                   
               
               
                 54790_4_1586 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235172-105235192 
                 UGAGGAACCUGUGGAAGAGG 
                 491 
               
               
                   
               
               
                 54790_4_1588 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235175-105235195 
                 AGCUGAGGAACCUGUGGAAG 
                 492 
               
               
                   
               
               
                 54790_4_1593 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235181-105235201 
                 GAAGGAAGCUGAGGAACCUG 
                 493 
               
               
                   
               
               
                 54790_4_1600 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235190-105235210 
                 UUUCCUUCUGAAGGAAGCUG 
                 494 
               
               
                   
               
               
                 54790_4_1606 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235199-105235219 
                 AGAGUGCUUUUUCCUUCUGA 
                 495 
               
               
                   
               
               
                 54790_4_1617 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235246-105235266 
                 UACUUUGGUUGGGGUAGUGG 
                 496 
               
               
                   
               
               
                 54790_4_1618 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235249-105235269 
                 UGUUACUUUGGUUGGGGUAG 
                 497 
               
               
                   
               
               
                 54790_4_1620 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235255-105235275 
                 GUGUUGUGUUACUUUGGUUG 
                 498 
               
               
                   
               
               
                 54790_4_1621 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235256-105235276 
                 AGUGUUGUGUUACUUUGGUU 
                 499 
               
               
                   
               
               
                 54790_4_1623 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235257-105235277 
                 AAGUGUUGUGUUACUUUGGU 
                 500 
               
               
                   
               
               
                 54790_4_1626 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235261-105235281 
                 UUAAAAGUGUUGUGUUACUU 
                 501 
               
               
                   
               
               
                 54790_4_1633 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235307-105235327 
                 CUCUGGGAAGGUGGUGCCUC 
                 502 
               
               
                   
               
               
                 54790_4_1634 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235316-105235336 
                 GGAUUAGGACUCUGGGAAGG 
                 503 
               
               
                   
               
               
                 54790_4_1635 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235319-105235339 
                 GAUGGAUUAGGACUCUGGGA 
                 504 
               
               
                   
               
               
                 54790_4_1636 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235323-105235343 
                 UGUAGAUGGAUUAGGACUCU 
                 505 
               
               
                   
               
               
                 54790_4_1638 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235324-105235344 
                 GUGUAGAUGGAUUAGGACUC 
                 506 
               
               
                   
               
               
                 54790_4_1641 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235331-105235351 
                 CAUACAUGUGUAGAUGGAUU 
                 507 
               
               
                   
               
               
                 54790_4_1643 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235337-105235357 
                 GGGCUGCAUACAUGUGUAGA 
                 508 
               
               
                   
               
               
                 54790_4_1647 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235357-105235377 
                 UUUCAGAAAGCAUCGGAGAA 
                 509 
               
               
                   
               
               
                 54790_4_1648 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235358-105235378 
                 CUUUCAGAAAGCAUCGGAGA 
                 510 
               
               
                   
               
               
                 54790_4_1653 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235364-105235384 
                 UGAGGCCUUUCAGAAAGCAU 
                 511 
               
               
                   
               
               
                 54790_4_1660 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235382-105235402 
                 CUGUUCACACAAUUAUUCUG 
                 512 
               
               
                   
               
               
                 54790_4_1668 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235439-105235459 
                 CUUGUUUUCUCAGAACACAA 
                 513 
               
               
                   
               
               
                 54790_4_1676 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235463-105235483 
                 UGCUUGAGGUGUUCUGACAU 
                 514 
               
               
                   
               
               
                 54790_4_1678 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235477-105235497 
                 AAAUUGGUGGGUUAUGCUUG 
                 515 
               
               
                   
               
               
                 54790_4_1680 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235489-105235509 
                 CACUGCUACCAAAAAUUGGU 
                 516 
               
               
                   
               
               
                 54790_4_1681 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235490-105235510 
                 CCACUGCUACCAAAAAUUGG 
                 517 
               
               
                   
               
               
                 54790_4_1683 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235493-105235513 
                 UCUCCACUGCUACCAAAAAU 
                 518 
               
               
                   
               
               
                 54790_4_1690 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235531-105235551 
                 CUUUGUUUCUCAUCAACUGC 
                 519 
               
               
                   
               
               
                 54790_4_1699 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235604-105235624 
                 UUCAGAUAGUGCUGUGUUGG 
                 520 
               
               
                   
               
               
                 54790_4_1700 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235605-105235625 
                 UUUCAGAUAGUGCUGUGUUG 
                 521 
               
               
                   
               
               
                 54790_4_1702 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235606-105235626 
                 GUUUCAGAUAGUGCUGUGUU 
                 522 
               
               
                   
               
               
                 54790_4_1703 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235607-105235627 
                 GGUUUCAGAUAGUGCUGUGU 
                 523 
               
               
                   
               
               
                 54790_4_1708 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235628-105235648 
                 GCCUUCAAUUCAAUCCAUCC 
                 524 
               
               
                   
               
               
                 54790_4_1711 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235650-105235670 
                 UUCCGCUUGGUGAAAACGAG 
                 525 
               
               
                   
               
               
                 54790_4_1712 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235651-105235671 
                 AUUCCGCUUGGUGAAAACGA 
                 526 
               
               
                   
               
               
                 54790_4_1713 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235652-105235672 
                 GAUUCCGCUUGGUGAAAACG 
                 527 
               
               
                   
               
               
                 54790_4_1722 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235663-105235683 
                 GUUUUAGAUGGGAUUCCGCU 
                 528 
               
               
                   
               
               
                 54790_4_1723 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235674-105235694 
                 UGCCUCAUUACGUUUUAGAU 
                 529 
               
               
                   
               
               
                 54790_4_1724 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235675-105235695 
                 AUGCCUCAUUACGUUUUAGA 
                 530 
               
               
                   
               
               
                 54790_4_1730 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235703-105235723 
                 GGUUGAUACUGAAGAAUUGA 
                 531 
               
               
                   
               
               
                 54790_4_1737 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235724-105235744 
                 GUCAUUUGAUUGGAGAGAUU 
                 532 
               
               
                   
               
               
                 54790_4_1738 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235725-105235745 
                 GGUCAUUUGAUUGGAGAGAU 
                 533 
               
               
                   
               
               
                 54790_4_1743 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235734-105235754 
                 UUGUUUGGAGGUCAUUUGAU 
                 534 
               
               
                   
               
               
                 54790_4_1749 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235746-105235766 
                 AUUUCCAGUGUAUUGUUUGG 
                 535 
               
               
                   
               
               
                 54790_4_1751 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235749-105235769 
                 GGAAUUUCCAGUGUAUUGUU 
                 536 
               
               
                   
               
               
                 54790_4_1756 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235770-105235790 
                 UGGGAGCCCCCCAGGCAUGU 
                 537 
               
               
                   
               
               
                 54790_4_1758 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235778-105235798 
                 GCUUGCCUUGGGAGCCCCCC 
                 538 
               
               
                   
               
               
                 54790_4_1763 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235789-105235809 
                 UCUGGGUGUAAGCUUGCCUU 
                 539 
               
               
                   
               
               
                 54790_4_1766 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235790-105235810 
                 UUCUGGGUGUAAGCUUGCCU 
                 540 
               
               
                   
               
               
                 54790_4_1769 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235806-105235826 
                 CUCCAGCUGUGUUGUUUUCU 
                 541 
               
               
                   
               
               
                 54790_4_1770 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235807-105235827 
                 GCUCCAGCUGUGUUGUUUUC 
                 542 
               
               
                   
               
               
                 54790_4_1779 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235846-105235866 
                 GCCCUUGAUUCAUUUCAACU 
                 543 
               
               
                   
               
               
                 54790_4_1782 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235872-105235892 
                 AUGUUGGUCCACUGUACCUU 
                 544 
               
               
                   
               
               
                 54790_4_1783 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235873-105235893 
                 GAUGUUGGUCCACUGUACCU 
                 545 
               
               
                   
               
               
                 54790_4_1790 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235888-105235908 
                 GUUUUUGGAACUGGAGAUGU 
                 546 
               
               
                   
               
               
                 54790_4_1791 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235897-105235917 
                 GGUGUGAGGGUUUUUGGAAC 
                 547 
               
               
                   
               
               
                 54790_4_1795 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235903-105235923 
                 GCACCUGGUGUGAGGGUUUU 
                 548 
               
               
                   
               
               
                 54790_4_1800 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235910-105235930 
                 GAGAAGUGCACCUGGUGUGA 
                 549 
               
               
                   
               
               
                 54790_4_1801 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235911-105235931 
                 GGAGAAGUGCACCUGGUGUG 
                 550 
               
               
                   
               
               
                 54790_4_1804 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235918-105235938 
                 CUGUUUUGGAGAAGUGCACC 
                 551 
               
               
                   
               
               
                 54790_4_1811 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235932-105235952 
                 UUUUGGUAAAUGGUCUGUUU 
                 552 
               
               
                   
               
               
                 54790_4_1813 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235942-105235962 
                 GCACAUGAGCUUUUGGUAAA 
                 553 
               
               
                   
               
               
                 54790_4_1814 
                 TET2 
                 EXON 
                 − 
                 chr4: 105235949-105235969 
                 AGUGACUGCACAUGAGCUUU 
                 554 
               
               
                   
               
               
                 54790_4_1828 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236010-105236030 
                 GGACAUAAGUUUUUCAGUUU 
                 555 
               
               
                   
               
               
                 54790_4_1829 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236011-105236031 
                 GGGACAUAAGUUUUUCAGUU 
                 556 
               
               
                   
               
               
                 54790_4_1836 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236031-105236051 
                 CAAGUGCUGUUUCAACACUG 
                 557 
               
               
                   
               
               
                 54790_4_1838 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236032-105236052 
                 UCAAGUGCUGUUUCAACACU 
                 558 
               
               
                   
               
               
                 54790_4_1839 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236033-105236053 
                 UUCAAGUGCUGUUUCAACAC 
                 559 
               
               
                   
               
               
                 54790_4_1846 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236078-105236098 
                 AAAAGGUGUGAGUUUGAAAA 
                 560 
               
               
                   
               
               
                 54790_4_1852 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236095-105236115 
                 UAUGAGGCUUAUGUUGCAAA 
                 561 
               
               
                   
               
               
                 54790_4_1856 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236111-105236131 
                 GUUUGUGCUGCCUGUUUAUG 
                 562 
               
               
                   
               
               
                 54790_4_1861 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236138-105236158 
                 GGGAGAUGUGAACUCUGGGA 
                 563 
               
               
                   
               
               
                 54790_4_1862 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236142-105236162 
                 UUGAGGGAGAUGUGAACUCU 
                 564 
               
               
                   
               
               
                 54790_4_1864 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236143-105236163 
                 UUUGAGGGAGAUGUGAACUC 
                 565 
               
               
                   
               
               
                 54790_4_1873 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236158-105236178 
                 GCUGCUGUUGCUGGUUUUGA 
                 566 
               
               
                   
               
               
                 54790_4_1875 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236159-105236179 
                 UGCUGCUGUUGCUGGUUUUG 
                 567 
               
               
                   
               
               
                 54790_4_1880 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236167-105236187 
                 GUAAUUUUUGCUGCUGUUGC 
                 568 
               
               
                   
               
               
                 54790_4_1892 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236215-105236235 
                 UUUGGGGGUGAGGAAAAGUC 
                 569 
               
               
                   
               
               
                 54790_4_1896 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236225-105236245 
                 UCAUUGUUGCUUUGGGGGUG 
                 570 
               
               
                   
               
               
                 54790_4_1901 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236230-105236250 
                 GCUGAUCAUUGUUGCUUUGG 
                 571 
               
               
                   
               
               
                 54790_4_1902 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236231-105236251 
                 UGCUGAUCAUUGUUGCUUUG 
                 572 
               
               
                   
               
               
                 54790_4_1904 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236232-105236252 
                 UUGCUGAUCAUUGUUGCUUU 
                 573 
               
               
                   
               
               
                 54790_4_1906 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236233-105236253 
                 UUUGCUGAUCAUUGUUGCUU 
                 574 
               
               
                   
               
               
                 54790_4_1914 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236275-105236295 
                 AACAUUCUUCCACUUUAGUC 
                 575 
               
               
                   
               
               
                 54790_4_1931 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236350-105236370 
                 GUACUUCCUCCAGUCCCAUU 
                 576 
               
               
                   
               
               
                 54790_4_1941 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236394-105236414 
                 UUUCAUGGUCUGACUAUAAG 
                 577 
               
               
                   
               
               
                 54790_4_1943 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236395-105236415 
                 AUUUCAUGGUCUGACUAUAA 
                 578 
               
               
                   
               
               
                 54790_4_1944 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236396-105236416 
                 GAUUUCAUGGUCUGACUAUA 
                 579 
               
               
                   
               
               
                 54790_4_1950 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236409-105236429 
                 UUUGCAUGCACUUGAUUUCA 
                 580 
               
               
                   
               
               
                 54790_4_1960 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236461-105236481 
                 GUUCUUUAUUCUCUGAAACU 
                 581 
               
               
                   
               
               
                 54790_4_1966 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236495-105236515 
                 UUGUUUCCUGCAAAAAGUUC 
                 582 
               
               
                   
               
               
                 54790_4_1972 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236520-105236540 
                 UUGCAUGUGAUGCAAGUUUU 
                 583 
               
               
                   
               
               
                 54790_4_1973 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236521-105236541 
                 AUUGCAUGUGAUGCAAGUUU 
                 584 
               
               
                   
               
               
                 54790_4_1982 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236549-105236569 
                 UGCUUUGGGAUCACAUUAUU 
                 585 
               
               
                   
               
               
                 54790_4_1984 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236563-105236583 
                 UGUGAAGAAGAUCUUGCUUU 
                 586 
               
               
                   
               
               
                 54790_4_1985 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236564-105236584 
                 CUGUGAAGAAGAUCUUGCUU 
                 587 
               
               
                   
               
               
                 54790_4_2009 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236653-105236673 
                 CUUGUUGACCAGACAUAUCU 
                 588 
               
               
                   
               
               
                 54790_4_2017 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236713-105236733 
                 GCACAGGAAAAACAUUUGCA 
                 589 
               
               
                   
               
               
                 54790_4_2019 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236729-105236749 
                 CUUCCUCCCUGGUCAGGCAC 
                 590 
               
               
                   
               
               
                 54790_4_2022 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236735-105236755 
                 GUGUGACUUCCUCCCUGGUC 
                 591 
               
               
                   
               
               
                 54790_4_2023 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236740-105236760 
                 UCUGAGUGUGACUUCCUCCC 
                 592 
               
               
                   
               
               
                 54790_4_2029 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236763-105236783 
                 UUGAGUGUCCUUCUGGGGAG 
                 593 
               
               
                   
               
               
                 54790_4_2030 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236764-105236784 
                 UUUGAGUGUCCUUCUGGGGA 
                 594 
               
               
                   
               
               
                 54790_4_2031 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236765-105236785 
                 UUUUGAGUGUCCUUCUGGGG 
                 595 
               
               
                   
               
               
                 54790_4_2034 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236768-105236788 
                 UGCUUUUGAGUGUCCUUCUG 
                 596 
               
               
                   
               
               
                 54790_4_2037 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236769-105236789 
                 AUGCUUUUGAGUGUCCUUCU 
                 597 
               
               
                   
               
               
                 54790_4_2039 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236770-105236790 
                 CAUGCUUUUGAGUGUCCUUC 
                 598 
               
               
                   
               
               
                 54790_4_2053 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236846-105236866 
                 CUAUGGCAAGACUCAGUUUG 
                 599 
               
               
                   
               
               
                 54790_4_2054 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236847-105236867 
                 ACUAUGGCAAGACUCAGUUU 
                 600 
               
               
                   
               
               
                 54790_4_2055 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236848-105236868 
                 GACUAUGGCAAGACUCAGUU 
                 601 
               
               
                   
               
               
                 54790_4_2060 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236863-105236883 
                 UUGGCCUGUGCAUCUGACUA 
                 602 
               
               
                   
               
               
                 54790_4_2063 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236882-105236902 
                 CAUCCAGGUUCCACCUUAAU 
                 603 
               
               
                   
               
               
                 54790_4_2064 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236897-105236917 
                 CAGGCAUGUGGCUUGCAUCC 
                 604 
               
               
                   
               
               
                 54790_4_2065 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236909-105236929 
                 GCUGUGUGCAUACAGGCAUG 
                 605 
               
               
                   
               
               
                 54790_4_2069 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236916-105236936 
                 UGGUGGUGCUGUGUGCAUAC 
                 606 
               
               
                   
               
               
                 54790_4_2077 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236933-105236953 
                 UUCCAUGUUUUGUUUUCUGG 
                 607 
               
               
                   
               
               
                 54790_4_2079 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236936-105236956 
                 UUUUUCCAUGUUUUGUUUUC 
                 608 
               
               
                   
               
               
                 54790_4_2085 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236978-105236998 
                 ACAUUAUCACAGCUUGCAGG 
                 609 
               
               
                   
               
               
                 54790_4_2089 
                 TET2 
                 EXON 
                 − 
                 chr4: 105236981-105237001 
                 UGCACAUUAUCACAGCUUGC 
                 610 
               
               
                   
               
               
                 54790_4_2092 
                 TET2 
                 EXON 
                 − 
                 chr4: 105237024-105237044 
                 CUGCUUCAGAUGCUGCUCCA 
                 611 
               
               
                   
               
               
                 54790_4_2096 
                 TET2 
                 EXON 
                 − 
                 chr4: 105237054-105237074 
                 CUUAUGGUCAAAUAACGACU 
                 612 
               
               
                   
               
               
                 54790_4_2099 
                 TET2 
                 EXON 
                 − 
                 chr4: 105237070-105237090 
                 AUUUGAGAGUAAGAGCCUUA 
                 613 
               
               
                   
               
               
                 54790_4_2112 
                 TET2 
                 EXON 
                 − 
                 chr4: 105237125-105237145 
                 UGUCUAGUCAAAACUGUGAC 
                 614 
               
               
                   
               
               
                 54790_4_2114 
                 TET2 
                 EXON 
                 − 
                 chr4: 105237150-105237170 
                 GCUAUCAAGUUCUGCAGCAG 
                 615 
               
               
                   
               
               
                 54790_4_2118 
                 TET2 
                 EXON 
                 − 
                 chr4: 105237172-105237192 
                 GCUGCUCUAAAGCUGGGGUG 
                 616 
               
               
                   
               
               
                 54790_4_2119 
                 TET2 
                 EXON 
                 − 
                 chr4: 105237177-105237197 
                 UGUUUGCUGCUCUAAAGCUG 
                 617 
               
               
                   
               
               
                 54790_4_2120 
                 TET2 
                 EXON 
                 − 
                 chr4: 105237178-105237198 
                 UUGUUUGCUGCUCUAAAGCU 
                 618 
               
               
                   
               
               
                 54790_4_2122 
                 TET2 
                 EXON 
                 − 
                 chr4: 105237179-105237199 
                 GUUGUUUGCUGCUCUAAAGC 
                 619 
               
               
                   
               
               
                 54790_4_2135 
                 TET2 
                 EXON 
                 − 
                 chr4: 105237218-105237238 
                 GAAGCAGCUGUUCUUUUGGU 
                 620 
               
               
                   
               
               
                 54790_4_2137 
                 TET2 
                 EXON 
                 − 
                 chr4: 105237222-105237242 
                 AACAGAAGCAGCUGUUCUUU 
                 621 
               
               
                   
               
               
                 54790_4_2148 
                 TET2 
                 EXON 
                 − 
                 chr4: 105237266-105237286 
                 GGAGUAUCUAGUAAUUUGGA 
                 622 
               
               
                   
               
               
                 54790_4_2153 
                 TET2 
                 EXON 
                 − 
                 chr4: 105237270-105237290 
                 UAUAGGAGUAUCUAGUAAUU 
                 623 
               
               
                   
               
               
                 54790_4_2156 
                 TET2 
                 EXON 
                 − 
                 chr4: 105237287-105237307 
                 GUAUCCAAUAAAUUUUUUAU 
                 624 
               
               
                   
               
               
                 54790_4_2160 
                 TET2 
                 EXON 
                 − 
                 chr4: 105237311-105237331 
                 AAAUCAUAUUGAGUCUUGAC 
                 625 
               
               
                   
               
               
                 54790_4_2163 
                 TET2 
                 EXON 
                 − 
                 chr4: 105237334-105237354 
                 UACCUACACAUCUGCAAGAU 
                 626 
               
               
                   
               
               
                 54790_4_2165 
                 TET2 
                 EXON 
                 − 
                 chr4: 105237335-105237355 
                 UUACCUACACAUCUGCAAGA 
                 627 
               
               
                   
               
               
                 54790_4_2170 
                 TET2 
                 EXON 
                 − 
                 chr4: 105237361-105237381 
                 CAUGUGUCUCAGUACAUUUC 
                 628 
               
               
                   
               
               
                 54790_4_2174 
                 TET2 
                 EXON 
                 − 
                 chr4: 105237392-105237412 
                 GAAGAUAAAUUUGCUAAUUC 
                 629 
               
               
                   
               
               
                 54790_4_2180 
                 TET2 
                 EXON 
                 − 
                 chr4: 105237429-105237449 
                 ACUCAAGAUUUAAAAAAAGA 
                 630 
               
               
                   
               
               
                 54790_4_2197 
                 TET2 
                 EXON 
                 − 
                 chr4: 105237510-105237530 
                 CUUUCACAAGACACAAGCAU 
                 631 
               
               
                   
               
               
                 54790_4_2206 
                 TET2 
                 EXON 
                 − 
                 chr4: 105237558-105237578 
                 GCACGAUUAUUUAAUUCUUU 
                 632 
               
               
                   
               
               
                 54790_4_2213 
                 TET2 
                 EXON 
                 − 
                 chr4: 105237593-105237613 
                 UUUUACAGGAUCUGAAGAGA 
                 633 
               
               
                   
               
               
                 54790_4_2215 
                 TET2 
                 EXON 
                 − 
                 chr4: 105237594-105237614 
                 AUUUUACAGGAUCUGAAGAG 
                 634 
               
               
                   
               
               
                 54790_4_2221 
                 TET2 
                 EXON 
                 − 
                 chr4: 105237607-105237627 
                 CAGAUACAUUCAAAUUUUAC 
                 635 
               
               
                   
               
               
                 54790_4_2225 
                 TET2 
                 EXON 
                 − 
                 chr4: 105237645-105237665 
                 UAAUAUACAAAGAGCUAAAU 
                 636 
               
               
                   
               
               
                 54790_4_2233 
                 TET2 
                 EXON 
                 − 
                 chr4: 105237671-105237691 
                 UGCUGCCUAGCUGUCUCUCC 
                 637 
               
               
                   
               
               
                 54790_4_2247 
                 TET2 
                 EXON 
                 − 
                 chr4: 105237727-105237747 
                 UUCGUACAUUAGACUGCCUA 
                 638 
               
               
                   
               
               
                 54790_4_2270 
                 TET2 
                 EXON 
                 − 
                 chr4: 105237874-105237894 
                 AAUGGAGAAAAGGAAACUUU 
                 639 
               
               
                   
               
               
                 54790_4_2274 
                 TET2 
                 EXON 
                 − 
                 chr4: 105237884-105237904 
                 CAAAUGUAUAAAUGGAGAAA 
                 640 
               
               
                   
               
               
                 54790_4_2277 
                 TET2 
                 EXON 
                 − 
                 chr4: 105237892-105237912 
                 CAACAUUCCAAAUGUAUAAA 
                 641 
               
               
                   
               
               
                 54790_4_2284 
                 TET2 
                 EXON 
                 − 
                 chr4: 105237936-105237956 
                 AGAUGAAAUUUUAGAGAAAA 
                 642 
               
               
                   
               
               
                 54790_4_2287 
                 TET2 
                 EXON 
                 − 
                 chr4: 105237937-105237957 
                 AAGAUGAAAUUUUAGAGAAA 
                 643 
               
               
                   
               
               
                 54790_4_2323 
                 TET2 
                 EXON 
                 − 
                 chr4: 105240511-105240531 
                 AGGGAAAACAUGGCACGGGU 
                 644 
               
               
                   
               
               
                 54790_4_2325 
                 TET2 
                 EXON 
                 − 
                 chr4: 105240515-105240535 
                 CAAGAGGGAAAACAUGGCAC 
                 645 
               
               
                   
               
               
                 54790_4_2326 
                 TET2 
                 EXON 
                 − 
                 chr4: 105240516-105240536 
                 GCAAGAGGGAAAACAUGGCA 
                 646 
               
               
                   
               
               
                 54790_4_2328 
                 TET2 
                 EXON 
                 − 
                 chr4: 105240521-105240541 
                 UCAUUGCAAGAGGGAAAACA 
                 647 
               
               
                   
               
               
                 54790_4_2330 
                 TET2 
                 EXON 
                 − 
                 chr4: 105240530-105240550 
                 UGGGGUAUCUCAUUGCAAGA 
                 648 
               
               
                   
               
               
                 54790_4_2331 
                 TET2 
                 EXON 
                 − 
                 chr4: 105240531-105240551 
                 GUGGGGUAUCUCAUUGCAAG 
                 649 
               
               
                   
               
               
                 54790_4_2336 
                 TET2 
                 EXON 
                 − 
                 chr4: 105240548-105240568 
                 CCAUCCUUCUACACAGUGUG 
                 650 
               
               
                   
               
               
                 54790_4_2337 
                 TET2 
                 EXON 
                 − 
                 chr4: 105240549-105240569 
                 UCCAUCCUUCUACACAGUGU 
                 651 
               
               
                   
               
               
                 54790_4_2338 
                 TET2 
                 EXON 
                 − 
                 chr4: 105240550-105240570 
                 CUCCAUCCUUCUACACAGUG 
                 652 
               
               
                   
               
               
                 54790_4_2342 
                 TET2 
                 EXON 
                 − 
                 chr4: 105240581-105240601 
                 CACACGCAAAGAGGGACAGU 
                 653 
               
               
                   
               
               
                 54790_4_2345 
                 TET2 
                 EXON 
                 − 
                 chr4: 105240589-105240609 
                 UUAAUAACCACACGCAAAGA 
                 654 
               
               
                   
               
               
                 54790_4_2347 
                 TET2 
                 EXON 
                 − 
                 chr4: 105240590-105240610 
                 CUUAAUAACCACACGCAAAG 
                 655 
               
               
                   
               
               
                 54790_4_2353 
                 TET2 
                 EXON 
                 − 
                 chr4: 105240616-105240636 
                 UGUGGUGUUUUAGCCCAGUG 
                 656 
               
               
                   
               
               
                 54790_4_2357 
                 TET2 
                 EXON 
                 − 
                 chr4: 105240634-105240654 
                 AAUAUUAUCUAUGAGAUGUG 
                 657 
               
               
                   
               
               
                 54790_4_2365 
                 TET2 
                 EXON 
                 − 
                 chr4: 105240693-105240713 
                 AAAAGUGGGAAGAUAGGGGU 
                 658 
               
               
                   
               
               
                 54790_4_2366 
                 TET2 
                 EXON 
                 − 
                 chr4: 105240694-105240714 
                 GAAAAGUGGGAAGAUAGGGG 
                 659 
               
               
                   
               
               
                 54790_4_2368 
                 TET2 
                 EXON 
                 − 
                 chr4: 105240697-105240717 
                 AUGGAAAAGUGGGAAGAUAG 
                 660 
               
               
                   
               
               
                 54790_4_2369 
                 TET2 
                 EXON 
                 − 
                 chr4: 105240698-105240718 
                 GAUGGAAAAGUGGGAAGAUA 
                 661 
               
               
                   
               
               
                 54790_4_2370 
                 TET2 
                 EXON 
                 − 
                 chr4: 105240699-105240719 
                 AGAUGGAAAAGUGGGAAGAU 
                 662 
               
               
                   
               
               
                 54790_4_2373 
                 TET2 
                 EXON 
                 − 
                 chr4: 105240707-105240727 
                 ACCAACAAAGAUGGAAAAGU 
                 663 
               
               
                   
               
               
                 54790_4_2377 
                 TET2 
                 EXON 
                 − 
                 chr4: 105240708-105240728 
                 AACCAACAAAGAUGGAAAAG 
                 664 
               
               
                   
               
               
                 54790_4_2380 
                 TET2 
                 EXON 
                 − 
                 chr4: 105240716-105240736 
                 CUGUUGCAAACCAACAAAGA 
                 665 
               
               
                   
               
               
                 54790_4_2382 
                 TET2 
                 EXON 
                 − 
                 chr4: 105240739-105240759 
                 GAGAGUCAGGCAAAAAGAAG 
                 666 
               
               
                   
               
               
                 54790_4_2383 
                 TET2 
                 EXON 
                 − 
                 chr4: 105240740-105240760 
                 GGAGAGUCAGGCAAAAAGAA 
                 667 
               
               
                   
               
               
                 54790_4_2384 
                 TET2 
                 EXON 
                 − 
                 chr4: 105240741-105240761 
                 UGGAGAGUCAGGCAAAAAGA 
                 668 
               
               
                   
               
               
                 54790_4_2389 
                 TET2 
                 EXON 
                 − 
                 chr4: 105240752-105240772 
                 AGAGAAAAUCCUGGAGAGUC 
                 669 
               
               
                   
               
               
                 54790_4_2393 
                 TET2 
                 EXON 
                 − 
                 chr4: 105240761-105240781 
                 UUUAUGAUGAGAGAAAAUCC 
                 670 
               
               
                   
               
               
                 54790_4_2422 
                 TET2 
                 EXON 
                 − 
                 chr4: 105240882-105240902 
                 UAUUGCUUGGUUUAUUGUCA 
                 671 
               
               
                   
               
               
                 54790_4_2424 
                 TET2 
                 EXON 
                 − 
                 chr4: 105240895-105240915 
                 CCCACCCCCAGAAUAUUGCU 
                 672 
               
               
                   
               
               
                 54790_4_2434 
                 TET2 
                 EXON 
                 − 
                 chr4: 105240954-105240974 
                 CUGGAAGCCUACCUAUUACU 
                 673 
               
               
                   
               
               
                 54790_4_2439 
                 TET2 
                 EXON 
                 − 
                 chr4: 105240973-105240993 
                 AAAAAACAUUUAAAGCUAAC 
                 674 
               
               
                   
               
               
                 54790_4_2446 
                 TET2 
                 EXON 
                 − 
                 chr4: 105241000-105241020 
                 UACAAUCCAAUUUUUUGAGC 
                 675 
               
               
                   
               
               
                 54790_4_2454 
                 TET2 
                 EXON 
                 − 
                 chr4: 105241052-105241072 
                 CUAAACAAAGAAUACAGUGA 
                 676 
               
               
                   
               
               
                 54790_4_2456 
                 TET2 
                 EXON 
                 − 
                 chr4: 105241053-105241073 
                 ACUAAACAAAGAAUACAGUG 
                 677 
               
               
                   
               
               
                 54790_4_2468 
                 TET2 
                 EXON 
                 − 
                 chr4: 105241107-105241127 
                 AUAUAUUACAUUUCAGAUAU 
                 678 
               
               
                   
               
               
                 54790_4_2469 
                 TET2 
                 EXON 
                 − 
                 chr4: 105241108-105241128 
                 AAUAUAUUACAUUUCAGAUA 
                 679 
               
               
                   
               
               
                 54790_4_2475 
                 TET2 
                 EXON 
                 − 
                 chr4: 105241136-105241156 
                 UAUAUGUACAUGCUGGUUGU 
                 680 
               
               
                   
               
               
                 54790_4_2477 
                 TET2 
                 EXON 
                 − 
                 chr4: 105241143-105241163 
                 AAUUAAGUAUAUGUACAUGC 
                 681 
               
               
                   
               
               
                 54790_4_2488 
                 TET2 
                 EXON 
                 − 
                 chr4: 105241193-105241213 
                 CUUUAAAAUGAGUAGAUUGA 
                 682 
               
               
                   
               
               
                 54790_4_2498 
                 TET2 
                 EXON 
                 − 
                 chr4: 105241245-105241265 
                 AACCCCCAACUACACAUUAA 
                 683 
               
               
                   
               
               
                 54790_4_2499 
                 TET2 
                 EXON 
                 − 
                 chr4: 105241246-105241266 
                 UAACCCCCAACUACACAUUA 
                 684 
               
               
                   
               
               
                 54790_4_2503 
                 TET2 
                 EXON 
                 − 
                 chr4: 105241285-105241305 
                 CUCAAUUAUACUAAAUAUAA 
                 685 
               
               
                   
               
               
                 54790_4_2519 
                 TET2 
                 EXON 
                 − 
                 chr4: 105241367-105241387 
                 GCUCCUAGAUGGGUAUAAAA 
                 686 
               
               
                   
               
               
                 54790_4_2522 
                 TET2 
                 EXON 
                 − 
                 chr4: 105241377-105241397 
                 AUUAGGACCUGCUCCUAGAU 
                 687 
               
               
                   
               
               
                 54790_4_2523 
                 TET2 
                 EXON 
                 − 
                 chr4: 105241378-105241398 
                 CAUUAGGACCUGCUCCUAGA 
                 688 
               
               
                   
               
               
                 54790_4_2527 
                 TET2 
                 EXON 
                 − 
                 chr4: 105241394-105241414 
                 UCUCUAAUAGCUGCCACAUU 
                 689 
               
               
                   
               
               
                 54790_4_2538 
                 TET2 
                 EXON 
                 − 
                 chr4: 105241470-105241490 
                 AAAAUUCUGACAUAUACAAA 
                 690 
               
               
                   
               
               
                 54790_4_2546 
                 TET2 
                 EXON 
                 − 
                 chr4: 105241494-105241514 
                 ACUGCUUUGUGUGUGAAGGC 
                 691 
               
               
                   
               
               
                 54790_4_2548 
                 TET2 
                 EXON 
                 − 
                 chr4: 105241498-105241518 
                 GUUUACUGCUUUGUGUGUGA 
                 692 
               
               
                   
               
               
                 54790_4_2555 
                 TET2 
                 EXON 
                 − 
                 chr4: 105241568-105241588 
                 AAUAGCACAGUGUGUAGUGU 
                 693 
               
               
                   
               
               
                 54790_4_2558 
                 TET2 
                 EXON 
                 − 
                 chr4: 105241593-105241613 
                 UGUCAAAUAUUGUGACUCUC 
                 694 
               
               
                   
               
               
                 54790_4_2563 
                 TET2 
                 EXON 
                 − 
                 chr4: 105241647-105241667 
                 UCUGGCAUCCAUCGCAAAGU 
                 695 
               
               
                   
               
               
                 54790_4_2564 
                 TET2 
                 EXON 
                 − 
                 chr4: 105241648-105241668 
                 UUCUGGCAUCCAUCGCAAAG 
                 696 
               
               
                   
               
               
                 54790_4_2568 
                 TET2 
                 EXON 
                 − 
                 chr4: 105241665-105241685 
                 CUGUUCAUGCCUGGGUUUUC 
                 697 
               
               
                   
               
               
                 54790_4_2569 
                 TET2 
                 EXON 
                 − 
                 chr4: 105241673-105241693 
                 GCCGAUUCCUGUUCAUGCCU 
                 698 
               
               
                   
               
               
                 54790_4_2570 
                 TET2 
                 EXON 
                 − 
                 chr4: 105241674-105241694 
                 GGCCGAUUCCUGUUCAUGCC 
                 699 
               
               
                   
               
               
                 54790_4_2573 
                 TET2 
                 EXON 
                 − 
                 chr4: 105241695-105241715 
                 CUUGUGGCUGGCAGCCUGGC 
                 700 
               
               
                   
               
               
                 54790_4_2574 
                 TET2 
                 EXON 
                 − 
                 chr4: 105241699-105241719 
                 GUACCUUGUGGCUGGCAGCC 
                 701 
               
               
                   
               
               
                 54790_4_2575 
                 TET2 
                 EXON 
                 − 
                 chr4: 105241707-105241727 
                 CUGUGCCAGUACCUUGUGGC 
                 702 
               
               
                   
               
               
                 54790_4_2577 
                 TET2 
                 EXON 
                 − 
                 chr4: 105241711-105241731 
                 GAGCCUGUGCCAGUACCUUG 
                 703 
               
               
                   
               
               
                 54790_4_2578 
                 TET2 
                 EXON 
                 − 
                 chr4: 105241733-105241753 
                 GCCAGAGUGGGACCUCUCGU 
                 704 
               
               
                   
               
               
                 54790_4_2582 
                 TET2 
                 EXON 
                 − 
                 chr4: 105241745-105241765 
                 UCAGGUGGGAAAGCCAGAGU 
                 705 
               
               
                   
               
               
                 54790_4_2585 
                 TET2 
                 EXON 
                 − 
                 chr4: 105241746-105241766 
                 AUCAGGUGGGAAAGCCAGAG 
                 706 
               
               
                   
               
               
                 54790_4_2591 
                 TET2 
                 EXON 
                 − 
                 chr4: 105241759-105241779 
                 UUGACACUUUAUUAUCAGGU 
                 707 
               
               
                   
               
               
                 54790_4_2595 
                 TET2 
                 EXON 
                 − 
                 chr4: 105241760-105241780 
                 UUUGACACUUUAUUAUCAGG 
                 708 
               
               
                   
               
               
                 54790_4_2598 
                 TET2 
                 EXON 
                 − 
                 chr4: 105241763-105241783 
                 UGCUUUGACACUUUAUUAUC 
                 709 
               
               
                   
               
               
                 54790_4_2609 
                 TET2 
                 EXON 
                 − 
                 chr4: 105241819-105241839 
                 ACUAGGUGAAUUUAAUUCAG 
                 710 
               
               
                   
               
               
                 54790_4_2613 
                 TET2 
                 EXON 
                 − 
                 chr4: 105241836-105241856 
                 AAGUACUCAUUUGCAACACU 
                 711 
               
               
                   
               
               
                 54790_4_2622 
                 TET2 
                 EXON 
                 − 
                 chr4: 105241878-105241898 
                 UCACACUUGCUCUCUUUUUA 
                 712 
               
               
                   
               
               
                 54790_4_2629 
                 TET2 
                 EXON 
                 − 
                 chr4: 105241939-105241959 
                 AUAGCGAAAAAAAAAAAAAA 
                 713 
               
               
                   
               
               
                 54790_4_2633 
                 TET2 
                 EXON 
                 − 
                 chr4: 105241986-105242006 
                 UCUUCUACAUGCAGGAGUAA 
                 714 
               
               
                   
               
               
                 54790_4_2635 
                 TET2 
                 EXON 
                 − 
                 chr4: 105241994-105242014 
                 CAUAAGAGUCUUCUACAUGC 
                 715 
               
               
                   
               
               
                 54790_4_2642 
                 TET2 
                 EXON 
                 − 
                 chr4: 105242038-105242058 
                 GCUGUAUAAAUUUAUAUGAA 
                 716 
               
               
                   
               
               
                 54790_4_2652 
                 TET2 
                 EXON 
                 − 
                 chr4: 105242086-105242106 
                 CUGCCUACCUCUUAAUGAAA 
                 717 
               
               
                   
               
               
                 54790_4_2663 
                 TET2 
                 EXON 
                 − 
                 chr4: 105242173-105242193 
                 AGAAAUGAAUAAUUUGGAAA 
                 718 
               
               
                   
               
               
                 54790_4_2665 
                 TET2 
                 EXON 
                 − 
                 chr4: 105242179-105242199 
                 UAAUUUAGAAAUGAAUAAUU 
                 719 
               
               
                   
               
               
                 54790_4_2679 
                 TET2 
                 EXON 
                 − 
                 chr4: 105242236-105242256 
                 GGAAAUUCACUAUUUCUGCC 
                 720 
               
               
                   
               
               
                 54790_4_2681 
                 TET2 
                 EXON 
                 − 
                 chr4: 105242257-105242277 
                 GUUGUUUUUUUUGGCACUUA 
                 721 
               
               
                   
               
               
                 54790_4_2683 
                 TET2 
                 EXON 
                 − 
                 chr4: 105242258-105242278 
                 UGUUGUUUUUUUUGGCACUU 
                 722 
               
               
                   
               
               
                 54790_4_2685 
                 TET2 
                 EXON 
                 − 
                 chr4: 105242266-105242286 
                 UGUUUUUUUGUUGUUUUUUU 
                 723 
               
               
                   
               
               
                 54790_4_2694 
                 TET2 
                 EXON 
                 − 
                 chr4: 105242360-105242380 
                 AUCCAGCCACAUGGAAAAUA 
                 724 
               
               
                   
               
               
                 54790_4_2697 
                 TET2 
                 EXON 
                 − 
                 chr4: 105242369-105242389 
                 AUAGUUAGUAUCCAGCCACA 
                 725 
               
               
                   
               
               
                 54790_4_2701 
                 TET2 
                 EXON 
                 − 
                 chr4: 105242395-105242415 
                 CACAAUUUAGAAAAGGAGGC 
                 726 
               
               
                   
               
               
                 54790_4_2702 
                 TET2 
                 EXON 
                 − 
                 chr4: 105242399-105242419 
                 GUCUCACAAUUUAGAAAAGG 
                 727 
               
               
                   
               
               
                 54790_4_2703 
                 TET2 
                 EXON 
                 − 
                 chr4: 105242402-105242422 
                 AAUGUCUCACAAUUUAGAAA 
                 728 
               
               
                   
               
               
                 54790_4_2721 
                 TET2 
                 EXON 
                 − 
                 chr4: 105242462-105242482 
                 UUUAGCUAUUUUAAAACUUG 
                 729 
               
               
                   
               
               
                 54790_4_2723 
                 TET2 
                 EXON 
                 − 
                 chr4: 105242463-105242483 
                 AUUUAGCUAUUUUAAAACUU 
                 730 
               
               
                   
               
               
                 54790_4_2726 
                 TET2 
                 EXON 
                 − 
                 chr4: 105242464-105242484 
                 AAUUUAGCUAUUUUAAAACU 
                 731 
               
               
                   
               
               
                 54790_4_2742 
                 TET2 
                 EXON 
                 − 
                 chr4: 105242539-105242559 
                 UUUCACAAAGCACAAAAUUC 
                 732 
               
               
                   
               
               
                 54790_4_2749 
                 TET2 
                 EXON 
                 − 
                 chr4: 105242583-105242603 
                 AAUUACAUGUGGGUGAAAAU 
                 733 
               
               
                   
               
               
                 54790_4_2752 
                 TET2 
                 EXON 
                 − 
                 chr4: 105242584-105242604 
                 AAAUUACAUGUGGGUGAAAA 
                 734 
               
               
                   
               
               
                 54790_4_2755 
                 TET2 
                 EXON 
                 − 
                 chr4: 105242593-105242613 
                 CUAUUUUGUAAAUUACAUGU 
                 735 
               
               
                   
               
               
                 54790_4_2756 
                 TET2 
                 EXON 
                 − 
                 chr4: 105242594-105242614 
                 ACUAUUUUGUAAAUUACAUG 
                 736 
               
               
                   
               
               
                 54790_4_2769 
                 TET2 
                 EXON 
                 − 
                 chr4: 105242669-105242689 
                 ACGCCAAGCAAACUGGAUAA 
                 737 
               
               
                   
               
               
                 54790_4_2772 
                 TET2 
                 EXON 
                 − 
                 chr4: 105242676-105242696 
                 CAGGUCUACGCCAAGCAAAC 
                 738 
               
               
                   
               
               
                 54790_4_2780 
                 TET2 
                 EXON 
                 − 
                 chr4: 105242695-105242715 
                 CGGUUUAUUAUUUUUUAAAC 
                 739 
               
               
                   
               
               
                 54790_4_2781 
                 TET2 
                 EXON 
                 − 
                 chr4: 105242715-105242735 
                 ACCACAUCCUGAGAAAUGAA 
                 740 
               
               
                   
               
               
                 54790_5_3 
                 TET2 
                 EXON 
                 + 
                 chr4: 105242816-105242836 
                 CUGUGGGUUUCUUUAAGGUU 
                 741 
               
               
                   
               
               
                 54790_5_7 
                 TET2 
                 EXON 
                 + 
                 chr4: 105242824-105242844 
                 UUCUUUAAGGUUUGGACAGA 
                 742 
               
               
                   
               
               
                 54790_5_8 
                 TET2 
                 EXON 
                 + 
                 chr4: 105242825-105242845 
                 UCUUUAAGGUUUGGACAGAA 
                 743 
               
               
                   
               
               
                 54790_5_15 
                 TET2 
                 EXON 
                 + 
                 chr4: 105242838-105242858 
                 GACAGAAGGGUAAAGCUAUU 
                 744 
               
               
                   
               
               
                 54790_5_20 
                 TET2 
                 EXON 
                 + 
                 chr4: 105242861-105242881 
                 AUUGAAAGAGUCAUCUAUAC 
                 745 
               
               
                   
               
               
                 54790_5_23 
                 TET2 
                 EXON 
                 + 
                 chr4: 105242870-105242890 
                 GUCAUCUAUACUGGUAAAGA 
                 746 
               
               
                   
               
               
                 54790_5_26 
                 TET2 
                 EXON 
                 + 
                 chr4: 105242884-105242904 
                 UAAAGAAGGCAAAAGUUCUC 
                 747 
               
               
                   
               
               
                 54790_5_27 
                 TET2 
                 EXON 
                 + 
                 chr4: 105242885-105242905 
                 AAAGAAGGCAAAAGUUCUCA 
                 748 
               
               
                   
               
               
                 54790_5_30 
                 TET2 
                 EXON 
                 + 
                 chr4: 105242904-105242924 
                 AGGGAUGUCCUAUUGCUAAG 
                 749 
               
               
                   
               
               
                 54790_5_31 
                 TET2 
                 EXON 
                 + 
                 chr4: 105242905-105242925 
                 GGGAUGUCCUAUUGCUAAGU 
                 750 
               
               
                   
               
               
                 54790_5_51 
                 TET2 
                 EXON 
                 − 
                 chr4: 105242915-105242935 
                 ACACUUACCCACUUAGCAAU 
                 751 
               
               
                   
               
               
                 54790_6_1 
                 TET2 
                 EXON 
                 + 
                 chr4: 105243550-105243570 
                 GGAAUGGUGAUCCACGCAGG 
                 752 
               
               
                   
               
               
                 54790_6_7 
                 TET2 
                 EXON 
                 + 
                 chr4: 105243589-105243609 
                 UGAAGAGAAGCUACUGUGUU 
                 753 
               
               
                   
               
               
                 54790_6_9 
                 TET2 
                 EXON 
                 + 
                 chr4: 105243594-105243614 
                 AGAAGCUACUGUGUUUGGUG 
                 754 
               
               
                   
               
               
                 54790_6_12 
                 TET2 
                 EXON 
                 + 
                 chr4: 105243595-105243615 
                 GAAGCUACUGUGUUUGGUGC 
                 755 
               
               
                   
               
               
                 54790_6_14 
                 TET2 
                 EXON 
                 + 
                 chr4: 105243605-105243625 
                 UGUUUGGUGCGGGAGCGAGC 
                 756 
               
               
                   
               
               
                 54790_6_18 
                 TET2 
                 EXON 
                 + 
                 chr4: 105243619-105243639 
                 GCGAGCUGGCCACACCUGUG 
                 757 
               
               
                   
               
               
                 54790_6_19 
                 TET2 
                 EXON 
                 + 
                 chr4: 105243646-105243666 
                 AGUGAUUGUGAUUCUCAUCC 
                 758 
               
               
                   
               
               
                 54790_6_21 
                 TET2 
                 EXON 
                 + 
                 chr4: 105243651-105243671 
                 UUGUGAUUCUCAUCCUGGUG 
                 759 
               
               
                   
               
               
                 54790_6_24 
                 TET2 
                 EXON 
                 + 
                 chr4: 105243652-105243672 
                 UGUGAUUCUCAUCCUGGUGU 
                 760 
               
               
                   
               
               
                 54790_6_27 
                 TET2 
                 EXON 
                 + 
                 chr4: 105243656-105243676 
                 AUUCUCAUCCUGGUGUGGGA 
                 761 
               
               
                   
               
               
                 54790_6_30 
                 TET2 
                 EXON 
                 + 
                 chr4: 105243673-105243693 
                 GGAAGGAAUCCCGCUGUCUC 
                 762 
               
               
                   
               
               
                 54790_6_32 
                 TET2 
                 EXON 
                 + 
                 chr4: 105243691-105243711 
                 UCUGGCUGACAAACUCUACU 
                 763 
               
               
                   
               
               
                 54790_6_37 
                 TET2 
                 EXON 
                 + 
                 chr4: 105243711-105243731 
                 CGGAGCUUACCGAGACGCUG 
                 764 
               
               
                   
               
               
                 54790_6_39 
                 TET2 
                 EXON 
                 + 
                 chr4: 105243719-105243739 
                 ACCGAGACGCUGAGGAAAUA 
                 765 
               
               
                   
               
               
                 54790_6_41 
                 TET2 
                 EXON 
                 + 
                 chr4: 105243738-105243758 
                 ACGGCACGCUCACCAAUCGC 
                 766 
               
               
                   
               
               
                 54790_6_48 
                 TET2 
                 EXON 
                 + 
                 chr4: 105243771-105243791 
                 AUGAAGAGUAAGUGAAGCCC 
                 767 
               
               
                   
               
               
                 54790_6_49 
                 TET2 
                 EXON 
                 + 
                 chr4: 105243772-105243792 
                 UGAAGAGUAAGUGAAGCCCA 
                 768 
               
               
                   
               
               
                 54790_6_51 
                 TET2 
                 EXON 
                 − 
                 chr4: 105243564-105243584 
                 GCUUCUGCGAACCACCUGCG 
                 769 
               
               
                   
               
               
                 54790_6_56 
                 TET2 
                 EXON 
                 − 
                 chr4: 105243631-105243651 
                 UCACUGCAGCCUCACAGGUG 
                 770 
               
               
                   
               
               
                 54790_6_57 
                 TET2 
                 EXON 
                 − 
                 chr4: 105243636-105243656 
                 CACAAUCACUGCAGCCUCAC 
                 771 
               
               
                   
               
               
                 54790_6_62 
                 TET2 
                 EXON 
                 − 
                 chr4: 105243667-105243687 
                 GCGGGAUUCCUUCCCACACC 
                 772 
               
               
                   
               
               
                 54790_6_66 
                 TET2 
                 EXON 
                 − 
                 chr4: 105243685-105243705 
                 GUUUGUCAGCCAGAGACAGC 
                 773 
               
               
                   
               
               
                 54790_6_67 
                 TET2 
                 EXON 
                 − 
                 chr4: 105243686-105243706 
                 AGUUUGUCAGCCAGAGACAG 
                 774 
               
               
                   
               
               
                 54790_6_75 
                 TET2 
                 EXON 
                 − 
                 chr4: 105243723-105243743 
                 GCCGUAUUUCCUCAGCGUCU 
                 775 
               
               
                   
               
               
                 54790_6_80 
                 TET2 
                 EXON 
                 − 
                 chr4: 105243753-105243773 
                 AUUCAAGGCACACCGGCGAU 
                 776 
               
               
                   
               
               
                 54790_6_82 
                 TET2 
                 EXON 
                 − 
                 chr4: 105243760-105243780 
                 ACUCUUCAUUCAAGGCACAC 
                 777 
               
               
                   
               
               
                 54790_6_84 
                 TET2 
                 EXON 
                 − 
                 chr4: 105243768-105243788 
                 CUUCACUUACUCUUCAUUCA 
                 778 
               
               
                   
               
               
                 54790_7_10 
                 TET2 
                 EXON 
                 + 
                 chr4: 105259615-105259635 
                 CAGGAGAACUUGCGCCUGUC 
                 779 
               
               
                   
               
               
                 54790_7_12 
                 TET2 
                 EXON 
                 + 
                 chr4: 105259616-105259636 
                 AGGAGAACUUGCGCCUGUCA 
                 780 
               
               
                   
               
               
                 54790_7_14 
                 TET2 
                 EXON 
                 + 
                 chr4: 105259617-105259637 
                 GGAGAACUUGCGCCUGUCAG 
                 781 
               
               
                   
               
               
                 54790_7_16 
                 TET2 
                 EXON 
                 + 
                 chr4: 105259621-105259641 
                 AACUUGCGCCUGUCAGGGGC 
                 782 
               
               
                   
               
               
                 54790_7_20 
                 TET2 
                 EXON 
                 + 
                 chr4: 105259637-105259657 
                 GGGCUGGAUCCAGAAACCUG 
                 783 
               
               
                   
               
               
                 54790_7_21 
                 TET2 
                 EXON 
                 + 
                 chr4: 105259655-105259675 
                 UGUGGUGCCUCCUUCUCUUU 
                 784 
               
               
                   
               
               
                 54790_7_23 
                 TET2 
                 EXON 
                 + 
                 chr4: 105259665-105259685 
                 CCUUCUCUUUUGGUUGUUCA 
                 785 
               
               
                   
               
               
                 54790_7_31 
                 TET2 
                 EXON 
                 + 
                 chr4: 105259682-105259702 
                 UCAUGGAGCAUGUACUACAA 
                 786 
               
               
                   
               
               
                 54790_7_35 
                 TET2 
                 EXON 
                 + 
                 chr4: 105259713-105259733 
                 UUGCCAGAAGCAAGAUCCCA 
                 787 
               
               
                   
               
               
                 54790_7_41 
                 TET2 
                 EXON 
                 + 
                 chr4: 105259730-105259750 
                 CCAAGGAAGUUUAAGCUGCU 
                 788 
               
               
                   
               
               
                 54790_7_42 
                 TET2 
                 EXON 
                 + 
                 chr4: 105259731-105259751 
                 CAAGGAAGUUUAAGCUGCUU 
                 789 
               
               
                   
               
               
                 54790_7_44 
                 TET2 
                 EXON 
                 + 
                 chr4: 105259732-105259752 
                 AAGGAAGUUUAAGCUGCUUG 
                 790 
               
               
                   
               
               
                 54790_7_48 
                 TET2 
                 EXON 
                 + 
                 chr4: 105259747-105259767 
                 GCUUGGGGAUGACCCAAAAG 
                 791 
               
               
                   
               
               
                 54790_7_53 
                 TET2 
                 EXON 
                 − 
                 chr4: 105259632-105259652 
                 UUCUGGAUCCAGCCCCUGAC 
                 792 
               
               
                   
               
               
                 54790_7_54 
                 TET2 
                 EXON 
                 − 
                 chr4: 105259649-105259669 
                 AAGGAGGCACCACAGGUUUC 
                 793 
               
               
                   
               
               
                 54790_7_56 
                 TET2 
                 EXON 
                 − 
                 chr4: 105259656-105259676 
                 AAAAGAGAAGGAGGCACCAC 
                 794 
               
               
                   
               
               
                 54790_7_57 
                 TET2 
                 EXON 
                 − 
                 chr4: 105259665-105259685 
                 UGAACAACCAAAAGAGAAGG 
                 795 
               
               
                   
               
               
                 54790_7_58 
                 TET2 
                 EXON 
                 − 
                 chr4: 105259668-105259688 
                 CCAUGAACAACCAAAAGAGA 
                 796 
               
               
                   
               
               
                 54790_7_72 
                 TET2 
                 EXON 
                 − 
                 chr4: 105259719-105259739 
                 CUUCCUUGGGAUCUUGCUUC 
                 797 
               
               
                   
               
               
                 54790_7_73 
                 TET2 
                 EXON 
                 − 
                 chr4: 105259732-105259752 
                 CAAGCAGCUUAAACUUCCUU 
                 798 
               
               
                   
               
               
                 54790_7_74 
                 TET2 
                 EXON 
                 − 
                 chr4: 105259733-105259753 
                 CCAAGCAGCUUAAACUUCCU 
                 799 
               
               
                   
               
               
                 54790_7_80 
                 TET2 
                 EXON 
                 − 
                 chr4: 105259762-105259782 
                 GAAGUAAACAAACCUCUUUU 
                 800 
               
               
                   
               
               
                 54790_7_81 
                 TET2 
                 EXON 
                 − 
                 chr4: 105259763-105259783 
                 GGAAGUAAACAAACCUCUUU 
                 801 
               
               
                   
               
               
                 54790_8_8 
                 TET2 
                 EXON 
                 + 
                 chr4: 105261748-105261768 
                 CUUUAUACAGGAAGAGAAAC 
                 802 
               
               
                   
               
               
                 54790_8_12 
                 TET2 
                 EXON 
                 + 
                 chr4: 105261781-105261801 
                 GCAAAACCUGUCCACUCUUA 
                 803 
               
               
                   
               
               
                 54790_8_18 
                 TET2 
                 EXON 
                 + 
                 chr4: 105261826-105261846 
                 ACCUGAUGCAUAUAAUAAUC 
                 804 
               
               
                   
               
               
                 54790_8_27 
                 TET2 
                 EXON 
                 − 
                 chr4: 105261790-105261810 
                 UUGGUGCCAUAAGAGUGGAC 
                 805 
               
               
                   
               
               
                 54790_8_30 
                 TET2 
                 EXON 
                 − 
                 chr4: 105261795-105261815 
                 AUAUGUUGGUGCCAUAAGAG 
                 806 
               
               
                   
               
               
                 54790_8_34 
                 TET2 
                 EXON 
                 − 
                 chr4: 105261809-105261829 
                 GGUGCAAGUUUCUUAUAUGU 
                 807 
               
               
                   
               
               
                 54790_8_38 
                 TET2 
                 EXON 
                 − 
                 chr4: 105261830-105261850 
                 ACCUGAUUAUUAUAUGCAUC 
                 808 
               
               
                   
               
               
                 54790_9_14 
                 TET2 
                 EXON 
                 + 
                 chr4: 105269623-105269643 
                 CAGAGCACCAGAGUGCCGUC 
                 809 
               
               
                   
               
               
                 54790_9_15 
                 TET2 
                 EXON 
                 + 
                 chr4: 105269624-105269644 
                 AGAGCACCAGAGUGCCGUCU 
                 810 
               
               
                   
               
               
                 54790_9_19 
                 TET2 
                 EXON 
                 + 
                 chr4: 105269632-105269652 
                 AGAGUGCCGUCUGGGUCUGA 
                 811 
               
               
                   
               
               
                 54790_9_20 
                 TET2 
                 EXON 
                 + 
                 chr4: 105269636-105269656 
                 UGCCGUCUGGGUCUGAAGGA 
                 812 
               
               
                   
               
               
                 54790_9_22 
                 TET2 
                 EXON 
                 + 
                 chr4: 105269651-105269671 
                 AAGGAAGGCCGUCCAUUCUC 
                 813 
               
               
                   
               
               
                 54790_9_24 
                 TET2 
                 EXON 
                 + 
                 chr4: 105269652-105269672 
                 AGGAAGGCCGUCCAUUCUCA 
                 814 
               
               
                   
               
               
                 54790_9_25 
                 TET2 
                 EXON 
                 + 
                 chr4: 105269653-105269673 
                 GGAAGGCCGUCCAUUCUCAG 
                 815 
               
               
                   
               
               
                 54790_9_27 
                 TET2 
                 EXON 
                 + 
                 chr4: 105269668-105269688 
                 CUCAGGGGUCACUGCAUGUU 
                 816 
               
               
                   
               
               
                 54790_9_35 
                 TET2 
                 EXON 
                 + 
                 chr4: 105269714-105269734 
                 GACUUGCACAACAUGCAGAA 
                 817 
               
               
                   
               
               
                 54790_9_37 
                 TET2 
                 EXON 
                 + 
                 chr4: 105269725-105269745 
                 CAUGCAGAAUGGCAGCACAU 
                 818 
               
               
                   
               
               
                 54790_9_39 
                 TET2 
                 EXON 
                 + 
                 chr4: 105269733-105269753 
                 AUGGCAGCACAUUGGUAAGU 
                 819 
               
               
                   
               
               
                 54790_9_40 
                 TET2 
                 EXON 
                 + 
                 chr4: 105269734-105269754 
                 UGGCAGCACAUUGGUAAGUU 
                 820 
               
               
                   
               
               
                 54790_9_43 
                 TET2 
                 EXON 
                 + 
                 chr4: 105269740-105269760 
                 CACAUUGGUAAGUUGGGCUG 
                 821 
               
               
                   
               
               
                 54790_9_49 
                 TET2 
                 EXON 
                 − 
                 chr4: 105269633-105269653 
                 UUCAGACCCAGACGGCACUC 
                 822 
               
               
                   
               
               
                 54790_9_50 
                 TET2 
                 EXON 
                 − 
                 chr4: 105269641-105269661 
                 GGCCUUCCUUCAGACCCAGA 
                 823 
               
               
                   
               
               
                 54790_9_51 
                 TET2 
                 EXON 
                 − 
                 chr4: 105269662-105269682 
                 CAGUGACCCCUGAGAAUGGA 
                 824 
               
               
                   
               
               
                 54790_9_52 
                 TET2 
                 EXON 
                 − 
                 chr4: 105269666-105269686 
                 CAUGCAGUGACCCCUGAGAA 
                 825 
               
               
                   
               
               
                 54790_9_61 
                 TET2 
                 EXON 
                 − 
                 chr4: 105269709-105269729 
                 CAUGUUGUGCAAGUCUCUGU 
                 826 
               
               
                   
               
               
                 54790_9_62 
                 TET2 
                 EXON 
                 − 
                 chr4: 105269710-105269730 
                 GCAUGUUGUGCAAGUCUCUG 
                 827 
               
               
                   
               
               
                 54790_10_10 
                 TET2 
                 EXON 
                 + 
                 chr4: 105272578-105272598 
                 AGAGAAGACAAUCGAGAAUU 
                 828 
               
               
                   
               
               
                 54790_10_13 
                 TET2 
                 EXON 
                 + 
                 chr4: 105272581-105272601 
                 GAAGACAAUCGAGAAUUUGG 
                 829 
               
               
                   
               
               
                 54790_10_16 
                 TET2 
                 EXON 
                 + 
                 chr4: 105272592-105272612 
                 AGAAUUUGGAGGAAAACCUG 
                 830 
               
               
                   
               
               
                 54790_10_23 
                 TET2 
                 EXON 
                 + 
                 chr4: 105272637-105272657 
                 UUUAUACAAAGUCUCUGACG 
                 831 
               
               
                   
               
               
                 54790_10_29 
                 TET2 
                 EXON 
                 + 
                 chr4: 105272647-105272667 
                 GUCUCUGACGUGGAUGAGUU 
                 832 
               
               
                   
               
               
                 54790_10_30 
                 TET2 
                 EXON 
                 + 
                 chr4: 105272648-105272668 
                 UCUCUGACGUGGAUGAGUUU 
                 833 
               
               
                   
               
               
                 54790_10_33 
                 TET2 
                 EXON 
                 + 
                 chr4: 105272655-105272675 
                 CGUGGAUGAGUUUGGGAGUG 
                 834 
               
               
                   
               
               
                 54790_10_36 
                 TET2 
                 EXON 
                 + 
                 chr4: 105272664-105272684 
                 GUUUGGGAGUGUGGAAGCUC 
                 835 
               
               
                   
               
               
                 54790_10_40 
                 TET2 
                 EXON 
                 + 
                 chr4: 105272667-105272687 
                 UGGGAGUGUGGAAGCUCAGG 
                 836 
               
               
                   
               
               
                 54790_10_46 
                 TET2 
                 EXON 
                 + 
                 chr4: 105272678-105272698 
                 AAGCUCAGGAGGAGAAAAAA 
                 837 
               
               
                   
               
               
                 54790_10_48 
                 TET2 
                 EXON 
                 + 
                 chr4: 105272683-105272703 
                 CAGGAGGAGAAAAAACGGAG 
                 838 
               
               
                   
               
               
                 54790_10_49 
                 TET2 
                 EXON 
                 + 
                 chr4: 105272694-105272714 
                 AAAACGGAGUGGUGCCAUUC 
                 839 
               
               
                   
               
               
                 54790_10_51 
                 TET2 
                 EXON 
                 + 
                 chr4: 105272711-105272731 
                 UUCAGGUACUGAGUUCUUUU 
                 840 
               
               
                   
               
               
                 54790_10_55 
                 TET2 
                 EXON 
                 + 
                 chr4: 105272723-105272743 
                 GUUCUUUUCGGCGAAAAGUC 
                 841 
               
               
                   
               
               
                 54790_10_64 
                 TET2 
                 EXON 
                 + 
                 chr4: 105272759-105272779 
                 CAGUCAAGACUUGCCGACAA 
                 842 
               
               
                   
               
               
                 54790_10_71 
                 TET2 
                 EXON 
                 + 
                 chr4: 105272805-105272825 
                 AGCUGAAAAGCUUUCCUCCC 
                 843 
               
               
                   
               
               
                 54790_10_78 
                 TET2 
                 EXON 
                 + 
                 chr4: 105272832-105272852 
                 CAGCUCAAAUAAAAAUGAAA 
                 844 
               
               
                   
               
               
                 54790_10_81 
                 TET2 
                 EXON 
                 + 
                 chr4: 105272880-105272900 
                 ACAAACUGAAAACGCAAGCC 
                 845 
               
               
                   
               
               
                 54790_10_82 
                 TET2 
                 EXON 
                 + 
                 chr4: 105272892-105272912 
                 CGCAAGCCAGGCUAAACAGU 
                 846 
               
               
                   
               
               
                 54790_10_83 
                 TET2 
                 EXON 
                 + 
                 chr4: 105272896-105272916 
                 AGCCAGGCUAAACAGUUGGC 
                 847 
               
               
                   
               
               
                 54790_10_85 
                 TET2 
                 EXON 
                 − 
                 chr4: 105272557-105272577 
                 GUGAGAGUGCAUACCUGGUA 
                 848 
               
               
                   
               
               
                 54790_10_87 
                 TET2 
                 EXON 
                 − 
                 chr4: 105272558-105272578 
                 AGUGAGAGUGCAUACCUGGU 
                 849 
               
               
                   
               
               
                 54790_10_91 
                 TET2 
                 EXON 
                 − 
                 chr4: 105272562-105272582 
                 CUCUAGUGAGAGUGCAUACC 
                 850 
               
               
                   
               
               
                 54790_10_99 
                 TET2 
                 EXON 
                 − 
                 chr4: 105272611-105272631 
                 ACGUGAAGCUGCUCAUCCUC 
                 851 
               
               
                   
               
               
                 54790_10_105 
                 TET2 
                 EXON 
                 − 
                 chr4: 105272638-105272658 
                 ACGUCAGAGACUUUGUAUAA 
                 852 
               
               
                   
               
               
                 54790_10_114 
                 TET2 
                 EXON 
                 − 
                 chr4: 105272711-105272731 
                 AAAAGAACUCAGUACCUGAA 
                 853 
               
               
                   
               
               
                 54790_10_127 
                 TET2 
                 EXON 
                 − 
                 chr4: 105272761-105272781 
                 CUUUGUCGGCAAGUCUUGAC 
                 854 
               
               
                   
               
               
                 54790_10_132 
                 TET2 
                 EXON 
                 − 
                 chr4: 105272775-105272795 
                 UGGCUUCUAGUUUCCUUUGU 
                 855 
               
               
                   
               
               
                 54790_10_136 
                 TET2 
                 EXON 
                 − 
                 chr4: 105272795-105272815 
                 CUUUUCAGCUGCAGCUUUCU 
                 856 
               
               
                   
               
               
                 54790_10_145 
                 TET2 
                 EXON 
                 − 
                 chr4: 105272822-105272842 
                 AUUUGAGCUGUUCUCCAGGG 
                 857 
               
               
                   
               
               
                 54790_10_147 
                 TET2 
                 EXON 
                 − 
                 chr4: 105272825-105272845 
                 UUUAUUUGAGCUGUUCUCCA 
                 858 
               
               
                   
               
               
                 54790_10_150 
                 TET2 
                 EXON 
                 − 
                 chr4: 105272826-105272846 
                 UUUUAUUUGAGCUGUUCUCC 
                 859 
               
               
                   
               
               
                 54790_10_167 
                 TET2 
                 EXON 
                 − 
                 chr4: 105272867-105272887 
                 AGUUUGUUUUGUACGUGAUG 
                 860 
               
               
                   
               
               
                 54790_10_168 
                 TET2 
                 EXON 
                 − 
                 chr4: 105272868-105272888 
                 CAGUUUGUUUUGUACGUGAU 
                 861 
               
               
                   
               
               
                 54790_10_169 
                 TET2 
                 EXON 
                 − 
                 chr4: 105272869-105272889 
                 UCAGUUUGUUUUGUACGUGA 
                 862 
               
               
                   
               
               
                 54790_10_177 
                 TET2 
                 EXON 
                 − 
                 chr4: 105272901-105272921 
                 UACCUGCCAACUGUUUAGCC 
                 863 
               
               
                   
               
               
                 54790_11_9 
                 TET2 
                 EXON 
                 + 
                 chr4: 105275178-105275198 
                 GUCAACUCUUAUUCUGCUUC 
                 864 
               
               
                   
               
               
                 54790_11_14 
                 TET2 
                 EXON 
                 + 
                 chr4: 105275203-105275223 
                 CCACCAAUCCAUACAUGAGA 
                 865 
               
               
                   
               
               
                 54790_11_19 
                 TET2 
                 EXON 
                 + 
                 chr4: 105275256-105275276 
                 UCACACACUUCAGAUAUCUA 
                 866 
               
               
                   
               
               
                 54790_11_24 
                 TET2 
                 EXON 
                 + 
                 chr4: 105275304-105275324 
                 UCCACCUCAUCUCAAGCUGC 
                 867 
               
               
                   
               
               
                 54790_11_34 
                 TET2 
                 EXON 
                 + 
                 chr4: 105275346-105275366 
                 AAUCCCAUGAACCCUUACCC 
                 868 
               
               
                   
               
               
                 54790_11_35 
                 TET2 
                 EXON 
                 + 
                 chr4: 105275347-105275367 
                 AUCCCAUGAACCCUUACCCU 
                 869 
               
               
                   
               
               
                 54790_11_44 
                 TET2 
                 EXON 
                 + 
                 chr4: 105275391-105275411 
                 UAUCCAUCAUAUCAAUGCAA 
                 870 
               
               
                   
               
               
                 54790_11_47 
                 TET2 
                 EXON 
                 + 
                 chr4: 105275405-105275425 
                 AUGCAAUGGAAACCUAUCAG 
                 871 
               
               
                   
               
               
                 54790_11_49 
                 TET2 
                 EXON 
                 + 
                 chr4: 105275426-105275446 
                 GGACAACUGCUCCCCAUAUC 
                 872 
               
               
                   
               
               
                 54790_11_50 
                 TET2 
                 EXON 
                 + 
                 chr4: 105275427-105275447 
                 GACAACUGCUCCCCAUAUCU 
                 873 
               
               
                   
               
               
                 54790_11_53 
                 TET2 
                 EXON 
                 + 
                 chr4: 105275456-105275476 
                 UUCUCCCCAGUCUCAGCCGA 
                 874 
               
               
                   
               
               
                 54790_11_55 
                 TET2 
                 EXON 
                 + 
                 chr4: 105275467-105275487 
                 CUCAGCCGAUGGAUCUGUAU 
                 875 
               
               
                   
               
               
                 54790_11_56 
                 TET2 
                 EXON 
                 + 
                 chr4: 105275533-105275553 
                 UCCAUACACUUUACCAGCCA 
                 876 
               
               
                   
               
               
                 54790_11_59 
                 TET2 
                 EXON 
                 + 
                 chr4: 105275538-105275558 
                 ACACUUUACCAGCCAAGGUU 
                 877 
               
               
                   
               
               
                 54790_11_65 
                 TET2 
                 EXON 
                 + 
                 chr4: 105275571-105275591 
                 AGUUUUACAUCUAAAUACUU 
                 878 
               
               
                   
               
               
                 54790_11_68 
                 TET2 
                 EXON 
                 + 
                 chr4: 105275577-105275597 
                 ACAUCUAAAUACUUAGGUUA 
                 879 
               
               
                   
               
               
                 54790_11_74 
                 TET2 
                 EXON 
                 + 
                 chr4: 105275594-105275614 
                 UUAUGGAAACCAAAAUAUGC 
                 880 
               
               
                   
               
               
                 54790_11_77 
                 TET2 
                 EXON 
                 + 
                 chr4: 105275595-105275615 
                 UAUGGAAACCAAAAUAUGCA 
                 881 
               
               
                   
               
               
                 54790_11_79 
                 TET2 
                 EXON 
                 + 
                 chr4: 105275601-105275621 
                 AACCAAAAUAUGCAGGGAGA 
                 882 
               
               
                   
               
               
                 54790_11_85 
                 TET2 
                 EXON 
                 + 
                 chr4: 105275643-105275663 
                 AGACCAAAUGUACAUCAUGU 
                 883 
               
               
                   
               
               
                 54790_11_86 
                 TET2 
                 EXON 
                 + 
                 chr4: 105275644-105275664 
                 GACCAAAUGUACAUCAUGUA 
                 884 
               
               
                   
               
               
                 54790_11_92 
                 TET2 
                 EXON 
                 + 
                 chr4: 105275675-105275695 
                 UCCUUAUCCCACUCAUGAGA 
                 885 
               
               
                   
               
               
                 54790_11_93 
                 TET2 
                 EXON 
                 + 
                 chr4: 105275679-105275699 
                 UAUCCCACUCAUGAGAUGGA 
                 886 
               
               
                   
               
               
                 54790_11_96 
                 TET2 
                 EXON 
                 + 
                 chr4: 105275690-105275710 
                 UGAGAUGGAUGGCCACUUCA 
                 887 
               
               
                   
               
               
                 54790_11_99 
                 TET2 
                 EXON 
                 + 
                 chr4: 105275691-105275711 
                 GAGAUGGAUGGCCACUUCAU 
                 888 
               
               
                   
               
               
                 54790_11_104 
                 TET2 
                 EXON 
                 + 
                 chr4: 105275735-105275755 
                 CAAUCUGAGCAAUCCAAACA 
                 889 
               
               
                   
               
               
                 54790_11_105 
                 TET2 
                 EXON 
                 + 
                 chr4: 105275748-105275768 
                 CCAAACAUGGACUAUAAAAA 
                 890 
               
               
                   
               
               
                 54790_11_110 
                 TET2 
                 EXON 
                 + 
                 chr4: 105275798-105275818 
                 CCAUAACUACAGUGCAGCUC 
                 891 
               
               
                   
               
               
                 54790_11_111 
                 TET2 
                 EXON 
                 + 
                 chr4: 105275799-105275819 
                 CAUAACUACAGUGCAGCUCC 
                 892 
               
               
                   
               
               
                 54790_11_116 
                 TET2 
                 EXON 
                 + 
                 chr4: 105275843-105275863 
                 UGCCCUGCAUCUCCAAAACA 
                 893 
               
               
                   
               
               
                 54790_11_120 
                 TET2 
                 EXON 
                 + 
                 chr4: 105275874-105275894 
                 AUGCUUUCCCACACAGCUAA 
                 894 
               
               
                   
               
               
                 54790_11_121 
                 TET2 
                 EXON 
                 + 
                 chr4: 105275875-105275895 
                 UGCUUUCCCACACAGCUAAU 
                 895 
               
               
                   
               
               
                 54790_11_129 
                 TET2 
                 EXON 
                 + 
                 chr4: 105275928-105275948 
                 GAUAGAACUGCUUGUGUCCA 
                 896 
               
               
                   
               
               
                 54790_11_131 
                 TET2 
                 EXON 
                 + 
                 chr4: 105275931-105275951 
                 AGAACUGCUUGUGUCCAAGG 
                 897 
               
               
                   
               
               
                 54790_11_133 
                 TET2 
                 EXON 
                 + 
                 chr4: 105275958-105275978 
                 CACAAAUUAAGUGAUGCUAA 
                 898 
               
               
                   
               
               
                 54790_11_137 
                 TET2 
                 EXON 
                 + 
                 chr4: 105275963-105275983 
                 AUUAAGUGAUGCUAAUGGUC 
                 899 
               
               
                   
               
               
                 54790_11_139 
                 TET2 
                 EXON 
                 + 
                 chr4: 105275978-105275998 
                 UGGUCAGGAAAAGCAGCCAU 
                 900 
               
               
                   
               
               
                 54790_11_141 
                 TET2 
                 EXON 
                 + 
                 chr4: 105275990-105276010 
                 GCAGCCAUUGGCACUAGUCC 
                 901 
               
               
                   
               
               
                 54790_11_142 
                 TET2 
                 EXON 
                 + 
                 chr4: 105275991-105276011 
                 CAGCCAUUGGCACUAGUCCA 
                 902 
               
               
                   
               
               
                 54790_11_143 
                 TET2 
                 EXON 
                 + 
                 chr4: 105275996-105276016 
                 AUUGGCACUAGUCCAGGGUG 
                 903 
               
               
                   
               
               
                 54790_11_145 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276003-105276023 
                 CUAGUCCAGGGUGUGGCUUC 
                 904 
               
               
                   
               
               
                 54790_11_148 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276011-105276031 
                 GGGUGUGGCUUCUGGUGCAG 
                 905 
               
               
                   
               
               
                 54790_11_150 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276023-105276043 
                 UGGUGCAGAGGACAACGAUG 
                 906 
               
               
                   
               
               
                 54790_11_152 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276028-105276048 
                 CAGAGGACAACGAUGAGGUC 
                 907 
               
               
                   
               
               
                 54790_11_156 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276053-105276073 
                 AGACAGCGAGCAGAGCUUUC 
                 908 
               
               
                   
               
               
                 54790_11_158 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276066-105276086 
                 AGCUUUCUGGAUCCUGACAU 
                 909 
               
               
                   
               
               
                 54790_11_160 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276067-105276087 
                 GCUUUCUGGAUCCUGACAUU 
                 910 
               
               
                   
               
               
                 54790_11_162 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276068-105276088 
                 CUUUCUGGAUCCUGACAUUG 
                 911 
               
               
                   
               
               
                 54790_11_165 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276069-105276089 
                 UUUCUGGAUCCUGACAUUGG 
                 912 
               
               
                   
               
               
                 54790_11_168 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276074-105276094 
                 GGAUCCUGACAUUGGGGGAG 
                 913 
               
               
                   
               
               
                 54790_11_169 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276080-105276100 
                 UGACAUUGGGGGAGUGGCCG 
                 914 
               
               
                   
               
               
                 54790_11_172 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276093-105276113 
                 GUGGCCGUGGCUCCAACUCA 
                 915 
               
               
                   
               
               
                 54790_11_173 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276094-105276114 
                 UGGCCGUGGCUCCAACUCAU 
                 916 
               
               
                   
               
               
                 54790_11_182 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276160-105276180 
                 CCCCUUUAAAGAAUCCCAAU 
                 917 
               
               
                   
               
               
                 54790_11_186 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276175-105276195 
                 CCAAUAGGAAUCACCCCACC 
                 918 
               
               
                   
               
               
                 54790_11_193 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276225-105276245 
                 AGCAUGAAUGAGCCAAAACA 
                 919 
               
               
                   
               
               
                 54790_11_194 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276230-105276250 
                 GAAUGAGCCAAAACAUGGCU 
                 920 
               
               
                   
               
               
                 54790_11_196 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276238-105276258 
                 CAAAACAUGGCUUGGCUCUU 
                 921 
               
               
                   
               
               
                 54790_11_199 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276239-105276259 
                 AAAACAUGGCUUGGCUCUUU 
                 922 
               
               
                   
               
               
                 54790_11_200 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276251-105276271 
                 GGCUCUUUGGGAAGCCAAAA 
                 923 
               
               
                   
               
               
                 54790_11_210 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276275-105276295 
                 UGAAAAAGCCCGUGAGAAAG 
                 924 
               
               
                   
               
               
                 54790_11_214 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276294-105276314 
                 GAGGAAGAGUGUGAAAAGUA 
                 925 
               
               
                   
               
               
                 54790_11_217 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276324-105276344 
                 UAUGUGCCUCAGAAAUCCCA 
                 926 
               
               
                   
               
               
                 54790_11_221 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276340-105276360 
                 CCCAUGGCAAAAAAGUGAAA 
                 927 
               
               
                   
               
               
                 54790_11_223 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276341-105276361 
                 CCAUGGCAAAAAAGUGAAAC 
                 928 
               
               
                   
               
               
                 54790_11_231 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276409-105276429 
                 UCAUCAAGUCUCUUGCCGAA 
                 929 
               
               
                   
               
               
                 54790_11_236 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276466-105276486 
                 CAUCUCCAUAUGCCUUCACU 
                 930 
               
               
                   
               
               
                 54790_11_237 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276467-105276487 
                 AUCUCCAUAUGCCUUCACUC 
                 931 
               
               
                   
               
               
                 54790_11_239 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276474-105276494 
                 UAUGCCUUCACUCGGGUCAC 
                 932 
               
               
                   
               
               
                 54790_11_240 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276475-105276495 
                 AUGCCUUCACUCGGGUCACA 
                 933 
               
               
                   
               
               
                 54790_11_243 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276515-105276535 
                 AUGAUAUCACCCCCUUUUGU 
                 934 
               
               
                   
               
               
                 54790_11_252 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276573-105276593 
                 GUAGUAUAGUUCUCAUGACG 
                 935 
               
               
                   
               
               
                 54790_11_253 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276574-105276594 
                 UAGUAUAGUUCUCAUGACGU 
                 936 
               
               
                   
               
               
                 54790_11_256 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276580-105276600 
                 AGUUCUCAUGACGUGGGCAG 
                 937 
               
               
                   
               
               
                 54790_11_258 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276581-105276601 
                 GUUCUCAUGACGUGGGCAGU 
                 938 
               
               
                   
               
               
                 54790_11_259 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276582-105276602 
                 UUCUCAUGACGUGGGCAGUG 
                 939 
               
               
                   
               
               
                 54790_11_262 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276587-105276607 
                 AUGACGUGGGCAGUGGGGAA 
                 940 
               
               
                   
               
               
                 54790_11_263 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276611-105276631 
                 CACAGUAUUCAUGACAAAUG 
                 941 
               
               
                   
               
               
                 54790_11_265 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276614-105276634 
                 AGUAUUCAUGACAAAUGUGG 
                 942 
               
               
                   
               
               
                 54790_11_267 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276615-105276635 
                 GUAUUCAUGACAAAUGUGGU 
                 943 
               
               
                   
               
               
                 54790_11_271 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276646-105276666 
                 CAGCUCACCAGCAACAAAAG 
                 944 
               
               
                   
               
               
                 54790_11_273 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276677-105276697 
                 CCAUAGCACUUAAUUUUCAC 
                 945 
               
               
                   
               
               
                 54790_11_275 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276688-105276708 
                 AAUUUUCACUGGCUCCCAAG 
                 946 
               
               
                   
               
               
                 54790_11_280 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276698-105276718 
                 GGCUCCCAAGUGGUCACAGA 
                 947 
               
               
                   
               
               
                 54790_11_283 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276706-105276726 
                 AGUGGUCACAGAUGGCAUCU 
                 948 
               
               
                   
               
               
                 54790_11_285 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276738-105276758 
                 AAGCAUUCUAUGCAAAAAGA 
                 949 
               
               
                   
               
               
                 54790_11_288 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276741-105276761 
                 CAUUCUAUGCAAAAAGAAGG 
                 950 
               
               
                   
               
               
                 54790_11_289 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276742-105276762 
                 AUUCUAUGCAAAAAGAAGGU 
                 951 
               
               
                   
               
               
                 54790_11_291 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276743-105276763 
                 UUCUAUGCAAAAAGAAGGUG 
                 952 
               
               
                   
               
               
                 54790_11_297 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276780-105276800 
                 CAAUUUACAUUUUUAAACAC 
                 953 
               
               
                   
               
               
                 54790_11_302 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276792-105276812 
                 UUAAACACUGGUUCUAUUAU 
                 954 
               
               
                   
               
               
                 54790_11_316 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276885-105276905 
                 AUAUCAAGUUUGCAUAGUCA 
                 955 
               
               
                   
               
               
                 54790_11_321 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276925-105276945 
                 UACUGUAGUAUUACAGUGAC 
                 956 
               
               
                   
               
               
                 54790_11_323 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276945-105276965 
                 AGGAAUCUUAAAAUACCAUC 
                 957 
               
               
                   
               
               
                 54790_11_329 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276975-105276995 
                 UAUAUGAUGUACUGAAAUAC 
                 958 
               
               
                   
               
               
                 54790_11_330 
                 TET2 
                 EXON 
                 + 
                 chr4: 105276983-105277003 
                 GUACUGAAAUACUGGAAUUA 
                 959 
               
               
                   
               
               
                 54790_11_344 
                 TET2 
                 EXON 
                 + 
                 chr4: 105277042-105277062 
                 UUAUUUAUCAAAAUAGCUAC 
                 960 
               
               
                   
               
               
                 54790_11_352 
                 TET2 
                 EXON 
                 + 
                 chr4: 105277058-105277078 
                 CUACAGGAAACAUGAAUAGC 
                 961 
               
               
                   
               
               
                 54790_11_356 
                 TET2 
                 EXON 
                 + 
                 chr4: 105277078-105277098 
                 AGGAAAACACUGAAUUUGUU 
                 962 
               
               
                   
               
               
                 54790_11_359 
                 TET2 
                 EXON 
                 + 
                 chr4: 105277094-105277114 
                 UGUUUGGAUGUUCUAAGAAA 
                 963 
               
               
                   
               
               
                 54790_11_367 
                 TET2 
                 EXON 
                 + 
                 chr4: 105277108-105277128 
                 AAGAAAUGGUGCUAAGAAAA 
                 964 
               
               
                   
               
               
                 54790_11_377 
                 TET2 
                 EXON 
                 + 
                 chr4: 105277187-105277207 
                 CUCCAGUGCCCUUGAAUAAU 
                 965 
               
               
                   
               
               
                 54790_11_378 
                 TET2 
                 EXON 
                 + 
                 chr4: 105277188-105277208 
                 UCCAGUGCCCUUGAAUAAUA 
                 966 
               
               
                   
               
               
                 54790_11_379 
                 TET2 
                 EXON 
                 + 
                 chr4: 105277189-105277209 
                 CCAGUGCCCUUGAAUAAUAG 
                 967 
               
               
                   
               
               
                 54790_11_393 
                 TET2 
                 EXON 
                 + 
                 chr4: 105277255-105277275 
                 CAAGCUUAGUUUUUAAAAUG 
                 968 
               
               
                   
               
               
                 54790_11_395 
                 TET2 
                 EXON 
                 + 
                 chr4: 105277267-105277287 
                 UUAAAAUGUGGACAUUUUAA 
                 969 
               
               
                   
               
               
                 54790_11_401 
                 TET2 
                 EXON 
                 + 
                 chr4: 105277274-105277294 
                 GUGGACAUUUUAAAGGCCUC 
                 970 
               
               
                   
               
               
                 54790_11_410 
                 TET2 
                 EXON 
                 + 
                 chr4: 105277304-105277324 
                 UCAUCCAGUGAAGUCCUUGU 
                 971 
               
               
                   
               
               
                 54790_11_419 
                 TET2 
                 EXON 
                 + 
                 chr4: 105277438-105277458 
                 UGACAACUUGAACAAUGCUA 
                 972 
               
               
                   
               
               
                 54790_11_437 
                 TET2 
                 EXON 
                 + 
                 chr4: 105277501-105277521 
                 AUGCAAAGUUGAUUUUUUUA 
                 973 
               
               
                   
               
               
                 54790_11_465 
                 TET2 
                 EXON 
                 + 
                 chr4: 105277599-105277619 
                 ACAGCCAGUUAAAUCCACCA 
                 974 
               
               
                   
               
               
                 54790_11_466 
                 TET2 
                 EXON 
                 + 
                 chr4: 105277600-105277620 
                 CAGCCAGUUAAAUCCACCAU 
                 975 
               
               
                   
               
               
                 54790_11_467 
                 TET2 
                 EXON 
                 + 
                 chr4: 105277601-105277621 
                 AGCCAGUUAAAUCCACCAUG 
                 976 
               
               
                   
               
               
                 54790_11_469 
                 TET2 
                 EXON 
                 + 
                 chr4: 105277609-105277629 
                 AAAUCCACCAUGGGGCUUAC 
                 977 
               
               
                   
               
               
                 54790_11_472 
                 TET2 
                 EXON 
                 + 
                 chr4: 105277617-105277637 
                 CAUGGGGCUUACUGGAUUCA 
                 978 
               
               
                   
               
               
                 54790_11_474 
                 TET2 
                 EXON 
                 + 
                 chr4: 105277618-105277638 
                 AUGGGGCUUACUGGAUUCAA 
                 979 
               
               
                   
               
               
                 54790_11_478 
                 TET2 
                 EXON 
                 + 
                 chr4: 105277649-105277669 
                 AGUCCACAAAACAUGUUUUC 
                 980 
               
               
                   
               
               
                 54790_11_492 
                 TET2 
                 EXON 
                 + 
                 chr4: 105277753-105277773 
                 AAGAAUUUUCUAUUAACUGC 
                 981 
               
               
                   
               
               
                 54790_11_503 
                 TET2 
                 EXON 
                 + 
                 chr4: 105277818-105277838 
                 CUGAAGCCUAUGCUAUUUUA 
                 982 
               
               
                   
               
               
                 54790_11_504 
                 TET2 
                 EXON 
                 + 
                 chr4: 105277826-105277846 
                 UAUGCUAUUUUAUGGAUCAU 
                 983 
               
               
                   
               
               
                 54790_11_511 
                 TET2 
                 EXON 
                 + 
                 chr4: 105277846-105277866 
                 AGGCUCUUCAGAGAACUGAA 
                 984 
               
               
                   
               
               
                 54790_11_524 
                 TET2 
                 EXON 
                 + 
                 chr4: 105277924-105277944 
                 UAAGUGUCCUCUUUAACAAG 
                 985 
               
               
                   
               
               
                 54790_11_532 
                 TET2 
                 EXON 
                 + 
                 chr4: 105277963-105277983 
                 CCUGCAUAAGAUGAAUAAAC 
                 986 
               
               
                   
               
               
                 54790_11_533 
                 TET2 
                 EXON 
                 + 
                 chr4: 105277964-105277984 
                 CUGCAUAAGAUGAAUAAACA 
                 987 
               
               
                   
               
               
                 54790_11_539 
                 TET2 
                 EXON 
                 + 
                 chr4: 105278008-105278028 
                 AGUUAAAAAGAAACAAAAAC 
                 988 
               
               
                   
               
               
                 54790_11_541 
                 TET2 
                 EXON 
                 + 
                 chr4: 105278015-105278035 
                 AAGAAACAAAAACAGGCAGC 
                 989 
               
               
                   
               
               
                 54790_11_542 
                 TET2 
                 EXON 
                 + 
                 chr4: 105278025-105278045 
                 AACAGGCAGCUGGUUUGCUG 
                 990 
               
               
                   
               
               
                 54790_11_543 
                 TET2 
                 EXON 
                 + 
                 chr4: 105278028-105278048 
                 AGGCAGCUGGUUUGCUGUGG 
                 991 
               
               
                   
               
               
                 54790_11_574 
                 TET2 
                 EXON 
                 + 
                 chr4: 105278210-105278230 
                 AAGCAGAAUUCACAUCAUGA 
                 992 
               
               
                   
               
               
                 54790_11_587 
                 TET2 
                 EXON 
                 + 
                 chr4: 105278310-105278330 
                 CAUAUACCUCAACACUAGUU 
                 993 
               
               
                   
               
               
                 54790_11_589 
                 TET2 
                 EXON 
                 + 
                 chr4: 105278317-105278337 
                 CUCAACACUAGUUUGGCAAU 
                 994 
               
               
                   
               
               
                 54790_11_627 
                 TET2 
                 EXON 
                 + 
                 chr4: 105278467-105278487 
                 CCUUUUUGUUCUAAAAAUUC 
                 995 
               
               
                   
               
               
                 54790_11_628 
                 TET2 
                 EXON 
                 + 
                 chr4: 105278468-105278488 
                 CUUUUUGUUCUAAAAAUUCA 
                 996 
               
               
                   
               
               
                 54790_11_637 
                 TET2 
                 EXON 
                 + 
                 chr4: 105278532-105278552 
                 UGUUUAUGUAAAAUUGUUGU 
                 997 
               
               
                   
               
               
                 54790_11_643 
                 TET2 
                 EXON 
                 + 
                 chr4: 105278556-105278576 
                 UAAUAAAUAUAUUCUUUGUC 
                 998 
               
               
                   
               
               
                 54790_11_645 
                 TET2 
                 EXON 
                 + 
                 chr4: 105278557-105278577 
                 AAUAAAUAUAUUCUUUGUCA 
                 999 
               
               
                   
               
               
                 54790_11_664 
                 TET2 
                 EXON 
                 + 
                 chr4: 105278640-105278660 
                 AACUAAUUUUGUAAAUCUGU 
                 1000 
               
               
                   
               
               
                 54790_11_679 
                 TET2 
                 EXON 
                 + 
                 chr4: 105278680-105278700 
                 AAAAGCAUUUUAAAAGUUUG 
                 1001 
               
               
                   
               
               
                 54790_11_686 
                 TET2 
                 EXON 
                 + 
                 chr4: 105278704-105278724 
                 AUCUUUUGACUGUUUCAAGC 
                 1002 
               
               
                   
               
               
                 54790_11_700 
                 TET2 
                 EXON 
                 + 
                 chr4: 105278748-105278768 
                 AGAAUGCACUGAGUUGAUAA 
                 1003 
               
               
                   
               
               
                 54790_11_701 
                 TET2 
                 EXON 
                 + 
                 chr4: 105278749-105278769 
                 GAAUGCACUGAGUUGAUAAA 
                 1004 
               
               
                   
               
               
                 54790_11_703 
                 TET2 
                 EXON 
                 + 
                 chr4: 105278762-105278782 
                 UGAUAAAGGGAAAAAUUGUA 
                 1005 
               
               
                   
               
               
                 54790_11_707 
                 TET2 
                 EXON 
                 + 
                 chr4: 105278766-105278786 
                 AAAGGGAAAAAUUGUAAGGC 
                 1006 
               
               
                   
               
               
                 54790_11_708 
                 TET2 
                 EXON 
                 + 
                 chr4: 105278773-105278793 
                 AAAAUUGUAAGGCAGGAGUU 
                 1007 
               
               
                   
               
               
                 54790_11_710 
                 TET2 
                 EXON 
                 + 
                 chr4: 105278780-105278800 
                 UAAGGCAGGAGUUUGGCAAG 
                 1008 
               
               
                   
               
               
                 54790_11_711 
                 TET2 
                 EXON 
                 + 
                 chr4: 105278787-105278807 
                 GGAGUUUGGCAAGUGGCUGU 
                 1009 
               
               
                   
               
               
                 54790_11_721 
                 TET2 
                 EXON 
                 + 
                 chr4: 105278846-105278866 
                 UUUGAUCCUGUAAUCACUGA 
                 1010 
               
               
                   
               
               
                 54790_11_728 
                 TET2 
                 EXON 
                 + 
                 chr4: 105278862-105278882 
                 CUGAAGGUACAUACUCCAUG 
                 1011 
               
               
                   
               
               
                 54790_11_729 
                 TET2 
                 EXON 
                 + 
                 chr4: 105278878-105278898 
                 CAUGUGGACUUCCCUUAAAC 
                 1012 
               
               
                   
               
               
                 54790_11_731 
                 TET2 
                 EXON 
                 + 
                 chr4: 105278892-105278912 
                 UUAAACAGGCAAACACCUAC 
                 1013 
               
               
                   
               
               
                 54790_11_733 
                 TET2 
                 EXON 
                 + 
                 chr4: 105278897-105278917 
                 CAGGCAAACACCUACAGGUA 
                 1014 
               
               
                   
               
               
                 54790_11_734 
                 TET2 
                 EXON 
                 + 
                 chr4: 105278927-105278947 
                 CAGAUUGUACAAUUACAUUU 
                 1015 
               
               
                   
               
               
                 54790_11_748 
                 TET2 
                 EXON 
                 + 
                 chr4: 105278978-105278998 
                 UAAAAUAAAUUCUUAAUCAG 
                 1016 
               
               
                   
               
               
                 54790_11_751 
                 TET2 
                 EXON 
                 + 
                 chr4: 105278981-105279001 
                 AAUAAAUUCUUAAUCAGAGG 
                 1017 
               
               
                   
               
               
                 54790_11_753 
                 TET2 
                 EXON 
                 + 
                 chr4: 105278988-105279008 
                 UCUUAAUCAGAGGAGGCCUU 
                 1018 
               
               
                   
               
               
                 54790_11_754 
                 TET2 
                 EXON 
                 + 
                 chr4: 105278989-105279009 
                 CUUAAUCAGAGGAGGCCUUU 
                 1019 
               
               
                   
               
               
                 54790_11_757 
                 TET2 
                 EXON 
                 + 
                 chr4: 105278998-105279018 
                 AGGAGGCCUUUGGGUUUUAU 
                 1020 
               
               
                   
               
               
                 54790_11_762 
                 TET2 
                 EXON 
                 + 
                 chr4: 105279017-105279037 
                 UUGGUCAAAUCUUUGUAAGC 
                 1021 
               
               
                   
               
               
                 54790_11_772 
                 TET2 
                 EXON 
                 + 
                 chr4: 105279052-105279072 
                 UAAAAAAUUUCUUGAAUUUG 
                 1022 
               
               
                   
               
               
                 54790_11_799 
                 TET2 
                 EXON 
                 + 
                 chr4: 105279173-105279193 
                 UUUGAUUACUACAUGUGCAU 
                 1023 
               
               
                   
               
               
                 54790_11_813 
                 TET2 
                 EXON 
                 + 
                 chr4: 105279240-105279260 
                 ACUGUCAUUUGUUAAACUGC 
                 1024 
               
               
                   
               
               
                 54790_11_818 
                 TET2 
                 EXON 
                 + 
                 chr4: 105279254-105279274 
                 AACUGCUGGCCAACAAGAAC 
                 1025 
               
               
                   
               
               
                 54790_11_822 
                 TET2 
                 EXON 
                 + 
                 chr4: 105279267-105279287 
                 CAAGAACAGGAAGUAUAGUU 
                 1026 
               
               
                   
               
               
                 54790_11_825 
                 TET2 
                 EXON 
                 + 
                 chr4: 105279268-105279288 
                 AAGAACAGGAAGUAUAGUUU 
                 1027 
               
               
                   
               
               
                 54790_11_827 
                 TET2 
                 EXON 
                 + 
                 chr4: 105279269-105279289 
                 AGAACAGGAAGUAUAGUUUG 
                 1028 
               
               
                   
               
               
                 54790_11_828 
                 TET2 
                 EXON 
                 + 
                 chr4: 105279270-105279290 
                 GAACAGGAAGUAUAGUUUGG 
                 1029 
               
               
                   
               
               
                 54790_11_829 
                 TET2 
                 EXON 
                 + 
                 chr4: 105279271-105279291 
                 AACAGGAAGUAUAGUUUGGG 
                 1030 
               
               
                   
               
               
                 54790_11_832 
                 TET2 
                 EXON 
                 + 
                 chr4: 105279275-105279295 
                 GGAAGUAUAGUUUGGGGGGU 
                 1031 
               
               
                   
               
               
                 54790_11_833 
                 TET2 
                 EXON 
                 + 
                 chr4: 105279276-105279296 
                 GAAGUAUAGUUUGGGGGGUU 
                 1032 
               
               
                   
               
               
                 54790_11_836 
                 TET2 
                 EXON 
                 + 
                 chr4: 105279277-105279297 
                 AAGUAUAGUUUGGGGGGUUG 
                 1033 
               
               
                   
               
               
                 54790_11_841 
                 TET2 
                 EXON 
                 + 
                 chr4: 105279292-105279312 
                 GGUUGGGGAGAGUUUACAUA 
                 1034 
               
               
                   
               
               
                 54790_11_851 
                 TET2 
                 EXON 
                 + 
                 chr4: 105279311-105279331 
                 AAGGAAGAGAAGAAAUUGAG 
                 1035 
               
               
                   
               
               
                 54790_11_859 
                 TET2 
                 EXON 
                 + 
                 chr4: 105279373-105279393 
                 CCUGCCUCAGUUAGAAUGAA 
                 1036 
               
               
                   
               
               
                 54790_11_864 
                 TET2 
                 EXON 
                 + 
                 chr4: 105279402-105279422 
                 GAUCUACAAUUUGCUAAUAU 
                 1037 
               
               
                   
               
               
                 54790_11_865 
                 TET2 
                 EXON 
                 + 
                 chr4: 105279411-105279431 
                 UUUGCUAAUAUAGGAAUAUC 
                 1038 
               
               
                   
               
               
                 54790_11_871 
                 TET2 
                 EXON 
                 + 
                 chr4: 105279449-105279469 
                 UACUUGAAAAUGCUUCUGAG 
                 1039 
               
               
                   
               
               
                 54790_11_886 
                 TET2 
                 EXON 
                 + 
                 chr4: 105279524-105279544 
                 CAGUUCACUUCUGAAGCUAG 
                 1040 
               
               
                   
               
               
                 54790_11_890 
                 TET2 
                 EXON 
                 + 
                 chr4: 105279538-105279558 
                 AGCUAGUGGUUAACUUGUGU 
                 1041 
               
               
                   
               
               
                 54790_11_912 
                 TET2 
                 EXON 
                 + 
                 chr4: 105279632-105279652 
                 UUUCAUUUUCAUGAGAUGUU 
                 1042 
               
               
                   
               
               
                 54790_11_920 
                 TET2 
                 EXON 
                 + 
                 chr4: 105279648-105279668 
                 UGUUUGGUUUAUAAGAUCUG 
                 1043 
               
               
                   
               
               
                 54790_11_921 
                 TET2 
                 EXON 
                 + 
                 chr4: 105279652-105279672 
                 UGGUUUAUAAGAUCUGAGGA 
                 1044 
               
               
                   
               
               
                 54790_11_928 
                 TET2 
                 EXON 
                 + 
                 chr4: 105279691-105279711 
                 UAUUGUAAUGUUAUGAAUGC 
                 1045 
               
               
                   
               
               
                 54790_11_954 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275038-105275058 
                 UCGCAAAAGUUCUGUGGACA 
                 1046 
               
               
                   
               
               
                 54790_11_955 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275039-105275059 
                 GUCGCAAAAGUUCUGUGGAC 
                 1047 
               
               
                   
               
               
                 54790_11_957 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275044-105275064 
                 ACAAAGUCGCAAAAGUUCUG 
                 1048 
               
               
                   
               
               
                 54790_11_960 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275165-105275185 
                 AGUUGACAGACUCUGUCUGA 
                 1049 
               
               
                   
               
               
                 54790_11_961 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275166-105275186 
                 GAGUUGACAGACUCUGUCUG 
                 1050 
               
               
                   
               
               
                 54790_11_970 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275206-105275226 
                 CCGUCUCAUGUAUGGAUUGG 
                 1051 
               
               
                   
               
               
                 54790_11_972 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275209-105275229 
                 GGGCCGUCUCAUGUAUGGAU 
                 1052 
               
               
                   
               
               
                 54790_11_973 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275214-105275234 
                 GGAUUGGGCCGUCUCAUGUA 
                 1053 
               
               
                   
               
               
                 54790_11_977 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275229-105275249 
                 GGAUAAGGACUAACUGGAUU 
                 1054 
               
               
                   
               
               
                 54790_11_978 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275230-105275250 
                 UGGAUAAGGACUAACUGGAU 
                 1055 
               
               
                   
               
               
                 54790_11_980 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275235-105275255 
                 GAGUUUGGAUAAGGACUAAC 
                 1056 
               
               
                   
               
               
                 54790_11_982 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275244-105275264 
                 GUGUGUGAAGAGUUUGGAUA 
                 1057 
               
               
                   
               
               
                 54790_11_984 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275250-105275270 
                 UCUGAAGUGUGUGAAGAGUU 
                 1058 
               
               
                   
               
               
                 54790_11_991 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275287-105275307 
                 GGAAUAGAAGUUCAUAGGGC 
                 1059 
               
               
                   
               
               
                 54790_11_992 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275291-105275311 
                 AGGUGGAAUAGAAGUUCAUA 
                 1060 
               
               
                   
               
               
                 54790_11_993 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275292-105275312 
                 GAGGUGGAAUAGAAGUUCAU 
                 1061 
               
               
                   
               
               
                 54790_11_999 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275308-105275328 
                 ACCUGCAGCUUGAGAUGAGG 
                 1062 
               
               
                   
               
               
                 54790_11_1001 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275311-105275331 
                 UGAACCUGCAGCUUGAGAUG 
                 1063 
               
               
                   
               
               
                 54790_11_1012 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275352-105275372 
                 AGCCCAGGGUAAGGGUUCAU 
                 1064 
               
               
                   
               
               
                 54790_11_1013 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275353-105275373 
                 AAGCCCAGGGUAAGGGUUCA 
                 1065 
               
               
                   
               
               
                 54790_11_1017 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275360-105275380 
                 GAUUCAAAAGCCCAGGGUAA 
                 1066 
               
               
                   
               
               
                 54790_11_1018 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275361-105275381 
                 UGAUUCAAAAGCCCAGGGUA 
                 1067 
               
               
                   
               
               
                 54790_11_1021 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275366-105275386 
                 UAUUCUGAUUCAAAAGCCCA 
                 1068 
               
               
                   
               
               
                 54790_11_1022 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275367-105275387 
                 GUAUUCUGAUUCAAAAGCCC 
                 1069 
               
               
                   
               
               
                 54790_11_1026 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275389-105275409 
                 GCAUUGAUAUGAUGGAUAUU 
                 1070 
               
               
                   
               
               
                 54790_11_1027 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275390-105275410 
                 UGCAUUGAUAUGAUGGAUAU 
                 1071 
               
               
                   
               
               
                 54790_11_1031 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275397-105275417 
                 UUUCCAUUGCAUUGAUAUGA 
                 1072 
               
               
                   
               
               
                 54790_11_1034 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275420-105275440 
                 GGGAGCAGUUGUCCACUGAU 
                 1073 
               
               
                   
               
               
                 54790_11_1035 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275440-105275460 
                 AGAAUAGGAACCCAGAUAUG 
                 1074 
               
               
                   
               
               
                 54790_11_1037 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275441-105275461 
                 GAGAAUAGGAACCCAGAUAU 
                 1075 
               
               
                   
               
               
                 54790_11_1040 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275442-105275462 
                 GGAGAAUAGGAACCCAGAUA 
                 1076 
               
               
                   
               
               
                 54790_11_1042 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275455-105275475 
                 CGGCUGAGACUGGGGAGAAU 
                 1077 
               
               
                   
               
               
                 54790_11_1046 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275463-105275483 
                 AGAUCCAUCGGCUGAGACUG 
                 1078 
               
               
                   
               
               
                 54790_11_1049 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275464-105275484 
                 CAGAUCCAUCGGCUGAGACU 
                 1079 
               
               
                   
               
               
                 54790_11_1050 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275465-105275485 
                 ACAGAUCCAUCGGCUGAGAC 
                 1080 
               
               
                   
               
               
                 54790_11_1055 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275475-105275495 
                 GGAUACCUAUACAGAUCCAU 
                 1081 
               
               
                   
               
               
                 54790_11_1058 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275496-105275516 
                 UUAGACAGAGGGUCUUGGCU 
                 1082 
               
               
                   
               
               
                 54790_11_1060 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275501-105275521 
                 UGAGCUUAGACAGAGGGUCU 
                 1083 
               
               
                   
               
               
                 54790_11_1061 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275507-105275527 
                 GUAGACUGAGCUUAGACAGA 
                 1084 
               
               
                   
               
               
                 54790_11_1062 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275508-105275528 
                 GGUAGACUGAGCUUAGACAG 
                 1085 
               
               
                   
               
               
                 54790_11_1067 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275529-105275549 
                 UGGUAAAGUGUAUGGAUGGG 
                 1086 
               
               
                   
               
               
                 54790_11_1068 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275532-105275552 
                 GGCUGGUAAAGUGUAUGGAU 
                 1087 
               
               
                   
               
               
                 54790_11_1069 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275533-105275553 
                 UGGCUGGUAAAGUGUAUGGA 
                 1088 
               
               
                   
               
               
                 54790_11_1072 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275537-105275557 
                 ACCUUGGCUGGUAAAGUGUA 
                 1089 
               
               
                   
               
               
                 54790_11_1075 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275549-105275569 
                 GGCUAUUUCCAAACCUUGGC 
                 1090 
               
               
                   
               
               
                 54790_11_1076 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275553-105275573 
                 CUCUGGCUAUUUCCAAACCU 
                 1091 
               
               
                   
               
               
                 54790_11_1079 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275570-105275590 
                 AGUAUUUAGAUGUAAAACUC 
                 1092 
               
               
                   
               
               
                 54790_11_1085 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275606-105275626 
                 AACCAUCUCCCUGCAUAUUU 
                 1093 
               
               
                   
               
               
                 54790_11_1089 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275641-105275661 
                 AUGAUGUACAUUUGGUCUAA 
                 1094 
               
               
                   
               
               
                 54790_11_1092 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275649-105275669 
                 UUCCCUACAUGAUGUACAUU 
                 1095 
               
               
                   
               
               
                 54790_11_1093 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275676-105275696 
                 AUCUCAUGAGUGGGAUAAGG 
                 1096 
               
               
                   
               
               
                 54790_11_1095 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275679-105275699 
                 UCCAUCUCAUGAGUGGGAUA 
                 1097 
               
               
                   
               
               
                 54790_11_1097 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275685-105275705 
                 UGGCCAUCCAUCUCAUGAGU 
                 1098 
               
               
                   
               
               
                 54790_11_1098 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275686-105275706 
                 GUGGCCAUCCAUCUCAUGAG 
                 1099 
               
               
                   
               
               
                 54790_11_1102 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275705-105275725 
                 UAGAGGUGGCUCCCAUGAAG 
                 1100 
               
               
                   
               
               
                 54790_11_1105 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275719-105275739 
                 AUUGGGUGGUAAUCUAGAGG 
                 1101 
               
               
                   
               
               
                 54790_11_1107 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275722-105275742 
                 CAGAUUGGGUGGUAAUCUAG 
                 1102 
               
               
                   
               
               
                 54790_11_1111 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275733-105275753 
                 UUUGGAUUGCUCAGAUUGGG 
                 1103 
               
               
                   
               
               
                 54790_11_1112 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275736-105275756 
                 AUGUUUGGAUUGCUCAGAUU 
                 1104 
               
               
                   
               
               
                 54790_11_1113 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275737-105275757 
                 CAUGUUUGGAUUGCUCAGAU 
                 1105 
               
               
                   
               
               
                 54790_11_1120 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275751-105275771 
                 CCAUUUUUAUAGUCCAUGUU 
                 1106 
               
               
                   
               
               
                 54790_11_1125 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275787-105275807 
                 UAGUUAUGGAUUAUGUGAGA 
                 1107 
               
               
                   
               
               
                 54790_11_1129 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275801-105275821 
                 CCGGAGCUGCACUGUAGUUA 
                 1108 
               
               
                   
               
               
                 54790_11_1133 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275820-105275840 
                 AGAGAGCUGUUGAACAUGCC 
                 1109 
               
               
                   
               
               
                 54790_11_1144 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275848-105275868 
                 CUCCUUGUUUUGGAGAUGCA 
                 1110 
               
               
                   
               
               
                 54790_11_1145 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275849-105275869 
                 UCUCCUUGUUUUGGAGAUGC 
                 1111 
               
               
                   
               
               
                 54790_11_1148 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275858-105275878 
                 GCAUGUCAUUCUCCUUGUUU 
                 1112 
               
               
                   
               
               
                 54790_11_1154 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275884-105275904 
                 UGAUAACCCAUUAGCUGUGU 
                 1113 
               
               
                   
               
               
                 54790_11_1155 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275885-105275905 
                 UUGAUAACCCAUUAGCUGUG 
                 1114 
               
               
                   
               
               
                 54790_11_1161 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275916-105275936 
                 GUUCUAUCAUGGUUAAGAGC 
                 1115 
               
               
                   
               
               
                 54790_11_1165 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275927-105275947 
                 GGACACAAGCAGUUCUAUCA 
                 1116 
               
               
                   
               
               
                 54790_11_1169 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275948-105275968 
                 UUAAUUUGUGUAAGCCUCCU 
                 1117 
               
               
                   
               
               
                 54790_11_1175 
                 TET2 
                 EXON 
                 − 
                 chr4: 105275997-105276017 
                 ACACCCUGGACUAGUGCCAA 
                 1118 
               
               
                   
               
               
                 54790_11_1176 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276011-105276031 
                 CUGCACCAGAAGCCACACCC 
                 1119 
               
               
                   
               
               
                 54790_11_1182 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276081-105276101 
                 ACGGCCACUCCCCCAAUGUC 
                 1120 
               
               
                   
               
               
                 54790_11_1186 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276100-105276120 
                 UGACCCAUGAGUUGGAGCCA 
                 1121 
               
               
                   
               
               
                 54790_11_1188 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276108-105276128 
                 AUGAGAAUUGACCCAUGAGU 
                 1122 
               
               
                   
               
               
                 54790_11_1200 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276157-105276177 
                 GGGAUUCUUUAAAGGGGUUG 
                 1123 
               
               
                   
               
               
                 54790_11_1202 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276163-105276183 
                 CCUAUUGGGAUUCUUUAAAG 
                 1124 
               
               
                   
               
               
                 54790_11_1203 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276164-105276184 
                 UCCUAUUGGGAUUCUUUAAA 
                 1125 
               
               
                   
               
               
                 54790_11_1205 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276165-105276185 
                 UUCCUAUUGGGAUUCUUUAA 
                 1126 
               
               
                   
               
               
                 54790_11_1207 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276177-105276197 
                 CUGGUGGGGUGAUUCCUAUU 
                 1127 
               
               
                   
               
               
                 54790_11_1209 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276178-105276198 
                 CCUGGUGGGGUGAUUCCUAU 
                 1128 
               
               
                   
               
               
                 54790_11_1211 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276191-105276211 
                 AGACGAGGGAGAUCCUGGUG 
                 1129 
               
               
                   
               
               
                 54790_11_1212 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276192-105276212 
                 AAGACGAGGGAGAUCCUGGU 
                 1130 
               
               
                   
               
               
                 54790_11_1214 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276193-105276213 
                 AAAGACGAGGGAGAUCCUGG 
                 1131 
               
               
                   
               
               
                 54790_11_1216 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276196-105276216 
                 GUAAAAGACGAGGGAGAUCC 
                 1132 
               
               
                   
               
               
                 54790_11_1219 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276205-105276225 
                 CUUAUGCUGGUAAAAGACGA 
                 1133 
               
               
                   
               
               
                 54790_11_1221 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276206-105276226 
                 UCUUAUGCUGGUAAAAGACG 
                 1134 
               
               
                   
               
               
                 54790_11_1228 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276218-105276238 
                 GCUCAUUCAUGCUCUUAUGC 
                 1135 
               
               
                   
               
               
                 54790_11_1230 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276240-105276260 
                 CAAAGAGCCAAGCCAUGUUU 
                 1136 
               
               
                   
               
               
                 54790_11_1241 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276268-105276288 
                 ACGGGCUUUUUCAGCCAUUU 
                 1137 
               
               
                   
               
               
                 54790_11_1246 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276286-105276306 
                 ACACUCUUCCUCUUUCUCAC 
                 1138 
               
               
                   
               
               
                 54790_11_1247 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276287-105276307 
                 CACACUCUUCCUCUUUCUCA 
                 1139 
               
               
                   
               
               
                 54790_11_1251 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276320-105276340 
                 AUUUCUGAGGCACAUAGUCU 
                 1140 
               
               
                   
               
               
                 54790_11_1252 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276321-105276341 
                 GAUUUCUGAGGCACAUAGUC 
                 1141 
               
               
                   
               
               
                 54790_11_1260 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276333-105276353 
                 UUUUUGCCAUGGGAUUUCUG 
                 1142 
               
               
                   
               
               
                 54790_11_1263 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276343-105276363 
                 CCGUUUCACUUUUUUGCCAU 
                 1143 
               
               
                   
               
               
                 54790_11_1265 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276344-105276364 
                 CCCGUUUCACUUUUUUGCCA 
                 1144 
               
               
                   
               
               
                 54790_11_1269 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276369-105276389 
                 GAAGUUUCAUGUGGCUCAGC 
                 1145 
               
               
                   
               
               
                 54790_11_1270 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276378-105276398 
                 GUGGGCUCUGAAGUUUCAUG 
                 1146 
               
               
                   
               
               
                 54790_11_1273 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276396-105276416 
                 UUGAUGAAACGCAGGUAAGU 
                 1147 
               
               
                   
               
               
                 54790_11_1274 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276397-105276417 
                 CUUGAUGAAACGCAGGUAAG 
                 1148 
               
               
                   
               
               
                 54790_11_1277 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276404-105276424 
                 CAAGAGACUUGAUGAAACGC 
                 1149 
               
               
                   
               
               
                 54790_11_1281 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276427-105276447 
                 GGUCACGGACAUGGUCCUUU 
                 1150 
               
               
                   
               
               
                 54790_11_1282 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276436-105276456 
                 GGAGUCUGUGGUCACGGACA 
                 1151 
               
               
                   
               
               
                 54790_11_1284 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276442-105276462 
                 UACUGUGGAGUCUGUGGUCA 
                 1152 
               
               
                   
               
               
                 54790_11_1286 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276448-105276468 
                 UGUAGUUACUGUGGAGUCUG 
                 1153 
               
               
                   
               
               
                 54790_11_1288 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276457-105276477 
                 AUAUGGAGAUGUAGUUACUG 
                 1154 
               
               
                   
               
               
                 54790_11_1290 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276474-105276494 
                 GUGACCCGAGUGAAGGCAUA 
                 1155 
               
               
                   
               
               
                 54790_11_1294 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276481-105276501 
                 AGGCCCUGUGACCCGAGUGA 
                 1156 
               
               
                   
               
               
                 54790_11_1297 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276501-105276521 
                 UAUCAUAUAUAUCUGUUGUA 
                 1157 
               
               
                   
               
               
                 54790_11_1300 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276527-105276547 
                 GUGAGGUAACCAACAAAAGG 
                 1158 
               
               
                   
               
               
                 54790_11_1301 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276528-105276548 
                 AGUGAGGUAACCAACAAAAG 
                 1159 
               
               
                   
               
               
                 54790_11_1303 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276529-105276549 
                 AAGUGAGGUAACCAACAAAA 
                 1160 
               
               
                   
               
               
                 54790_11_1305 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276530-105276550 
                 CAAGUGAGGUAACCAACAAA 
                 1161 
               
               
                   
               
               
                 54790_11_1310 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276544-105276564 
                 GGUUGUGGUCUUUUCAAGUG 
                 1162 
               
               
                   
               
               
                 54790_11_1312 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276559-105276579 
                 UACUACUGACAGGUUGGUUG 
                 1163 
               
               
                   
               
               
                 54790_11_1313 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276565-105276585 
                 GAACUAUACUACUGACAGGU 
                 1164 
               
               
                   
               
               
                 54790_11_1314 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276569-105276589 
                 AUGAGAACUAUACUACUGAC 
                 1165 
               
               
                   
               
               
                 54790_11_1331 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276646-105276666 
                 CUUUUGUUGCUGGUGAGCUG 
                 1166 
               
               
                   
               
               
                 54790_11_1334 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276656-105276676 
                 AAGAUAACCUCUUUUGUUGC 
                 1167 
               
               
                   
               
               
                 54790_11_1336 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276680-105276700 
                 CCAGUGAAAAUUAAGUGCUA 
                 1168 
               
               
                   
               
               
                 54790_11_1339 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276705-105276725 
                 GAUGCCAUCUGUGACCACUU 
                 1169 
               
               
                   
               
               
                 54790_11_1344 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276706-105276726 
                 AGAUGCCAUCUGUGACCACU 
                 1170 
               
               
                   
               
               
                 54790_11_1354 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276738-105276758 
                 UCUUUUUGCAUAGAAUGCUU 
                 1171 
               
               
                   
               
               
                 54790_11_1363 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276780-105276800 
                 GUGUUUAAAAAUGUAAAUUG 
                 1172 
               
               
                   
               
               
                 54790_11_1370 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276841-105276861 
                 AGAGUUGUAAGCGGGGGGGG 
                 1173 
               
               
                   
               
               
                 54790_11_1371 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276842-105276862 
                 UAGAGUUGUAAGCGGGGGGG 
                 1174 
               
               
                   
               
               
                 54790_11_1374 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276843-105276863 
                 GUAGAGUUGUAAGCGGGGGG 
                 1175 
               
               
                   
               
               
                 54790_11_1376 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276844-105276864 
                 UGUAGAGUUGUAAGCGGGGG 
                 1176 
               
               
                   
               
               
                 54790_11_1378 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276845-105276865 
                 GUGUAGAGUUGUAAGCGGGG 
                 1177 
               
               
                   
               
               
                 54790_11_1379 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276846-105276866 
                 UGUGUAGAGUUGUAAGCGGG 
                 1178 
               
               
                   
               
               
                 54790_11_1382 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276847-105276867 
                 AUGUGUAGAGUUGUAAGCGG 
                 1179 
               
               
                   
               
               
                 54790_11_1383 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276848-105276868 
                 GAUGUGUAGAGUUGUAAGCG 
                 1180 
               
               
                   
               
               
                 54790_11_1386 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276849-105276869 
                 AGAUGUGUAGAGUUGUAAGC 
                 1181 
               
               
                   
               
               
                 54790_11_1388 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276850-105276870 
                 CAGAUGUGUAGAGUUGUAAG 
                 1182 
               
               
                   
               
               
                 54790_11_1394 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276876-105276896 
                 AAACUUGAUAUUAUUAAAAG 
                 1183 
               
               
                   
               
               
                 54790_11_1406 
                 TET2 
                 EXON 
                 − 
                 chr4: 105276963-105276983 
                 AUCAUAUAUUCAGCACCAGA 
                 1184 
               
               
                   
               
               
                 54790_11_1440 
                 TET2 
                 EXON 
                 − 
                 chr4: 105277160-105277180 
                 AUAGCAUCUUGAUGAUAUAA 
                 1185 
               
               
                   
               
               
                 54790_11_1444 
                 TET2 
                 EXON 
                 − 
                 chr4: 105277192-105277212 
                 CCCCUAUUAUUCAAGGGCAC 
                 1186 
               
               
                   
               
               
                 54790_11_1446 
                 TET2 
                 EXON 
                 − 
                 chr4: 105277198-105277218 
                 AAGGUACCCCUAUUAUUCAA 
                 1187 
               
               
                   
               
               
                 54790_11_1447 
                 TET2 
                 EXON 
                 − 
                 chr4: 105277199-105277219 
                 AAAGGUACCCCUAUUAUUCA 
                 1188 
               
               
                   
               
               
                 54790_11_1451 
                 TET2 
                 EXON 
                 − 
                 chr4: 105277217-105277237 
                 UGAUAAAAACUUGAAUGAAA 
                 1189 
               
               
                   
               
               
                 54790_11_1457 
                 TET2 
                 EXON 
                 − 
                 chr4: 105277246-105277266 
                 AACUAAGCUUGUGUAAGAAU 
                 1190 
               
               
                   
               
               
                 54790_11_1463 
                 TET2 
                 EXON 
                 − 
                 chr4: 105277293-105277313 
                 ACUGGAUGAGCAAAAUCCAG 
                 1191 
               
               
                   
               
               
                 54790_11_1469 
                 TET2 
                 EXON 
                 − 
                 chr4: 105277311-105277331 
                 UUGUCCUACAAGGACUUCAC 
                 1192 
               
               
                   
               
               
                 54790_11_1471 
                 TET2 
                 EXON 
                 − 
                 chr4: 105277321-105277341 
                 AUAUCGUUUAUUGUCCUACA 
                 1193 
               
               
                   
               
               
                 54790_11_1478 
                 TET2 
                 EXON 
                 − 
                 chr4: 105277432-105277452 
                 UGUUCAAGUUGUCAAAGCUU 
                 1194 
               
               
                   
               
               
                 54790_11_1501 
                 TET2 
                 EXON 
                 − 
                 chr4: 105277563-105277583 
                 AUUGCUCAUCAGCAGAUGCA 
                 1195 
               
               
                   
               
               
                 54790_11_1505 
                 TET2 
                 EXON 
                 − 
                 chr4: 105277591-105277611 
                 UUAACUGGCUGUGUUAAAAA 
                 1196 
               
               
                   
               
               
                 54790_11_1507 
                 TET2 
                 EXON 
                 − 
                 chr4: 105277606-105277626 
                 AGCCCCAUGGUGGAUUUAAC 
                 1197 
               
               
                   
               
               
                 54790_11_1509 
                 TET2 
                 EXON 
                 − 
                 chr4: 105277616-105277636 
                 GAAUCCAGUAAGCCCCAUGG 
                 1198 
               
               
                   
               
               
                 54790_11_1512 
                 TET2 
                 EXON 
                 − 
                 chr4: 105277619-105277639 
                 CUUGAAUCCAGUAAGCCCCA 
                 1199 
               
               
                   
               
               
                 54790_11_1517 
                 TET2 
                 EXON 
                 − 
                 chr4: 105277655-105277675 
                 GCACCAGAAAACAUGUUUUG 
                 1200 
               
               
                   
               
               
                 54790_11_1547 
                 TET2 
                 EXON 
                 − 
                 chr4: 105277827-105277847 
                 UAUGAUCCAUAAAAUAGCAU 
                 1201 
               
               
                   
               
               
                 54790_11_1553 
                 TET2 
                 EXON 
                 − 
                 chr4: 105277879-105277899 
                 GUACAUAAUUAUCAACACAA 
                 1202 
               
               
                   
               
               
                 54790_11_1558 
                 TET2 
                 EXON 
                 − 
                 chr4: 105277934-105277954 
                 GCUCAAUCCUCUUGUUAAAG 
                 1203 
               
               
                   
               
               
                 54790_11_1565 
                 TET2 
                 EXON 
                 − 
                 chr4: 105277966-105277986 
                 CCUGUUUAUUCAUCUUAUGC 
                 1204 
               
               
                   
               
               
                 54790_11_1574 
                 TET2 
                 EXON 
                 − 
                 chr4: 105277996-105278016 
                 UUUUAACUGACAGAUUCACA 
                 1205 
               
               
                   
               
               
                 54790_11_1613 
                 TET2 
                 EXON 
                 − 
                 chr4: 105278246-105278266 
                 CAUUAUGAUAUAUUUGUAGC 
                 1206 
               
               
                   
               
               
                 54790_11_1621 
                 TET2 
                 EXON 
                 − 
                 chr4: 105278304-105278324 
                 UGUUGAGGUAUAUGACAAGU 
                 1207 
               
               
                   
               
               
                 54790_11_1624 
                 TET2 
                 EXON 
                 − 
                 chr4: 105278319-105278339 
                 CUAUUGCCAAACUAGUGUUG 
                 1208 
               
               
                   
               
               
                 54790_11_1630 
                 TET2 
                 EXON 
                 − 
                 chr4: 105278373-105278393 
                 AAGGACUUGGAAAAAAAUGA 
                 1209 
               
               
                   
               
               
                 54790_11_1636 
                 TET2 
                 EXON 
                 − 
                 chr4: 105278386-105278406 
                 UAACAAUAAAAAAAAGGACU 
                 1210 
               
               
                   
               
               
                 54790_11_1643 
                 TET2 
                 EXON 
                 − 
                 chr4: 105278392-105278412 
                 UUUUUUUAACAAUAAAAAAA 
                 1211 
               
               
                   
               
               
                 54790_11_1647 
                 TET2 
                 EXON 
                 − 
                 chr4: 105278423-105278443 
                 AGAAAUCAAGUAUUGAAAAA 
                 1212 
               
               
                   
               
               
                 54790_11_1658 
                 TET2 
                 EXON 
                 − 
                 chr4: 105278470-105278490 
                 CCUGAAUUUUUAGAACAAAA 
                 1213 
               
               
                   
               
               
                 54790_11_1667 
                 TET2 
                 EXON 
                 − 
                 chr4: 105278513-105278533 
                 ACAGGUGACAUGUUGGCAUA 
                 1214 
               
               
                   
               
               
                 54790_11_1669 
                 TET2 
                 EXON 
                 − 
                 chr4: 105278514-105278534 
                 CACAGGUGACAUGUUGGCAU 
                 1215 
               
               
                   
               
               
                 54790_11_1674 
                 TET2 
                 EXON 
                 − 
                 chr4: 105278520-105278540 
                 CAUAAACACAGGUGACAUGU 
                 1216 
               
               
                   
               
               
                 54790_11_1675 
                 TET2 
                 EXON 
                 − 
                 chr4: 105278531-105278551 
                 CAACAAUUUUACAUAAACAC 
                 1217 
               
               
                   
               
               
                 54790_11_1682 
                 TET2 
                 EXON 
                 − 
                 chr4: 105278589-105278609 
                 AGAAGGGAUUCAAAAUAAAA 
                 1218 
               
               
                   
               
               
                 54790_11_1683 
                 TET2 
                 EXON 
                 − 
                 chr4: 105278590-105278610 
                 UAGAAGGGAUUCAAAAUAAA 
                 1219 
               
               
                   
               
               
                 54790_11_1685 
                 TET2 
                 EXON 
                 − 
                 chr4: 105278605-105278625 
                 CAUGUACAAGUAAAAUAGAA 
                 1220 
               
               
                   
               
               
                 54790_11_1687 
                 TET2 
                 EXON 
                 − 
                 chr4: 105278606-105278626 
                 ACAUGUACAAGUAAAAUAGA 
                 1221 
               
               
                   
               
               
                 54790_11_1733 
                 TET2 
                 EXON 
                 − 
                 chr4: 105278813-105278833 
                 GAGAGUUACAAGUAAGUCUC 
                 1222 
               
               
                   
               
               
                 54790_11_1739 
                 TET2 
                 EXON 
                 − 
                 chr4: 105278855-105278875 
                 UAUGUACCUUCAGUGAUUAC 
                 1223 
               
               
                   
               
               
                 54790_11_1746 
                 TET2 
                 EXON 
                 − 
                 chr4: 105278880-105278900 
                 CUGUUUAAGGGAAGUCCACA 
                 1224 
               
               
                   
               
               
                 54790_11_1749 
                 TET2 
                 EXON 
                 − 
                 chr4: 105278892-105278912 
                 GUAGGUGUUUGCCUGUUUAA 
                 1225 
               
               
                   
               
               
                 54790_11_1751 
                 TET2 
                 EXON 
                 − 
                 chr4: 105278893-105278913 
                 UGUAGGUGUUUGCCUGUUUA 
                 1226 
               
               
                   
               
               
                 54790_11_1754 
                 TET2 
                 EXON 
                 − 
                 chr4: 105278910-105278930 
                 CUGUUGCACACCAUACCUGU 
                 1227 
               
               
                   
               
               
                 54790_11_1758 
                 TET2 
                 EXON 
                 − 
                 chr4: 105278953-105278973 
                 UAGUAAGCAAAAAUGUAUUU 
                 1228 
               
               
                   
               
               
                 54790_11_1768 
                 TET2 
                 EXON 
                 − 
                 chr4: 105279007-105279027 
                 AUUUGACCAAUAAAACCCAA 
                 1229 
               
               
                   
               
               
                 54790_11_1784 
                 TET2 
                 EXON 
                 − 
                 chr4: 105279084-105279104 
                 UUUUGGAAAUGUUUGCAAAU 
                 1230 
               
               
                   
               
               
                 54790_11_1789 
                 TET2 
                 EXON 
                 − 
                 chr4: 105279101-105279121 
                 GUAAGCAAAGCAAACAUUUU 
                 1231 
               
               
                   
               
               
                 54790_11_1792 
                 TET2 
                 EXON 
                 − 
                 chr4: 105279127-105279147 
                 CAAAAAACAUUAAAAUCAUG 
                 1232 
               
               
                   
               
               
                 54790_11_1796 
                 TET2 
                 EXON 
                 − 
                 chr4: 105279154-105279174 
                 AUGUUUGGGGCUAGAUAUUA 
                 1233 
               
               
                   
               
               
                 54790_11_1797 
                 TET2 
                 EXON 
                 − 
                 chr4: 105279167-105279187 
                 AUGUAGUAAUCAAAUGUUUG 
                 1234 
               
               
                   
               
               
                 54790_11_1798 
                 TET2 
                 EXON 
                 − 
                 chr4: 105279168-105279188 
                 CAUGUAGUAAUCAAAUGUUU 
                 1235 
               
               
                   
               
               
                 54790_11_1800 
                 TET2 
                 EXON 
                 − 
                 chr4: 105279169-105279189 
                 ACAUGUAGUAAUCAAAUGUU 
                 1236 
               
               
                   
               
               
                 54790_11_1803 
                 TET2 
                 EXON 
                 − 
                 chr4: 105279212-105279232 
                 CAGAAAUCAAAUAUUAAGAA 
                 1237 
               
               
                   
               
               
                 54790_11_1809 
                 TET2 
                 EXON 
                 − 
                 chr4: 105279240-105279260 
                 GCAGUUUAACAAAUGACAGU 
                 1238 
               
               
                   
               
               
                 54790_11_1814 
                 TET2 
                 EXON 
                 − 
                 chr4: 105279266-105279286 
                 ACUAUACUUCCUGUUCUUGU 
                 1239 
               
               
                   
               
               
                 54790_11_1832 
                 TET2 
                 EXON 
                 − 
                 chr4: 105279376-105279396 
                 CCAUUCAUUCUAACUGAGGC 
                 1240 
               
               
                   
               
               
                 54790_11_1833 
                 TET2 
                 EXON 
                 − 
                 chr4: 105279380-105279400 
                 CUUUCCAUUCAUUCUAACUG 
                 1241 
               
               
                   
               
               
                 54790_11_1841 
                 TET2 
                 EXON 
                 − 
                 chr4: 105279449-105279469 
                 CUCAGAAGCAUUUUCAAGUA 
                 1242 
               
               
                   
               
               
                 54790_11_1877 
                 TET2 
                 EXON 
                 − 
                 chr4: 105279748-105279768 
                 AACACUCACAUAGCAUUAUC 
                 1243 
               
               
                   
               
            
           
         
       
     
     TALEN Gene Editing Systems 
     TALENs are produced artificially by fusing a TAL effector DNA binding domain to a DNA cleavage domain. Transcription activator-like effects (TALEs) can be engineered to bind any desired DNA sequence, including a portion of the HLA or TCR gene. By combining an engineered TALE with a DNA cleavage domain, a restriction enzyme can be produced which is specific to any desired DNA sequence, including a HLA or TCR sequence. These can then be introduced into a cell, wherein they can be used for genome editing. Boch (2011) Nature Biotech. 29: 135-6; and Boch et al. (2009) Science 326: 1509-12; Moscou et al. (2009) Science 326: 3501. 
     TALEs are proteins secreted by  Xanthomonas  bacteria. The DNA binding domain contains a repeated, highly conserved 33-34 amino acid sequence, with the exception of the 12th and 13th amino acids. These two positions are highly variable, showing a strong correlation with specific nucleotide recognition. They can thus be engineered to bind to a desired DNA sequence. 
     To produce a TALEN, a TALE protein is fused to a nuclease (N), which is, for example, a wild-type or mutated FokI endonuclease. Several mutations to FokI have been made for its use in TALENs; these, for example, improve cleavage specificity or activity. Cermak et al. (2011) Nucl. Acids Res. 39: e82; Miller et al. (2011) Nature Biotech. 29: 143-8; Hockemeyer et al. (2011) Nature Biotech. 29: 731-734; Wood et al. (2011) Science 333: 307; Doyon et al. (2010) Nature Methods 8: 74-79; Szczepek et al. (2007) Nature Biotech. 25: 786-793; and Guo et al. (2010) J. Mol. Biol. 200: 96. 
     The FokI domain functions as a dimer, requiring two constructs with unique DNA binding domains for sites in the target genome with proper orientation and spacing. Both the number of amino acid residues between the TALE DNA binding domain and the FokI cleavage domain and the number of bases between the two individual TALEN binding sites appear to be important parameters for achieving high levels of activity. Miller et al. (2011) Nature Biotech. 29: 143-8. 
     A Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, TALEN can be used inside a cell to produce a double-stranded break (DSB). A mutation can be introduced at the break site if the repair mechanisms improperly repair the break via non-homologous end joining. For example, improper repair may introduce a frame shift mutation. Alternatively, foreign DNA can be introduced into the cell along with the TALEN, e.g., DNA encoding a CAR, e.g., as described herein; depending on the sequences of the foreign DNA and chromosomal sequence, this process can be used to integrate the DNA encoding the CAR, e.g., as described herein, at or near the site targeted by the TALEN. As shown herein, in the examples, but without being bound by theory, such integration may lead to the expression of the CAR as well as disruption of the Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, gene. Such foreign DNA molecule is referred to herein as “template DNA.” In embodiments, the template DNA further comprises homology arms 5′ to, 3′ to, or both 5′ and 3′ to the nucleic acid of the template DNA which encodes the molecule or molecules of interest (e.g., which encodes a CAR described herein), wherein said homology arms are complementary to genomic DNA sequence flanking the target sequence. 
     TALENs specific to sequences in Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, can be constructed using any method known in the art, including various schemes using modular components. Zhang et al. (2011) Nature Biotech. 29: 149-53; Geibler et al. (2011) PLoS ONE 6: e19509; U.S. Pat. Nos. 8,420,782; 8,470,973, the contents of which are hereby incorporated by reference in their entirety. 
     Zinc Finger Nuclease to Inhibit Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2 
     “ZFN” or “Zinc Finger Nuclease” refer to a zinc finger nuclease, an artificial nuclease which can be used to modify, e.g., delete one or more nucleic acids of, a desired nucleic acid sequence, e.g., Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2. 
     Like a TALEN, a ZFN comprises a FokI nuclease domain (or derivative thereof) fused to a DNA-binding domain. In the case of a ZFN, the DNA-binding domain comprises one or more zinc fingers. Carroll et al. (2011) Genetics Society of America 188: 773-782; and Kim et al. (1996) Proc. Natl. Acad. Sci. USA 93: 1156-1160. 
     A zinc finger is a small protein structural motif stabilized by one or more zinc ions. A zinc finger can comprise, for example, Cys2His2, and can recognize an approximately 3-bp sequence. Various zinc fingers of known specificity can be combined to produce multi-finger polypeptides which recognize about 6, 9, 12, 15 or 18-bp sequences. Various selection and modular assembly techniques are available to generate zinc fingers (and combinations thereof) recognizing specific sequences, including phage display, yeast one-hybrid systems, bacterial one-hybrid and two-hybrid systems, and mammalian cells. 
     Like a TALEN, a ZFN must dimerize to cleave DNA. Thus, a pair of ZFNs are required to target non-palindromic DNA sites. The two individual ZFNs must bind opposite strands of the DNA with their nucleases properly spaced apart. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95: 10570-5. 
     Also like a TALEN, a ZFN can create a double-stranded break in the DNA, which can create a frame-shift mutation if improperly repaired, leading to a decrease in the expression and amount of Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, in a cell. ZFNs can also be used with homologous recombination to mutate the Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, gene, or to introduce nucleic acid encoding a CAR at a site at or near the targeted sequence. As discussed above, the nucleci acid encoding a CAR may be introduced as part of a template DNA. In embodiments, the template DNA further comprises homology arms 5′ to, 3′ to, or both 5′ and 3′ to the nucleic acid of the template DNA which encodes the molecule or molecules of interest (e.g., which encodes a CAR described herein), wherein said homology arms are complementary to genomic DNA sequence flanking the target sequence. 
     ZFNs specific to sequences in the Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, gene can be constructed using any method known in the art. See, e.g., Provasi (2011) Nature Med. 18: 807-815; Torikai (2013) Blood 122: 1341-1349; Cathomen et al. (2008) Mol. Ther. 16: 1200-7; and Guo et al. (2010) J. Mol. Biol. 400: 96; U.S. Patent Publication 2011/0158957; and U.S. Patent Publication 2012/0060230, the contents of which are hereby incorporated by reference in their entirety. In embodiments, The ZFN gene editing system may also comprise nucleic acid encoding one or more components of the ZFN gene editing system, e.g., a ZFN gene editing system targeted to Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2. 
     Without being bound by theory, it is believed that use of gene editing systems (e.g., CRISPR/Cas gene editing systems) which target Tet, e.g., Tet1, Tet2, and/or Tet3, e.g., Tet2, may allow one to inhibit one or more functions of Tet, e.g., Tet1, Tet2, and/or Tet3, e.g., Tet2, by, for example, causing an editing event which results in expression of a truncated Tet, e.g., Tet1, Tet2, and/or Tet3, e.g., Tet2. Again, without being bound by theory, such truncated Tet, e.g., Tet1, Tet2, and/or Tet3, e.g., Tet2 proteins may preserve one or more functions of the Tet, e.g., Tet1, Tet2, and/or Tet3, e.g., Tet2 (e.g., a scaffolding function), while inhibiting one or more other functions of the Tet, e.g., Tet1, Tet2, and/or Tet3, e.g., Tet2 (e.g., a catalytic function), and as such, may be preferable. Gene editing systems which target a late exon or intron of a Tet gene, e.g., Tet1, Tet2, and/or Tet3 gene, e.g., Tet2 gene, may be particularly preferred in this regard. In an aspect, the gene editing system Tet inhibitor, e.g., Tet1, Tet2, and/or Tet3 inhibitor, e.g., Tet2 inhibitor of the invention targets a late exon or intron of the tet gene. In an aspect, the gene editing system Tet inhibitor, e.g., Tet1, Tet2, and/or Tet3 inhibitor, e.g., Tet2 inhibitor of the invention targets an exon or intron downstream of exon 8. In an aspect, the gene editing system Tet inhibitor, e.g., Tet1, Tet2, and/or Tet3 inhibitor, e.g., Tet2 inhibitor, targets exon 8 or exon 9, e.g., exon 9, of the tet2 gene. 
     Without being bound by theory, it may also be preferable in other embodiments to target an early exon or intron of Tet gene, e.g., Tet1, Tet2, and/or Tet3 gene, e.g., Tet2 gene, for example, to introduce a premature stop codon in the targeted gene which results in no expression of the gene product, or expression of a completely non-functional gene product. Gene editing systems which target an early exon or intron of a Tet gene, e.g., Tet1, Tet2, and/or Tet3 gene, e.g., Tet2 gene, may be particularly preferred in this regard. In an aspect, the gene editing system Tet inhibitor, e.g., Tet1, Tet2, and/or Tet3 inhibitor, e.g., Tet2 inhibitor of the invention targets an early exon or intron of the tet gene. In an aspect, the gene editing system Tet inhibitor, e.g., Tet1, Tet2, and/or Tet3 inhibitor, e.g., Tet2 inhibitor of the invention targets an exon or intron upstream of exon 4. In embodiments, the gene editing system Tet inhibitor, e.g., Tet1, Tet2, and/or Tet3 inhibitor, e.g., Tet2 inhibitor, targets exon 1, exon 2, or exon 3, e.g., exon 3, of the tet2 gene. 
     Without being bound by theory, it may also be preferable in other embodiments to target a sequence of a Tet gene, e.g., Tet1, Tet2, and/or Tet3 gene, e.g., Tet2 gene, that is specific to one or more isoforms of the tet (e.g., tet2 gene) but does not affect one or more other isoforms of the tet (e.g., tet2). In embodiments, it may be preferable to specifically target isoforms of the tet (e.g., tet2) which contain a catalytic domain. 
     dsRNA, e.g., siRNA or shRNA, Inhibitors of Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2 
     According to the present invention, double stranded RNA (“dsRNA”), e.g., siRNA or shRNA can be used as Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, inhibitors. Also contemplated by the present invention are the uses of nucleic acid encoding said dsRNA Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, inhibitors. 
     In an embodiment, the Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, inhibitor is a nucleic acid, e.g., a dsRNA, e.g., a siRNA or shRNA specific for nucleic acid encoding Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, e.g., genomic DNA or mRNA encoding Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2. 
     An aspect of the invention provides a composition comprising a dsRNA, e.g., a siRNA or shRNA, comprising at least 15 contiguous nucleotides, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 contiguous nucleotides, e.g., 21 contiguous nucleotides, which are complementary (e.g., 100% complementary) to a sequence of a Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, nucleic acid sequence (e.g., genomic DNA or mRNA encoding Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2). In embodiments, the at least 15 contiguous nucleotides, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 contiguous nucleotides, e.g., 21 contiguous nucleotides, include contiguous nucleotides of a Target sequence of shRNA or Nucleic Acid encoding Tet2 shRNA listed in table 4. It is understood that some of the target sequences and/or shRNA molecules are presented as DNA, but the dsRNA agents targeting these sequences or comprising these sequences can be RNA, or any nucleotide, modified nucleotide or substitute disclosed herein and/or known in the art, provided that the molecule can still mediate RNA interference. 
     In an embodiment, a nucleic acid molecule that encodes a dsRNA molecule that inhibits expression of Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, is operably linked to a promoter, e.g., a H1- or a U6-derived promoter such that the dsRNA molecule that inhibits expression of Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, is expressed within a CAR-expressing cell. See e.g., Tiscornia G., “Development of Lentiviral Vectors Expressing siRNA,” Chapter 3, in  Gene Transfer: Delivery and Expression of DNA and RNA  (eds. Friedmann and Rossi). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA, 2007; Brummelkamp T R, et al. (2002) Science 296: 550-553; Miyagishi M, et al. (2002) Nat. Biotechnol. 19: 497-500. In an embodiment the nucleic acid molecule that encodes a dsRNA molecule that inhibits Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, is present on the same vector, e.g., a lentiviral vector, that comprises a nucleic acid molecule that encodes a component, e.g., all of the components, of the CAR. In such an embodiment, the nucleic acid molecule that encodes a dsRNA molecule that inhibits Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, is located on the vector, e.g., the lentiviral vector, 5′- or 3′- to the nucleic acid that encodes a component, e.g., all of the components, of the CAR. The nucleic acid molecule that encodes a dsRNA molecule that inhibits expression of Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, can be transcribed in the same or different direction as the nucleic acid that encodes a component, e.g., all of the components, of the CAR. In an embodiment the nucleic acid molecule that encodes a dsRNA molecule that inhibits expression of Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, is present on a vector other than the vector that comprises a nucleic acid molecule that encodes a component, e.g., all of the components, of the CAR. In an embodiment, the nucleic acid molecule that encodes a dsRNA molecule that inhibits expression of Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, is transiently expressed within a CAR-expressing cell. In an embodiment, the nucleic acid molecule that encodes a dsRNA molecule that inhibits expression of Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, is stably integrated into the genome of a CAR-expressing cell. 
     Examples of nucleic acid sequences that encode shRNA sequences are provided below. The Target Sequence refers to the sequence within the Tet2 genomic DNA (or surrounding DNA). The nucleic acid encoding Tet2 shRNA encodes shRNA molecules useful in the present invention. In embodiments, the Tet2 inhibitor is an siRNA or shRNA specific for a Target sequence listed below, or specific for its mRNA complement. In embodiments, the Tet2 inhibitor is a shRNA encoded by the Nucleic Acid encoding Tet2 shRNA of the table 4 below. In embodiments, the Tet2 inhibitor is nucleic acid comprising by the Nucleic Acid encoding Tet2 shRNA of the table 4 below, e.g., which is under the control of a U6 or H1 promoter such that a Tet2 shRNA is produced. In embodiments, the invention provides a siRNA or shRNA comprising sequence which is the RNA analog (i.e., all T nucleic acid residues replaced with U nucleic acid residues) of the Target sequence of shRNA, e.g., the Target sequence of shRNA of any of the shRNAs of Table 4. 
     
       
         
           
               
               
               
               
             
               
                 TABLE 4 
               
               
                   
               
               
                   
                   
                 Target sequence 
                   
               
               
                   
                 SHRNA_NAME 
                 of shRNA 
                 Nucleic Acid encoding Tet2 shRNA 
               
               
                   
               
             
            
               
                 TET2 
                 TET2- 
                 CACATGGCGTTTA 
                 CACATGGCGTTTATCCAGAAT 
               
               
                   
                 3838_76472_insert 
                 TCCAGAAT (SEQ 
                 CTCGAGATTCTGGATAAACGCCATGT 
               
               
                   
                 (TET2 shRNA #1) 
                 ID NO: 1244) 
                 GTTTTTTGAATTCGCACCAGCACGCT 
               
               
                   
                   
                   
                 ACGCACACACAGTACACACACTGACG 
               
               
                   
                   
                   
                 TTTCGCCGTCTTC (SEQ ID NO: 1253) 
               
               
                   
               
               
                 TET2 
                 TET2_NM_017628.4_25616_concept 
                 CAGATGCACAGGC 
                 GAAGACGCACCGGCAGATGTACAGG 
               
               
                   
                 (TET2 shRNA #2) 
                 CAATTAAG (SEQ 
                 CTAATTAAGGTTAATATTCATAGCCTT 
               
               
                   
                   
                 ID NO: 1245) 
                 AATTGGCCTGTGCATCTGTTTTTTGAA 
               
               
                   
                   
                   
                 TTCGCACCAGCACGCTACGCAACACG 
               
               
                   
                   
                   
                 TCAACCAGTGTCAGTGTTTCGCCGT 
               
               
                   
                   
                   
                 (SEQ ID NO: 1254) 
               
               
                   
               
               
                 TET2 
                 TET2_NM_017628.4_25625_concept 
                 GAGCTGCTGAATT 
                 GAAGACGCACCGGGAGCTGCTGAAT 
               
               
                   
                 (TET2 shRNA #3) 
                 CAACTAGA (SEQ 
                 TCAATTAGAGTTAATATTCATAGCTCT 
               
               
                   
                   
                 ID NO: 1246) 
                 AGTTGAATTCAGCAGCTCTTTTTTGA 
               
               
                   
                   
                   
                 ATTCGCACCAGCACGCTACGCATGCA 
               
               
                   
                   
                   
                 GTCAACCAGTGTCAACCATTCGCCGT 
               
               
                   
                   
                   
                 (SEQ ID NO: 1255) 
               
               
                   
               
               
                 TET2 
                 TET2- 
                 CAGATCGCCATAA 
                 CAGATCGCCATAACATAAATACTCGA 
               
               
                   
                 6571_76471_target 
                 CATAAATA (SEQ ID 
                 GTATTTATGTTATGGCGATCTGTTTTT 
               
               
                   
                 (TET2 shRNA #4) 
                 NO: 1247) 
                 TGAATTCGCACCAGCACGCTACGCAT 
               
               
                   
                   
                   
                 GACCAGTACACACACTGCATGTTCGC 
               
               
                   
                   
                   
                 CGTCTTC (SEQ ID NO: 1256) 
               
               
                   
               
               
                 TET2 
                 TET2_NM_017628.4_25619_target 
                 GACCATGGAGCAG 
                 GAAGACGCACCGGGACCATGGAGTA 
               
               
                   
                 (TET2 shRNA #5) 
                 CATCTGAA (SEQ 
                 GCATTTGAAGTTAATATTCATAGCTTC 
               
               
                   
                   
                 ID NO: 1248) 
                 AGATGCTGCTCCATGGTCTTTTTTGA 
               
               
                   
                   
                   
                 ATTCGCACCAGCACGCTACGCATGGT 
               
               
                   
                   
                   
                 GTCAACCAGTGTCAGTTGTTCGCCGT 
               
               
                   
                   
                   
                 (SEQ ID NO: 1257) 
               
               
                   
               
               
                 TET2 
                 TET2 shRNA #6 
                 GCCAAGTCATTATT 
                 GCCAAGTCATTATTTGACCATCTCGA 
               
               
                   
                   
                 TGACCAT (SEQ ID 
                 GATGGTCAAATAATGACTTGGCTTTT 
               
               
                   
                   
                 NO: 1249) 
                 TTGA (SEQ ID NO: 1258) 
               
               
                   
               
               
                 TET2 
                 TET2 shRNA #7 
                 CCTCAGAGATATT 
                 CCTCAGAGATATTGTGGGTTTCTCGA 
               
               
                   
                   
                 GTGGGTTT (SEQ 
                 GAAACCCACAATATCTCTGAGGTTTT 
               
               
                   
                   
                 ID NO: 1250) 
                 TTGA (SEQ ID NO: 1259) 
               
               
                   
               
               
                 TET2 
                 TET2 shRNA #8 
                 GGGTAAGCCAAGA 
                 GGGTAAGCCAAGAAAGAAACTCGAG 
               
               
                   
                   
                 AAGAAA (SEQ ID 
                 TTTCTTTCTTGGCTTACCCTTTTTTGA 
               
               
                   
                   
                 NO: 1251) 
                 (SEQ ID NO: 1260) 
               
               
                   
               
               
                 TET2 
                 TET2 8 long 
                 GGGTAAGCCAAGA 
                 GAAGACGCACCGGGGGTAAGCCAAG 
               
               
                   
                 (TET2 shRNA #9) 
                 AAGAAA (SEQ ID 
                 AAAGAAAGTTAATATTCATAGCTTTC 
               
               
                   
                   
                 NO: 1252) 
                 TTTCTTGGCTTACCCTTTTTTGAATTC 
               
               
                   
                   
                   
                 GCACCAGCACGCTACGCAACACGTCA 
               
               
                   
                   
                   
                 ACCAGTGTCAGTGTTTCGCCGT (SEQ 
               
               
                   
                   
                   
                 ID NO: 1261) 
               
               
                   
               
            
           
         
       
     
     Additional dsRNA inhibitor of Tet2, e.g., shRNA and siRNA molecules can be designed and tested using methods known in the art and as described herein. In embodiments, the dsRNA Tet2 inhibitor, e.g., shRNA or siRNA, targets a sequence of SEQ TD NO: 1358. In embodiments, the dsRNA Tet2 inhibitor, e.g., shRNA or siRNA, targets a sequence of SEQ ID NO: 1359. In embodiments, the dsRNA Tet2 inhibitor, e.g., shRNA or siRNA, targets a sequence of SEQ ID NO: 1360. In embodiments, the dsRNA Tet2 inhibitor, e.g., shRNA or siRNA, targets a sequence of SEQ ID NO: 1361. In embodiments, the dsRNA Tet2 inhibitor, e.g., shRNA or siRNA, targets a sequence of SEQ ID NO: 1362. In embodiments, the dsRNA Tet2 inhibitor, e.g., shRNA or siRNA, targets a sequence of SEQ ID NO: 1363. In embodiments, the dsRNA Tet2 inhibitor, e.g., shRNA or siRNA, targets a sequence of an mRNA encoding Tet2. 
     In embodiments, the inhibitor is a nucleic acid, e.g., DNA, encoding a dsRNA Tet2 inhibitor, e.g., shRNA or siRNA, of any of the above embodiments. In embodiments, the nucleic acid, e.g., DNA, is disposed on a vector, e.g., any conventional expression system, e.g., as described herein, e.g., a lentiviral vector. 
     Without being bound by theory, a dsRNA TET inhibitor (e.g., siRNA or shRNA) which targets a sequence of a Tet mRNA, e.g., Tet1, Tet2, and/or Tet3 gene, e.g., Tet2 mRNA, that is specific to one or more isoforms of tet (e.g., tet2) but does not affect one or more other isoforms of tet (e.g., tet2) (for example, due to targeting a unique splice junction, or targeting a domain which is present in one or more isoforms of tet, e.g., tet2, but is not present in one or more other isoforms of tet, e.g., tet2). In embodiments, it may be preferable to specifically target isoforms of the tet (e.g., tet2) which contain a catalytic domain. 
     Small Molecules 
     Tet Inhibitors 
     In embodiments, a Tet inhibitor is a small molecule that inhibits expression and/or a function of Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2. 
     Tet2 Inhibitors 
     In embodiments, a Tet2 inhibitor is a small molecule that inhibits Tet2 expression and/or function. For example, a Tet2 inhibitor according to the present invention is 2-hydroxyglutarate (CAS #2889-31-8). 
     In another example, a Tet2 inhibitor according to the present invention has the following structure: 
     
       
         
         
             
             
         
       
     
     In another example, a Tet2 inhibitor according to the present invention is N-[3-[7-(2,5-Dimethyl-2H-pyrazol-3-ylamino)-1-methyl-2-oxo-1,4-dihydro-2H-pyrimido[4,5-d]pyrimidin-3-yl]-4-methylphenyl]-3-trifluoromethyl-benzamide (CAS #839707-37-8), and has the following structure: 
     
       
         
         
             
             
         
       
     
     In another example, a Tet2 inhibitor according to the present invention is 2-[(2,6-dichloro-3-methylphenyl)amino]benzoic acid (CAS #644-62-2), and has the following structure: 
     
       
         
         
             
             
         
       
     
     In embodiments, the Tet2 inhibitor of the present invention is a pharmaceutically acceptable salt of any of the foregoing. 
     HDAC Inhibitors 
     Any known HDAC inhibitors can be used according to the present invention. Non-limiting examples of HDAC inhibitors include Voninostat (Zolinza®); Romidepsin (Istodax®); Treichostatin A (TSA); Oxamflatin; Vorinostat (Zolinza®, Suberoylanilide hydroxamic acid); Pyroxamide (syberoyl-3-aminopyridineamide hydroxamic acid); Trapoxin A (RF-1023A); Trapoxin B (RF-10238); Cyclo[(αS,2S)-α-amino-η-oxo-2-oxiraneoctanoyl-O-methyl-D-tyrosyl-L-isoleucyl-L-prolyl] (Cyl-1); Cyclo[(αS,2S)-α-amino-η-oxo-2-oxiraneoctanoyl-O-methyl-D-tyrosyl-L-isoleucyl-(2S)-2-piperidinecarbonyl] (Cyl-2); Cyclic[L-alanyl-D-alanyl-(2S)-η-oxo-L-α-aminooxiraneoctanoyl-D-prolyl] (HC-toxin); Cyclo[(αS,2S)-α-amino-η-oxo-2-oxiraneoctanoyl-D-phenylalanyl-L-leucyl-(2S)-2-piperidinecarbonyl] (WF-3161); Chlamydocin ((S)-Cyclic(2-methylalanyl-L-phenylalanyl-D-prolyl-η-oxo-L-α-aminooxiraneoctanoyl); Apicidin (Cyclo(8-oxo-L-2-aminodecanoyl-1-methoxy-L-tryptophyl-L-isoleucyl-D-2-piperidinecarbonyl); Romidepsin (Istodax®, FR-901228); 4-Phenylbutyrate; Spiruchostatin A; Mylproin (Valproic acid); Entinostat (MS-275, N-(2-Aminophenyl)-4-[N-(pyridine-3-yl-methoxycarbonyl)-amino-methyl]-benzamide); Depudecin (4,5:8,9-dianhydro-1,2,6,7,11-pentadeoxy-D-threo-D-ido-Undeca-1,6-dienitol); 4-(Acetylamino)-N-(2-aminophenyl)-benzamide (also known as CI-994); N1-(2-Aminophenyl)-N8-phenyl-octanediamide (also known as BML-210); 4-(Dimethylamino)-N-(7-(hydroxyamino)-7-oxoheptyl)benzamide (also known as M344); (E)-3-(4-(((2-(1H-indol-3-yl)ethyl)(2-hydroxyethyl)amino)-methyl)phenyl)-N-hydroxyacrylamide (NVP-LAQ824); Panobinostat (Farydak®); Mocetinostat, and Belinostat. 
     Proteins 
     Dominant Negative Tet2 
     According to the present invention, dominant negative Tet2 isoforms, and nucleic acid encoding said dominant negative Tet2, can be used as Tet2 inhibitors. In embodiments, the dominant negative Tet2 lacks catalytic function of Tet2. An example of a dominant negative Tet2 is a protein comprising or consisting of SEQ ID NO: 1357 with the mutation R1261G, according to the numbering of SEQ ID NO: 1357. An example of a dominant negative Tet2 is a protein comprising or consisting of SEQ ID NO: 1357 with the mutation R1262A, according to the numbering of SEQ ID NO: 1357. An example of a dominant negative Tet2 is a protein comprising or consisting of SEQ ID NO: 1357 with the mutation S1290A, according to the numbering of SEQ ID NO: 1357. An example of a dominant negative Tet2 is a protein comprising or consisting of SEQ ID NO: 1357 with the mutation WSMYYN (amino acids 1291-1296 of SEQ ID NO: 1357) to GGSGGS (SEQ ID NO: 67), according to the numbering of SEQ ID NO: 1357. An example of a dominant negative Tet2 is a protein comprising or consisting of SEQ ID NO: 1357 with the mutation M1293A and Y1294A, according to the numbering of SEQ ID NO: 1357. An example of a dominant negative Tet2 is a protein comprising or consisting of SEQ ID NO: 1357 with the mutation Y1295A, according to the numbering of SEQ ID NO: 1357. An example of a dominant negative Tet2 is a protein comprising or consisting of SEQ ID NO: 1357 with the mutation S1303N, according to the numbering of SEQ ID NO: 1357. An example of a dominant negative Tet2 is a protein comprising or consisting of SEQ ID NO: 1357 with the mutation H1382Y, according to the numbering of SEQ ID NO: 1357. An example of a dominant negative Tet2 is a protein comprising or consisting of SEQ ID NO: 1357 with the mutation D1384A, according to the numbering of SEQ ID NO: 1357. An example of a dominant negative Tet2 is a protein comprising or consisting of SEQ ID NO: 1357 with the mutation D1384V, according to the numbering of SEQ ID NO: 1357. In embodiments, the dominant negative Tet2 may include combinations of any of the aforementioned mutations. Such mutations are additionally described in, for example, Chen et al., Nature, 493:561-564 (2013); Hu et al, Cell, 155:1545-1555 (2013), the contents of which are hereby incorporated by reference in their entirety. 
     Dominant Negative Tet2 Binding Partners 
     Without being bound by theory, it is believed that Tet2 interacts, e.g., binds, with one or more HDAC, e.g., one or more HDAC expressed in immune effector cells, e.g., in T cells, and that such Tet2:HDAC complexes may contribute to Tet2 activity in the cell. In embodiments, a Tet2 inhibitor of the invention is a dominant negative Tet2 binding partner, e.g., a dominant negative Tet2-binding HDAC. In other embodiments, a Tet2 inhibitor of the invention comprises nucleic acid encoding a dominant negative Tet2 binding partner, e.g., a dominant negative Tet2-binding HDAC. 
     Vectors Encoding Tet2 Inhibitors 
     As described herein, the invention provides vectors, e.g., as described herein, which encode Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, inhibitors, such as the gene editing systems, shRNA or siRNA inhibitors or dominant negative inhibitors of Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2 (e.g., as described herein). 
     In embodiments further comprising, for example, a CAR, the nucleic acid may further comprise sequence encoding a CAR, e.g., as described herein. In some embodiments, the invention provides a vector comprising a nucleic acid sequence encoding a Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2 inhibitor described herein and comprising a nucleic acid sequence encoding a CAR molecule described herein. In embodiments, nucleic acid sequences are disposed on separate vectors. In other embodiments, the two or more nucleic acid sequences are encoded by a single nucleic molecule in the same frame and as a single polypeptide chain. In this aspect, the two or more CARs can, e.g., be separated by one or more peptide cleavage sites. (e.g., an auto-cleavage site or a substrate for an intracellular protease). Examples of peptide cleavage sites include the following, wherein the GSG residues are optional: 
     
       
         
           
               
            
               
                 T2A: 
               
               
                 (SEQ ID NO: 68) 
               
               
                 (GSG) E G R G S L L T C G D V E E N P G P 
               
               
                   
               
               
                 P2A: 
               
               
                 (SEQ ID NO: 69) 
               
               
                 (GSG) A T N F S L L K Q A G D V E E N P G P 
               
               
                   
               
               
                 E2A: 
               
               
                 (SEQ ID NO: 70) 
               
               
                 (GSG) Q C T N Y A L L K L A G D V E S N P G P 
               
               
                   
               
               
                 F2A: 
               
               
                 (SEQ ID NO: 71) 
               
               
                 (GSG) V K Q T L N F D L L K L A G D V E S N P G P. 
               
            
           
         
       
     
     These peptide cleavage sites are referred to collectively herein as “2A sites.” In embodiments, the vector comprises nucleic acid sequence encoding a CAR described herein and nucleic acid sequence encoding a shRNA or siRNA Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, Inhibitor described herein. In embodiments, the vector comprises nucleic acid sequence encoding a CAR described herein and nucleic acid sequence encoding a genome editing system (e.g., a CRISPR/Cas system) Tet e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, Inhibitor described herein. 
     Methods of Use of Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, Inhibitors 
     The invention provides methods of increasing the therapeutic efficacy of a CAR-expressing cell, e.g., a cell expressing a CAR as described herein, e.g., a CAR19-expressing cell (e.g., CTL019), comprising a step of decreasing the level of 5-hydroxymethylcytosine in said cell. In embodiments, the method comprises reducing or eliminating the function or expression of Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2. In embodiments, the method comprises contacting said cells with a Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, inhibitor as described herein. 
     The invention further provides methods of manufacturing a CAR-expressing cell, e.g., a CAR-expressing cell having improved function (e.g., having improved efficacy, e.g., tumor targeting, or proliferation) comprising the step of reducing or eliminating the expression or function of Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, in said cell. In embodiments, the method comprises contacting said cells with a Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, inhibitor as described herein. In embodiments, the contacting is done ex vivo. In embodiments, the contacting is done in vivo. In embodiments, the contacting is done prior to, simultaneously with, or after said cells are modified to express a CAR, e.g., a CAR as described herein. 
     In embodiments, the invention provides a method for inhibiting a function or expression of Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, in a CAR-expressing cell, e.g., a cell expressing a CAR as described herein, e.g., a CAR19-expressing cell (e.g., CTL019-expressing cell), the method comprising a step of decreasing the level of 5-hydroxymethylcytosine in said cell. In embodiments, the method comprises reducing or eliminating the function or expression of Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2. In embodiments, the method comprises contacting said cells with a Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, inhibitor as described herein. 
     In one embodiment, the invention provides a method, e.g., a method described above, comprises introducing nucleic acid encoding a CAR into a cell, e.g., an immune effector cell, e.g., a T cell, at a site within the Tet gene, or its regulatory elements, such that expression of Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, is disrupted. Integration at a site within the Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, gene may be accomplished, for example, using a Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2-targeting gene editing system as described above. 
     In one embodiment, the invention provides a method, e.g., a method described above, comprising a step of introducing into the cell a gene editing system, e.g., a CRISPR/Cas gene editing system which targets Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, e.g., a CRISPR/Cas system comprising a gRNA which has a targeting sequence complementary to a target sequence of the Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2, gene. In embodiments, the CRISPR/Cas system is introduced into said cell as a ribonuclear protein complex of gRNA and Cas enzyme, e.g., is introduced via electroporation. In one embodiment, the method comprises introducing nucleic acid encoding one or more of the components of the CRISPR/Cas system into said cell. In one embodiment, said nucleic acid is disposed on the vector encoding a CAR, e.g., a CAR as described herein. 
     In one embodiment, the invention provides a method, e.g., a method described above, comprising a step of introducing into the cell an inhibitory dsRNA, e.g., a shRNA or siRNA, which targets Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2. In one embodiment, the method comprises introducing into said cell nucleic acid encoding an inhibitory dsRNA, e.g., a shRNA or siRNA, which targets Tet, e.g., Tet1, Tet2 and/or Tet3, e.g., Tet2. In one embodiment, said nucleic acid is disposed on the vector encoding a CAR, e.g., a CAR as described herein. 
     Additional components of CARs and CAR T cells, and methods pertaining to the invention are described below. 
     Provided herein are compositions of matter and methods of use for the treatment of a disease such as cancer using immune effector cells (e.g., T cells, NK cells) engineered with CARs of the invention. 
     In one aspect, the invention provides a number of chimeric antigen receptors (CAR) comprising an antigen binding domain (e.g., antibody or antibody fragment, TCR or TCR fragment) engineered for specific binding to a tumor antigen, e.g., a tumor antigen described herein. In one aspect, the invention provides an immune effector cell (e.g., T cell, NK cell) engineered to express a CAR, wherein the engineered immune effector cell exhibits an anticancer property. In one aspect, a cell is transformed with the CAR and the CAR is expressed on the cell surface. In some embodiments, the cell (e.g., T cell, NK cell) is transduced with a viral vector encoding a CAR. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some such embodiments, the cell may stably express the CAR. In another embodiment, the cell (e.g., T cell, NK cell) is transfected with a nucleic acid, e.g., mRNA, cDNA, DNA, encoding a CAR. In some such embodiments, the cell may transiently express the CAR. 
     In one aspect, the antigen binding domain of a CAR described herein is a scFv antibody fragment. In one aspect, such antibody fragments are functional in that they retain the equivalent binding affinity, e.g., they bind the same antigen with comparable affinity, as the IgG antibody from which it is derived. In other embodiments, the antibody fragment has a lower binding affinity, e.g., it binds the same antigen with a lower binding affinity than the antibody from which it is derived, but is functional in that it provides a biological response described herein. In one embodiment, the CAR molecule comprises an antibody fragment that has a binding affinity KD of 10 −4  M to 10 −8  M, e.g., 10 −5  M to 10 −7  M, e.g., 10 −6  M or 10 −7  M, for the target antigen. In one embodiment, the antibody fragment has a binding affinity that is at least five-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold or 1,000-fold less than a reference antibody, e.g., an antibody described herein. 
     In one aspect such antibody fragments are functional in that they provide a biological response that can include, but is not limited to, activation of an immune response, inhibition of signal-transduction origination from its target antigen, inhibition of kinase activity, and the like, as will be understood by a skilled artisan. 
     In one aspect, the antigen binding domain of the CAR is a scFv antibody fragment that is humanized compared to the murine sequence of the scFv from which it is derived. 
     In one aspect, the antigen binding domain of a CAR of the invention (e.g., a scFv) is encoded by a nucleic acid molecule whose sequence has been codon optimized for expression in a mammalian cell. In one aspect, entire CAR construct of the invention is encoded by a nucleic acid molecule whose entire sequence has been codon optimized for expression in a mammalian cell. Codon optimization refers to the discovery that the frequency of occurrence of synonymous codons (i.e., codons that code for the same amino acid) in coding DNA is biased in different species. Such codon degeneracy allows an identical polypeptide to be encoded by a variety of nucleotide sequences. A variety of codon optimization methods is known in the art, and include, e.g., methods disclosed in at least U.S. Pat. Nos. 5,786,464 and 6,114,148. 
     In one aspect, the CARs of the invention combine an antigen binding domain of a specific antibody with an intracellular signaling molecule. For example, in some aspects, the intracellular signaling molecule includes, but is not limited to, CD3-zeta chain, 4-1BB and CD28 signaling modules and combinations thereof. In one aspect, the antigen binding domain binds to a tumor antigen as described herein. 
     Furthermore, the present invention provides CARs and CAR-expressing cells and their use in medicaments or methods for treating, among other diseases, cancer or any malignancy or autoimmune diseases involving cells or tissues which express a tumor antigen as described herein. 
     In one aspect, the CAR of the invention can be used to eradicate a normal cell that express a tumor antigen as described herein, thereby applicable for use as a cellular conditioning therapy prior to cell transplantation. In one aspect, the normal cell that expresses a tumor antigen as described herein is a normal stem cell and the cell transplantation is a stem cell transplantation. 
     In one aspect, the invention provides an immune effector cell (e.g., T cell, NK cell) engineered to express a chimeric antigen receptor (CAR), wherein the engineered immune effector cell exhibits an antitumor property. A preferred antigen is a cancer associated antigen (i.e., tumor antigen) described herein. In one aspect, the antigen binding domain of the CAR comprises a partially humanized antibody fragment. In one aspect, the antigen binding domain of the CAR comprises a partially humanized scFv. Accordingly, the invention provides CARs that comprises a humanized antigen binding domain and is engineered into a cell, e.g., a T cell or a NIK cell, and methods of their use for adoptive therapy. 
     In one aspect, the CARs of the invention comprise at least one intracellular domain selected from the group of a CD137 (4-1BB) signaling domain, a CD28 signaling domain, a CD27 signal domain, a CD3zeta signal domain, and any combination thereof. In one aspect, the CARs of the invention comprise at least one intracellular signaling domain is from one or more costimulatory molecule(s) other than a CD137 (4-1BB) or CD28. 
     Sequences of some examples of various components of CARs of the instant invention is listed in Table 1, where aa stands for amino acids, and na stands for nucleic acids that encode the corresponding peptide. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Sequences of various components of CAR (aa—amino acids, 
               
               
                 na—nucleic acids that encodes the corresponding protein) 
               
            
           
           
               
               
               
               
            
               
                 SEQ 
                   
                   
                 Corresp. 
               
               
                 ID 
                   
                   
                 To 
               
               
                 NO 
                 description 
                 Sequence 
                 huCD19 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 1 
                 EF-1 
                 CGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCAC 
                 100 
               
               
                   
                 promoter 
                 AGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGC 
                   
               
               
                   
                   
                 CTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTA 
                   
               
               
                   
                   
                 CTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGT 
                   
               
               
                   
                   
                 GCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAG 
                   
               
               
                   
                   
                 AACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTA 
                   
               
               
                   
                   
                 CGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAG 
                   
               
               
                   
                   
                 TACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGA 
                   
               
               
                   
                   
                 GTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTT 
                   
               
               
                   
                   
                 GAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGG 
                   
               
               
                   
                   
                 CACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAA 
                   
               
               
                   
                   
                 AATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTT 
                   
               
               
                   
                   
                 GTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGC 
                   
               
               
                   
                   
                 CGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGC 
                   
               
               
                   
                   
                 GAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTA 
                   
               
               
                   
                   
                 GTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGT 
                   
               
               
                   
                   
                 GTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGT 
                   
               
               
                   
                   
                 TGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGC 
                   
               
               
                   
                   
                 TCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCA 
                   
               
               
                   
                   
                 CCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATG 
                   
               
               
                   
                   
                 TGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCT 
                   
               
               
                   
                   
                 CGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTAT 
                   
               
               
                   
                   
                 GCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGC 
                   
               
               
                   
                   
                 CAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGT 
                   
               
               
                   
                   
                 TTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTT 
                   
               
               
                   
                   
                 TTCTTCCATTTCAGGTGTCGTGA 
                   
               
               
                   
               
               
                 2 
                 Leader (aa) 
                 MALPVTALLLPLALLLHAARP 
                 13 
               
               
                   
               
               
                 3 
                 Leader (na) 
                 ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCT 
                 54 
               
               
                   
                   
                 GCATGCCGCTAGACCC 
                   
               
               
                   
               
               
                 4 
                 CD 8 hinge 
                 TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD 
                 14 
               
               
                   
                 (aa) 
                   
                   
               
               
                   
               
               
                 5 
                 CD8 hinge 
                 ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCG 
                 55 
               
               
                   
                 (na) 
                 CGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGC 
                   
               
               
                   
                   
                 GGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT 
                   
               
               
                   
               
               
                 6 
                 Ig4 hinge 
                 ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ 
                 102 
               
               
                   
                 (aa) 
                 EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLN 
                   
               
               
                   
                   
                 GKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL 
                   
               
               
                   
                   
                 TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKS 
                   
               
               
                   
                   
                 RWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKM 
                   
               
               
                   
               
               
                 7 
                 Ig4 hinge 
                 GAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTT 
                 103 
               
               
                   
                 (na) 
                 CCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACA 
                   
               
               
                   
                   
                 CCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGA 
                   
               
               
                   
                   
                 CGTGTCCCAGGAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGAC 
                   
               
               
                   
                   
                 GGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAG 
                   
               
               
                   
                   
                 TTCAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCA 
                   
               
               
                   
                   
                 GGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAG 
                   
               
               
                   
                   
                 GGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCC 
                   
               
               
                   
                   
                 AGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGA 
                   
               
               
                   
                   
                 GATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCT 
                   
               
               
                   
                   
                 ACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCG 
                   
               
               
                   
                   
                 AGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAG 
                   
               
               
                   
                   
                 CTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAG 
                   
               
               
                   
                   
                 GAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACA 
                   
               
               
                   
                   
                 ACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGATG 
                   
               
               
                   
               
               
                 8 
                 IgD hinge 
                 RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEK 
                 47 
               
               
                   
                 (aa) 
                 EKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGS 
                   
               
               
                   
                   
                 DLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWN 
                   
               
               
                   
                   
                 AGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASW 
                   
               
               
                   
                   
                 LLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSV 
                   
               
               
                   
                   
                 LRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDH 
                   
               
               
                   
               
               
                 9 
                 IgD hinge 
                 AGGTGGCCCGAAAGTCCCAAGGCCCAGGCATCTAGTGTTCCTACTGC 
                 48 
               
               
                   
                 (na) 
                 ACAGCCCCAGGCAGAAGGCAGCCTAGCCAAAGCTACTACTGCACCT 
                   
               
               
                   
                   
                 GCCACTACGCGCAATACTGGCCGTGGCGGGGAGGAGAAGAAAAAG 
                   
               
               
                   
                   
                 GAGAAAGAGAAAGAAGAACAGGAAGAGAGGGAGACCAAGACCCCT 
                   
               
               
                   
                   
                 GAATGTCCATCCCATACCCAGCCGCTGGGCGTCTATCTCTTGACTCCC 
                   
               
               
                   
                   
                 GCAGTACAGGACTTGTGGCTTAGAGATAAGGCCACCTTTACATGTTT 
                   
               
               
                   
                   
                 CGTCGTGGGCTCTGACCTGAAGGATGCCCATTTGACTTGGGAGGTT 
                   
               
               
                   
                   
                 GCCGGAAAGGTACCCACAGGGGGGGTTGAGGAAGGGTTGCTGGAG 
                   
               
               
                   
                   
                 CGCCATTCCAATGGCTCTCAGAGCCAGCACTCAAGACTCACCCTTCC 
                   
               
               
                   
                   
                 GAGATCCCTGTGGAACGCCGGGACCTCTGTCACATGTACTCTAAATC 
                   
               
               
                   
                   
                 ATCCTAGCCTGCCCCCACAGCGTCTGATGGCCCTTAGAGAGCCAGCC 
                   
               
               
                   
                   
                 GCCCAGGCACCAGTTAAGCTTAGCCTGAATCTGCTCGCCAGTAGTGA 
                   
               
               
                   
                   
                 TCCCCCAGAGGCCGCCAGCTGGCTCTTATGCGAAGTGTCCGGCTTTA 
                   
               
               
                   
                   
                 GCCCGCCCAACATCTTGCTCATGTGGCTGGAGGACCAGCGAGAAGT 
                   
               
               
                   
                   
                 GAACACCAGCGGCTTCGCTCCAGCCCGGCCCCCACCCCAGCCGGGTT 
                   
               
               
                   
                   
                 CTACCACATTCTGGGCCTGGAGTGTCTTAAGGGTCCCAGCACCACCT 
                   
               
               
                   
                   
                 AGCCCCCAGCCAGCCACATACACCTGTGTTGTGTCCCATGAAGATAG 
                   
               
               
                   
                   
                 CAGGACCCTGCTAAATGCTTCTAGGAGTCTGGAGGTTTCCTACGTGA 
                   
               
               
                   
                   
                 CTGACCATT 
                   
               
               
                   
               
               
                 10 
                 GS 
                 GGGGSGGGGS 
                 49 
               
               
                   
                 hinge/linker 
                   
                   
               
               
                   
                 (aa) 
                   
                   
               
               
                   
               
               
                 11 
                 GS 
                 GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC 
                 50 
               
               
                   
                 hinge/linker 
                   
                   
               
               
                   
                 (na) 
                   
                   
               
               
                   
               
               
                 12 
                 CD8TM (aa) 
                 IYIWAPLAGTCGVLLLSLVITLYC 
                 15 
               
               
                   
               
               
                 13 
                 CD8 TM (na) 
                 ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCT 
                 56 
               
               
                   
                   
                 GTCACTGGTTATCACCCTTTACTGC 
                   
               
               
                   
               
               
                 14 
                 4-1BB 
                 KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 
                 16 
               
               
                   
                 intracellular 
                   
                   
               
               
                   
                 domain (aa) 
                   
                   
               
               
                   
               
               
                 15 
                 4-1BB 
                 AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTAT 
                 60 
               
               
                   
                 intracellular 
                 GAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGA 
                   
               
               
                   
                 domain (na) 
                 TTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG 
                   
               
               
                   
               
               
                 16 
                 CD27 (aa) 
                 QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP 
                 51 
               
               
                   
               
               
                 17 
                 CD27 (na) 
                 AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGA 
                 52 
               
               
                   
                   
                 CTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCC 
                   
               
               
                   
                   
                 CCACCACGCGACTTCGCAGCCTATCGCTCC 
                   
               
               
                   
               
               
                 18 
                 CD3-zeta 
                 RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG 
                 17 
               
               
                   
                 (aa) 
                 KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLS 
                   
               
               
                   
                   
                 TATKDTYDALHMQALPPR 
                   
               
               
                   
               
               
                 19 
                 CD3-zeta 
                 AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAG 
                 101 
               
               
                   
                 (na) 
                 GGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGG 
                   
               
               
                   
                   
                 AGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGG 
                   
               
               
                   
                   
                 GGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGA 
                   
               
               
                   
                   
                 ACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGAT 
                   
               
               
                   
                   
                 GAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCA 
                   
               
               
                   
                   
                 GGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGC 
                   
               
               
                   
                   
                 AGGCCCTGCCCCCTCGC 
                   
               
               
                   
               
               
                 20 
                 CD3-zeta 
                 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG 
                 43 
               
               
                   
                 (aa) 
                 KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLS 
                   
               
               
                   
                   
                 TATKDTYDALHMQALPPR 
                   
               
               
                   
               
               
                 21 
                 CD3-zeta 
                 AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAG 
                 44 
               
               
                   
                 (na) 
                 GGCCAG 
                   
               
               
                   
                   
                 AACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACG 
                   
               
               
                   
                   
                 ATGTTT 
                   
               
               
                   
                   
                 TGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGA 
                   
               
               
                   
                   
                 GAAGGA 
                   
               
               
                   
                   
                 AGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGAT 
                   
               
               
                   
                   
                 GGCGG 
                   
               
               
                   
                   
                 AGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCA 
                   
               
               
                   
                   
                 AGGGGC 
                   
               
               
                   
                   
                 ACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTAC 
                   
               
               
                   
                   
                 GACGC 
                   
               
               
                   
                   
                 CCTTCACATGCAGGCCCTGCCCCCTCGC 
                   
               
               
                   
               
               
                 22 
                 linker 
                 GGGGS 
                 18 
               
               
                   
               
               
                 23 
                 linker 
                 GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC 
                 50 
               
               
                   
               
               
                 24 
                 PD-1 
                 Pgwfldspdrpwnpptfspallvvtegdnatftcsfsntsesfvlnwyrmspsnqtdkl 
                   
               
               
                   
                 extracellular 
                 aafpedrsqpgqdcrfrvtqlpngrdfhmsvvrarrndsgtylcgaislapkaqikeslra 
                   
               
               
                   
                 domain (aa) 
                 elrvterraevptahpspsprpagqfqtlv 
                   
               
               
                   
               
               
                 25 
                 PD-1 
                 Cccggatggtttctggactctccggatcgcccgtggaatcccccaaccttctcaccggcact 
                   
               
               
                   
                 extracellular 
                 cttggttgtgactgagggcgataatgcgaccttcacgtgctcgttctccaacacctccgaat 
                   
               
               
                   
                 domain (na) 
                 cattcgtgctgaactggtaccgcatgagcccgtcaaaccagaccgacaagctcgccgcgtt 
                   
               
               
                   
                   
                 tccggaagatcggtcgcaaccgggacaggattgtcggttccgcgtgactcaactgccgaat 
                   
               
               
                   
                   
                 ggcagagacttccacatgagcgtggtccgcgctaggcgaaacgactccgggacctacctg 
                   
               
               
                   
                   
                 tgcggagccatctcgctggcgcctaaggcccaaatcaaagagagcttgagggccgaactg 
                   
               
               
                   
                   
                 agagtgaccgagcgcagagctgaggtgccaactgcacatccatccccatcgcctcggcct 
                   
               
               
                   
                   
                 gcggggcagtttcagaccctggtc 
                   
               
               
                   
               
               
                 26 
                 PD-1 CAR 
                 Malpvtalllplalllhaarppgwfldspdrpwnpptfspallvvtegdnatftcsfsntse 
                   
               
               
                   
                 (aa) with 
                 sfvlnwyrmspsnqtdklaafpedrsqpgqdcrfrvtqlpngrdfhmsvvrarrndsgt 
                   
               
               
                   
                 signal 
                 ylcgaislapkaqikeslraelrvterraevptahpspsprpagqfqtlvtttpaprpptpa 
                   
               
               
                   
                   
                 ptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkklly 
                   
               
               
                   
                   
                 ifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnl 
                   
               
               
                   
                   
                 grreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrg 
                   
               
               
                   
                   
                 kghdglyqglstatkdtydalhmqalppr 
                   
               
               
                   
               
               
                 27 
                 PD-1 CAR 
                 Atggccctccctgtcactgccctgcttctccccctcgcactcctgctccacgccgctagacca 
                   
               
               
                   
                 (na) 
                 cccggatggtttctggactctccggatcgcccgtggaatcccccaaccttctcaccggcact 
                   
               
               
                   
                   
                 cttggttgtgactgagggcgataatgcgaccttcacgtgctcgttctccaacacctccgaat 
                   
               
               
                   
                   
                 cattcgtgctgaactggtaccgcatgagcccgtcaaaccagaccgacaagctcgccgcgtt 
                   
               
               
                   
                   
                 tccggaagatcggtcgcaaccgggacaggattgtcggttccgcgtgactcaactgccgaat 
                   
               
               
                   
                   
                 ggcagagacttccacatgagcgtggtccgcgctaggcgaaacgactccgggacctacctg 
                   
               
               
                   
                   
                 tgcggagccatctcgctggcgcctaaggcccaaatcaaagagagcttgagggccgaactg 
                   
               
               
                   
                   
                 agagtgaccgagcgcagagctgaggtgccaactgcacatccatccccatcgcctcggcct 
                   
               
               
                   
                   
                 gcggggcagtttcagaccctggtcacgaccactccggcgccgcgcccaccgactccggcc 
                   
               
               
                   
                   
                 ccaactatcgcgagccagcccctgtcgctgaggccggaagcatgccgccctgccgccgga 
                   
               
               
                   
                   
                 ggtgctgtgcatacccggggattggacttcgcatgcgacatctacatttgggctcctctcgc 
                   
               
               
                   
                   
                 cggaacttgtggcgtgctccttctgtccctggtcatcaccctgtactgcaagcggggtcgga 
                   
               
               
                   
                   
                 aaaagcttctgtacattttcaagcagcccttcatgaggcccgtgcaaaccacccaggagga 
                   
               
               
                   
                   
                 ggacggttgctcctgccggttccccgaagaggaagaaggaggttgcgagctgcgcgtgaa 
                   
               
               
                   
                   
                 gttctcccggagcgccgacgcccccgcctataagcagggccagaaccagctgtacaacga 
                   
               
               
                   
                   
                 actgaacctgggacggcgggaagagtacgatgtgctggacaagcggcgcggccgggacc 
                   
               
               
                   
                   
                 ccgaaatgggcgggaagcctagaagaaagaaccctcaggaaggcctgtataacgagctg 
                   
               
               
                   
                   
                 cagaaggacaagatggccgaggcctactccgaaattgggatgaagggagagcggcgga 
                   
               
               
                   
                   
                 ggggaaaggggcacgacggcctgtaccaaggactgtccaccgccaccaaggacacatac 
                   
               
               
                   
                   
                 gatgccctgcacatgcaggcccttccccctcgc 
                   
               
               
                   
               
               
                 28 
                 linker 
                 (Gly-Gly-Gly-Ser)n, where n = 1-10 
                 105 
               
               
                   
               
               
                 29 
                 linker 
                 (Gly4 Ser)4 
                 106 
               
               
                   
               
               
                 30 
                 linker 
                 (Gly4 Ser)3 
                 107 
               
               
                   
               
               
                 31 
                 linker 
                 (Gly3Ser) 
                 108 
               
               
                   
               
               
                 32 
                 polyA 
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                 118 
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
               
               
                 33 
                 polyA 
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                 104 
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
               
               
                 34 
                 polyA 
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                 109 
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
               
               
                 35 
                 polyA 
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                 110 
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
               
               
                 36 
                 polyA 
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                 111 
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
                   
                 tttttttttt tttttttttt tttttttttt 
                   
               
               
                   
               
               
                 37 
                 polyA 
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                 112 
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
               
               
                 38 
                 polyA 
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                 113 
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
                   
                 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 
                   
               
               
                   
               
               
                 39 
                 PD1 CAR 
                 
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     Cancer Associated Antigens 
     The present invention provides immune effector cells (e.g., T cells, NK cells) that are engineered to contain one or more CARs that direct the immune effector cells to cancer. This is achieved through an antigen binding domain on the CAR that is specific for a cancer associated antigen. There are two classes of cancer associated antigens (tumor antigens) that can be targeted by the CARs of the instant invention: (1) cancer associated antigens that are expressed on the surface of cancer cells; and (2) cancer associated antigens that itself is intracelluar, however, a fragment of such antigen (peptide) is presented on the surface of the cancer cells by MHC (major histocompatibility complex). 
     Accordingly, the present invention provides CARs that target the following cancer associated antigens (tumor antigens): CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1 (CLECL1), CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-11Ra, PSCA, VEGFR2, LewisY, CD24, PDGFR-beta, PRSS21, SSEA-4, CD20, Folate receptor alpha, ERBB2 (Her2/neu), MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1/CD248, TEM7R, CLDN6, TSHR, GPRC5D, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, legumain, HPV E6, E7, MAGE-A1, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1/Galectin 8, MelanA/MART1, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor, Cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LTLRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, and IGLL1. 
     Tumor-Supporting Antigens 
     A CAR described herein can comprise an antigen binding domain (e.g., antibody or antibody fragment, TCR or TCR fragment) that binds to a tumor-supporting antigen (e.g., a tumor-supporting antigen as described herein). In some embodiments, the tumor-supporting antigen is an antigen present on a stromal cell or a myeloid-derived suppressor cell (MDSC). Stromal cells can secrete growth factors to promote cell division in the microenvironment. MDSC cells can inhibit T cell proliferation and activation. Without wishing to be bound by theory, in some embodiments, the CAR-expressing cells destroy the tumor-supporting cells, thereby indirectly inhibiting tumor growth or survival. 
     In embodiments, the stromal cell antigen is chosen from one or more of: bone marrow stromal cell antigen 2 (BST2), fibroblast activation protein (FAP) and tenascin. In an embodiment, the FAP-specific antibody is, competes for binding with, or has the same CDRs as, sibrotuzumab. In embodiments, the MDSC antigen is chosen from one or more of: CD33, CD11b, C14, CD15, and CD66b. Accordingly, in some embodiments, the tumor-supporting antigen is chosen from one or more of: bone marrow stromal cell antigen 2 (BST2), fibroblast activation protein (FAP) or tenascin, CD33, CD11b, C14, CD15, and CD66b. 
     Chimeric Antigen Receptor (CAR) 
     The present invention encompasses a recombinant DNA construct comprising sequences encoding a CAR, wherein the CAR comprises an antigen binding domain (e.g., antibody or antibody fragment, TCR or TCR fragment) that binds specifically to a cancer associated antigen described herein, wherein the sequence of the antigen binding domain is contiguous with and in the same reading frame as a nucleic acid sequence encoding an intracellular signaling domain. The intracellular signaling domain can comprise a costimulatory signaling domain and/or a primary signaling domain, e.g., a zeta chain. The costimulatory signaling domain refers to a portion of the CAR comprising at least a portion of the intracellular domain of a costimulatory molecule. 
     In specific aspects, a CAR construct of the invention comprises a scFv domain, wherein the scFv may be preceded by an optional leader sequence such as provided in SEQ ID NO: 2, and followed by an optional hinge sequence such as provided in SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 or SEQ ID NO:10, a transmembrane region such as provided in SEQ ID NO:12, an intracellular signalling domain that includes SEQ ID NO:14 or SEQ ID NO:16 and a CD3 zeta sequence that includes SEQ ID NO:18 or SEQ ID NO:20, e.g., wherein the domains are contiguous with and in the same reading frame to form a single fusion protein. 
     In one aspect, an exemplary CAR constructs comprise an optional leader sequence (e.g., a leader sequence described herein), an extracellular antigen binding domain (e.g., an antigen binding domain described herein), a hinge (e.g., a hinge region described herein), a transmembrane domain (e.g., a transmembrane domain described herein), and an intracellular stimulatory domain (e.g., an intracellular stimulatory domain described herein). In one aspect, an exemplary CAR construct comprises an optional leader sequence (e.g., a leader sequence described herein), an extracellular antigen binding domain (e.g., an antigen binding domain described herein), a hinge (e.g., a hinge region described herein), a transmembrane domain (e.g., a transmembrane domain described herein), an intracellular costimulatory signaling domain (e.g., a costimulatory signaling domain described herein) and/or an intracellular primary signaling domain (e.g., a primary signaling domain described herein). 
     An exemplary leader sequence is provided as SEQ ID NO: 2. An exemplary hinge/spacer sequence is provided as SEQ ID NO: 4 or SEQ ID NO:6 or SEQ ID NO:8 or SEQ ID NO:10. An exemplary transmembrane domain sequence is provided as SEQ ID NO:12. An exemplary sequence of the intracellular signaling domain of the 4-1BB protein is provided as SEQ ID NO: 14. An exemplary sequence of the intracellular signaling domain of CD27 is provided as SEQ ID NO:16. An exemplary CD3zeta domain sequence is provided as SEQ ID NO: 18 or SEQ ID NO:20. 
     In one aspect, the present invention encompasses a recombinant nucleic acid construct comprising a nucleic acid molecule encoding a CAR, wherein the nucleic acid molecule comprises the nucleic acid sequence encoding an antigen binding domain, e.g., described herein, that is contiguous with and in the same reading frame as a nucleic acid sequence encoding an intracellular signaling domain. 
     In one aspect, the present invention encompasses a recombinant nucleic acid construct comprising a nucleic acid molecule encoding a CAR, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding an antigen binding domain, wherein the sequence is contiguous with and in the same reading frame as the nucleic acid sequence encoding an intracellular signaling domain. An exemplary intracellular signaling domain that can be used in the CAR includes, but is not limited to, one or more intracellular signaling domains of, e.g., CD3-zeta, CD28, CD27, 4-1BB, and the like. In some instances, the CAR can comprise any combination of CD3-zeta, CD28, 4-1BB, and the like. 
     The nucleic acid sequences coding for the desired molecules can be obtained using recombinant methods known in the art, such as, for example by screening libraries from cells expressing the nucleic acid molecule, by deriving the nucleic acid molecule from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques. Alternatively, the nucleic acid of interest can be produced synthetically, rather than cloned. 
     The present invention includes retroviral and lentiviral vector constructs expressing a CAR that can be directly transduced into a cell. 
     The present invention also includes an RNA construct that can be directly transfected into a cell. A method for generating mRNA for use in transfection involves in vitro transcription (IVT) of a template with specially designed primers, followed by polyA addition, to produce a construct containing 3′ and 5′ untranslated sequence (“UTR”) (e.g., a 3′ and/or 5′ UTR described herein), a 5′ cap (e.g., a 5′ cap described herein) and/or Internal Ribosome Entry Site (IRES) (e.g., an IRES described herein), the nucleic acid to be expressed, and a polyA tail, typically 50-2000 bases in length (SEQ ID NO:32). RNA so produced can efficiently transfect different kinds of cells. In one embodiment, the template includes sequences for the CAR. In an embodiment, an RNA CAR vector is transduced into a cell, e.g., a T cell or a NK cell, by electroporation. 
     Antigen Binding Domain 
     In one aspect, the CAR of the invention comprises a target-specific binding element otherwise referred to as an antigen binding domain. The choice of moiety depends upon the type and number of ligands that define the surface of a target cell. For example, the antigen binding domain may be chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state. Thus, examples of cell surface markers that may act as ligands for the antigen binding domain in a CAR of the invention include those associated with viral, bacterial and parasitic infections, autoimmune disease and cancer cells. 
     In one aspect, the CAR-mediated T-cell response can be directed to an antigen of interest by way of engineering an antigen binding domain that specifically binds a desired antigen into the CAR. 
     In one aspect, the portion of the CAR comprising the antigen binding domain comprises an antigen binding domain that targets a tumor antigen, e.g., a tumor antigen described herein. 
     The antigen binding domain can be any domain that binds to the antigen including but not limited to a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, and a functional fragment thereof, including but not limited to a single-domain antibody such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain (VHH) of camelid derived nanobody, and to an alternative scaffold known in the art to function as antigen binding domain, such as a recombinant fibronectin domain, a T cell receptor (TCR), or a fragment there of, e.g., single chain TCR, and the like. In some instances, it is beneficial for the antigen binding domain to be derived from the same species in which the CAR will ultimately be used in. For example, for use in humans, it may be beneficial for the antigen binding domain of the CAR to comprise human or humanized residues for the antigen binding domain of an antibody or antibody fragment. 
     In one embodiment, the CD19 CAR is a CD19 CAR described in U.S. Pat. Nos. 8,399,645; 7,446,190; Xu et al., Leuk Lymphoma. 2013 54(2):255-260(2012); Cruz et al., Blood 122(17):2965-2973 (2013); Brentjens et al., Blood, 118(18):4817-4828 (2011); Kochenderfer et al., Blood 116(20):4099-102 (2010); Kochenderfer et al., Blood 122 (25):4129-39(2013); or 16th Annu Meet Am Soc Gen Cell Ther (ASGCT) (May 15-18, Salt Lake City) 2013, Abst 10 (each of which is herein incorporated by reference in their entirety). In one embodiment, an antigen binding domain against CD19 is an antigen binding portion, e.g., CDRs, of a CAR, antibody or antigen-binding fragment thereof described in, e.g., PCT publication WO2012/079000 (incorporated herein by reference in its entirety). In one embodiment, an antigen binding domain against CD19 is an antigen binding portion, e.g., CDRs, of a CAR, antibody or antigen-binding fragment thereof described in, e.g., PCT publication WO2014/153270; Kochenderfer, J. N. et al., J. Immunother. 32 (7), 689-702 (2009); Kochenderfer, J. N., et al., Blood, 116 (20), 4099-4102 (2010); PCT publication WO2014/031687; Bejcek, Cancer Research, 55, 2346-2351, 1995; or U.S. Pat. No. 7,446,190 (each of which is herein incorporated by reference in their entirety). 
     In one embodiment, the antigen binding domain against mesothelin is or may be derived from an antigen binding domain, e.g., CDRs, scFv, or VH and VL, of an antibody, antigen-binding fragment or CAR described in, e.g., PCT publication WO2015/090230 (In one embodiment the CAR is a CAR described in WO2015/090230, the contents of which are incorporated herein in their entirety). In embodiments, the antigen binding domain against mesothelin is or is derived from an antigen binding portion, e.g., CDRs, scFv, or VH and VL, of an antibody, antigen-binding fragment, or CAR described in, e.g., PCT publication WO1997/025068, WO1999/028471, WO2005/014652, WO2006/099141, WO2009/045957, WO2009/068204, WO2013/142034, WO2013/040557, or WO2013/063419 (each of which is herein incorporated by reference in their entirety). 
     In one embodiment, an antigen binding domain against CD123 is or is derived from an antigen binding portion, e.g., CDRs, scFv or VH and VL, of an antibody, antigen-binding fragment or CAR described in, e.g., PCT publication WO2014/130635 (incorporated herein by reference in its entirety). In one embodiment, an antigen binding domain against CD123 is or is derived from an antigen binding portion, e.g., CDRs, scFv or VH and VL, of an antibody, antigen-binding fragment or CAR described in, e.g., PCT publication WO2016/028896 (incorporated herein by reference in its entirety); in embodiments, the CAR is a CAR described in WO2016/028896. In one embodiment, an antigen binding domain against CD123 is or is derived from an antigen binding portion, e.g., CDRs, scFv, or VL and VH, of an antibody, antigen-binding fragment, or CAR described in, e.g., PCT publication WO1997/024373, WO2008/127735 (e.g., a CD123 binding domain of 26292, 32701, 37716 or 32703), WO2014/138805 (e.g., a CD123 binding domain of CSL362), WO2014/138819, WO2013/173820, WO2014/144622, WO2001/66139, WO2010/126066 (e.g., the CD123 binding domain of any of Old4, Old5, Old17, Old19, New102, or Old6), WO2014/144622, or US2009/0252742 (each of which is incorporated herein by reference in its entirety). 
     In one embodiment, an antigen binding domain against CD22 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Haso et al., Blood, 121(7): 1165-1174 (2013); Wayne et al., Clin Cancer Res 16(6): 1894-1903 (2010); Kato et al., Leuk Res 37(1):83-88 (2013); Creative BioMart (creativebiomart.net): MOM-18047-S(P). 
     In one embodiment, an antigen binding domain against CS-1 is an antigen binding portion, e.g., CDRs, of Elotuzumab (BMS), see e.g., Tai et al., 2008, Blood 112(4):1329-37; Tai et al., 2007, Blood. 110(5):1656-63. 
     In one embodiment, an antigen binding domain against CLL-1 is an antigen binding portion, e.g., CDRs or VH and VL, of an antibody, antigen-binding fragment or CAR described in, e.g., PCT publication WO2016/014535, the contents of which are incorporated herein in their entirety. In one embodiment, an antigen binding domain against CLL-1 is an antigen binding portion, e.g., CDRs, of an antibody available from R&amp;D, ebiosciences, Abcam, for example, PE-CLL1-hu Cat #353604 (BioLegend); and PE-CLL1 (CLEC12A) Cat #562566 (BD). 
     In one embodiment, an antigen binding domain against CD33 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Bross et al., Clin Cancer Res 7(6):1490-1496 (2001) (Gemtuzumab Ozogamicin, hP67.6), Caron et al., Cancer Res 52(24):6761-6767 (1992) (Lintuzumab, HuM195), Lapusan et al., Invest New Drugs 30(3):1121-1131 (2012) (AVE9633), Aigner et al., Leukemia 27(5): 1107-1115 (2013) (AMG330, CD33 BiTE), Dutour et al., Adv hematol 2012:683065 (2012), and Pizzitola et al., Leukemia doi:10.1038/Lue.2014.62 (2014). Exemplary CAR molecules that target CD33 are described herein, and are provided in WO2016/014576, e.g., in Table 2 of WO2016/014576 (incorporated by reference in its entirety). 
     In one embodiment, an antigen binding domain against GD2 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Mujoo et al., Cancer Res. 47(4):1098-1104 (1987); Cheung et al., Cancer Res 45(6):2642-2649 (1985), Cheung et al., J Clin Oncol 5(9):1430-1440 (1987), Cheung et al., J Clin Oncol 16(9):3053-3060 (1998), Handgretinger et al., Cancer Immunol Immunother 35(3):199-204 (1992). In some embodiments, an antigen binding domain against GD2 is an antigen binding portion of an antibody selected from mAb 14.18, 14G2a, ch14.18, hu14.18, 3F8, hu3F8, 3G6, 8B6, 60C3, 10B8, ME36.1, and 8H9, see e.g., WO2012033885, WO2013040371, WO2013192294, WO2013061273, WO2013123061, WO2013074916, and WO201385552. In some embodiments, an antigen binding domain against GD2 is an antigen binding portion of an antibody described in US Publication No.: 20100150910 or PCT Publication No.: WO 2011160119. 
     In one embodiment, an antigen binding domain against BCMA is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., WO2012163805, WO200112812, and WO2003062401. In embodiments, additional exemplary BCMA CAR constructs are generated using an antigen binding domain, e.g., CDRs, scFv, or VH and VL sequences from PCT Publication WO2012/0163805 (the contents of which are hereby incorporated by reference in its entirety). In embodiments, additional exemplary BCMA CAR constructs are generated using an antigen binding domain, e.g., CDRs, scFv, or VH and VL sequences from PCT Publication WO2016/014565 (the contents of which are hereby incorporated by reference in its entirety). In embodiments, additional exemplary BCMA CAR constructs are generated using an antigen binding domain, e.g., CDRs, scFv, or VH and VL sequences from PCT Publication WO2014/122144 (the contents of which are hereby incorporated by reference in its entirety). In embodiments, additional exemplary BCMA CAR constructs are generated using the CAR molecules, and/or the BCMA binding domains (e.g., CDRs, scFv, or VH and VL sequences) from PCT Publication WO2016/014789 (the contents of which are hereby incorporated by reference in its entirety). In embodiments, additional exemplary BCMA CAR constructs are generated using the CAR molecules, and/or the BCMA binding domains (e.g., CDRs, scFv, or VH and VL sequences) from PCT Publication WO2014/089335 (the contents of which are hereby incorporated by reference in its entirety). In embodiments, additional exemplary BCMA CAR constructs are generated using the CAR molecules, and/or the BCMA binding domains (e.g., CDRs, scFv, or VH and VL sequences) from PCT Publication WO2014/140248 (the contents of which are hereby incorporated by reference in its entirety). 
     In one embodiment, an antigen binding domain against Tn antigen is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., US 2014/0178365, U.S. Pat. No. 8,440,798, Brooks et al., PNAS 107(22):10056-10061 (2010), and Stone et al., Oncolmmunology 1(6):863-873(2012). 
     In one embodiment, an antigen binding domain against PSMA is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Parker et al., Protein Expr Purif 89(2):136-145 (2013), US 20110268656 (J591 ScFv); Frigerio et al, European J Cancer 49(9):2223-2232 (2013) (scFvD2B); WO 2006125481 (mAbs 3/A12, 3/E7 and 3/F11) and single chain antibody fragments (scFv A5 and D7). 
     In one embodiment, an antigen binding domain against ROR1 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Hudecek et al., Clin Cancer Res 19(12):3153-3164 (2013); WO 2011159847; and US20130101607. 
     In one embodiment, an antigen binding domain against FLT3 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., WO2011076922, U.S. Pat. No. 5,777,084, EP0754230, US20090297529, and several commercial catalog antibodies (R&amp;D, ebiosciences, Abcam). 
     In one embodiment, an antigen binding domain against TAG72 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Hombach et al., Gastroenterology 113(4):1163-1170 (1997); and Abcam ab691. 
     In one embodiment, an antigen binding domain against FAP is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Ostermann et al., Clinical Cancer Research 14:4584-4592 (2008) (FAP5), US Pat. Publication No. 2009/0304718; sibrotuzumab (see e.g., Hofheinz et al., Oncology Research and Treatment 26(1), 2003); and Tran et al., J Exp Med 210(6):1125-1135 (2013). 
     In one embodiment, an antigen binding domain against CD38 is an antigen binding portion, e.g., CDRs, of daratumumab (see, e.g., Groen et al., Blood 116(21):1261-1262 (2010); MOR202 (see, e.g., U.S. Pat. No. 8,263,746); or antibodies described in U.S. Pat. No. 8,362,211. 
     In one embodiment, an antigen binding domain against CD44v6 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Casucci et al., Blood 122(20):3461-3472 (2013). 
     In one embodiment, an antigen binding domain against CEA is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Chmielewski et al., Gastoenterology 143(4):1095-1107 (2012). 
     In one embodiment, an antigen binding domain against EPCAM is an antigen binding portion, e.g., CDRS, of an antibody selected from MT110, EpCAM-CD3 bispecific Ab (see, e.g., clinicaltrials.gov/ct2/show/NCT00635596); Edrecolomab; 3622W94; ING-1; and adecatumumab (MT201). 
     In one embodiment, an antigen binding domain against PRSS21 is an antigen binding portion, e.g., CDRs, of an antibody described in U.S. Pat. No. 8,080,650. 
     In one embodiment, an antigen binding domain against B7H3 is an antigen binding portion, e.g., CDRs, of an antibody MGA271 (Macrogenics). 
     In one embodiment, an antigen binding domain against KIT is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., U.S. Pat. No. 7,915,391, US20120288506, and several commercial catalog antibodies. 
     In one embodiment, an antigen binding domain against IL-13Ra2 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., WO2008/146911, WO2004087758, several commercial catalog antibodies, and WO2004087758. 
     In one embodiment, an antigen binding domain against CD30 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., U.S. Pat. No. 7,090,843 B1, and EP0805871. 
     In one embodiment, an antigen binding domain against GD3 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., U.S. Pat. Nos. 7,253,263; 8,207,308; US 20120276046; EP1013761; WO2005035577; and U.S. Pat. No. 6,437,098. 
     In one embodiment, an antigen binding domain against CD171 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Hong et al., J Immunother 37(2):93-104 (2014). 
     In one embodiment, an antigen binding domain against IL-11Ra is an antigen binding portion, e.g., CDRs, of an antibody available from Abcam (cat #ab55262) or Novus Biologicals (cat #EPR5446). In another embodiment, an antigen binding domain again IL-11Ra is a peptide, see, e.g., Huang et al., Cancer Res 72(1):271-281 (2012). 
     In one embodiment, an antigen binding domain against PSCA is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Morgenroth et al., Prostate 67(10):1121-1131 (2007) (scFv 7F5); Nejatollahi et al., J of Oncology 2013(2013), article ID 839831 (scFv C5-II); and US Pat Publication No. 20090311181. 
     In one embodiment, an antigen binding domain against VEGFR2 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Chinnasamy et al., J Clin Invest 120(11):3953-3968 (2010). 
     In one embodiment, an antigen binding domain against LewisY is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Kelly et al., Cancer Biother Radiopharm 23(4):411-423 (2008) (hu3S193 Ab (scFvs)); Dolezal et al., Protein Engineering 16(1):47-56 (2003) (NC10 scFv). 
     In one embodiment, an antigen binding domain against CD24 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Maliar et al., Gastroenterology 143(5):1375-1384 (2012). 
     In one embodiment, an antigen binding domain against PDGFR-beta is an antigen binding portion, e.g., CDRs, of an antibody Abcam ab32570. 
     In one embodiment, an antigen binding domain against SSEA-4 is an antigen binding portion, e.g., CDRs, of antibody MC813 (Cell Signaling), or other commercially available antibodies. 
     In one embodiment, an antigen binding domain against CD20 is an antigen binding portion, e.g., CDRs, of the antibody Rituximab, Ofatumumab, Ocrelizumab, Veltuzumab, or GA101. 
     In one embodiment, an antigen binding domain against Folate receptor alpha is an antigen binding portion, e.g., CDRs, of the antibody IMGN853, or an antibody described in US20120009181; U.S. Pat. No. 4,851,332, LK26: U.S. Pat. No. 5,952,484. 
     In one embodiment, an antigen binding domain against ERBB2 (Her2/neu) is an antigen binding portion, e.g., CDRs, of the antibody trastuzumab, or pertuzumab. 
     In one embodiment, an antigen binding domain against MUC1 is an antigen binding portion, e.g., CDRs, of the antibody SAR566658. 
     In one embodiment, the antigen binding domain against EGFR is antigen binding portion, e.g., CDRs, of the antibody cetuximab, panitumumab, zalutumumab, nimotuzumab, or matuzumab. In one embodiment, the antigen binding domain against EGFRvIII is or may be derived from an antigen binding domain, e.g., CDRs, scFv, or VH and VL, of an antibody, antigen-binding fragment or CAR described in, e.g., PCT publication WO2014/130657 (In one embodiment the CAR is a CAR described in WO2014/130657, the contents of which are incorporated herein in their entirety). 
     In one embodiment, an antigen binding domain against NCAM is an antigen binding portion, e.g., CDRs, of the antibody clone 2-2B: MAB5324 (EMD Millipore) 
     In one embodiment, an antigen binding domain against Ephrin B2 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Abengozar et al., Blood 119(19):4565-4576 (2012). 
     In one embodiment, an antigen binding domain against IGF-I receptor is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., U.S. Pat. No. 8,344,112 B2; EP2322550 A1; WO 2006/138315, or PCT/US2006/022995. 
     In one embodiment, an antigen binding domain against CAIX is an antigen binding portion, e.g., CDRs, of the antibody clone 303123 (R&amp;D Systems). 
     In one embodiment, an antigen binding domain against LMP2 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., U.S. Pat. No. 7,410,640, or US20050129701. 
     In one embodiment, an antigen binding domain against gp100 is an antigen binding portion, e.g., CDRs, of the antibody HM41B45, NKIbetaB, or an antibody described in WO2013165940, or US20130295007 In one embodiment, an antigen binding domain against tyrosinase is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., U.S. Pat. No. 5,843,674; or U.S. Ser. No. 19/950,504048. 
     In one embodiment, an antigen binding domain against EphA2 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Yu et al., Mol Ther 22(1):102-111 (2014). 
     In one embodiment, an antigen binding domain against GD3 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., U.S. Pat. Nos. 7,253,263; 8,207,308; US 20120276046; EP1013761 A3; 20120276046; WO2005035577; or U.S. Pat. No. 6,437,098. 
     In one embodiment, an antigen binding domain against fucosyl GM1 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., US20100297138; or WO2007/067992. 
     In one embodiment, an antigen binding domain against sLe is an antigen binding portion, e.g., CDRs, of the antibody G193 (for lewis Y), see Scott A M et al, Cancer Res 60: 3254-61 (2000), also as described in Neeson et al, J Immunol May 2013 190 (Meeting Abstract Supplement) 177.10. 
     In one embodiment, an antigen binding domain against GM3 is an antigen binding portion, e.g., CDRs, of the antibody CA 2523449 (mAb 14F7). 
     In one embodiment, an antigen binding domain against HMWMAA is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Kmiecik et al., Oncoimmunology 3(1):e27185 (2014) (PMID: 24575382) (mAb9.2.27); U.S. Pat. No. 6,528,481; WO2010033866; or US 20140004124. 
     In one embodiment, an antigen binding domain against o-acetyl-GD2 is an antigen binding portion, e.g., CDRs, of the antibody 8B6. 
     In one embodiment, an antigen binding domain against TEM1/CD248 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Marty et al., Cancer Lett 235(2):298-308 (2006); Zhao et al., J Immunol Methods 363(2):221-232 (2011). 
     In one embodiment, an antigen binding domain against CLDN6 is an antigen binding portion, e.g., CDRs, of the antibody IMAB027 (Ganymed Pharmaceuticals), see e.g., clinicaltrial.gov/show/NCT02054351. 
     In one embodiment, an antigen binding domain against TSHR is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., U.S. Pat. Nos. 8,603,466; 8,501,415; or U.S. Pat. No. 8,309,693. 
     In one embodiment, an antigen binding domain against GPRC5D is an antigen binding portion, e.g., CDRs, of the antibody FAB6300A (R&amp;D Systems); or LS-A4180 (Lifespan Biosciences). 
     In one embodiment, an antigen binding domain against CD97 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., U.S. Pat. No. 6,846,911; de Groot et al., J Immunol 183(6):4127-4134 (2009); or an antibody from R&amp;D:MAB3734. 
     In one embodiment, an antigen binding domain against ALK is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Mino-Kenudson et al., Clin Cancer Res 16(5):1561-1571 (2010). 
     In one embodiment, an antigen binding domain against polysialic acid is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Nagae et al., J Biol Chem 288(47):33784-33796 (2013). 
     In one embodiment, an antigen binding domain against PLAC1 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Ghods et al., Biotechnol Appl Biochem 2013 doi:10.1002/bab.1177. 
     In one embodiment, an antigen binding domain against GloboH is an antigen binding portion of the antibody VK9; or an antibody described in, e.g., Kudryashov V et al, Glycoconj J.15(3):243-9 (1998), Lou et al., Proc Natl Acad Sci USA 111(7):2482-2487 (2014); MBr1: Bremer E-G et al. J Biol Chem 259:14773-14777 (1984). 
     In one embodiment, an antigen binding domain against NY-BR-1 is an antigen binding portion, e.g., CDRs of an antibody described in, e.g., Jager et al., Appl Immunohistochem Mol Morphol 15(1):77-83 (2007). 
     In one embodiment, an antigen binding domain against WT-1 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Dao et al., Sci Transl Med 5(176):176ra33 (2013); or WO2012/135854. 
     In one embodiment, an antigen binding domain against MAGE-A1 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Willemsen et al., J Immunol 174(12):7853-7858 (2005) (TCR-like scFv). 
     In one embodiment, an antigen binding domain against sperm protein 17 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Song et al., Target Oncol 2013 Aug. 14 (PMID: 23943313); Song et al., Med Oncol 29(4):2923-2931 (2012). 
     In one embodiment, an antigen binding domain against Tie 2 is an antigen binding portion, e.g., CDRs, of the antibody AB33 (Cell Signaling Technology). 
     In one embodiment, an antigen binding domain against MAD-CT-2 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., PMID: 2450952; U.S. Pat. No. 7,635,753. 
     In one embodiment, an antigen binding domain against Fos-related antigen 1 is an antigen binding portion, e.g., CDRs, of the antibody 12F9 (Novus Biologicals). 
     In one embodiment, an antigen binding domain against MelanA/MART1 is an antigen binding portion, e.g., CDRs, of an antibody described in, EP2514766 A2; or U.S. Pat. No. 7,749,719. 
     In one embodiment, an antigen binding domain against sarcoma translocation breakpoints is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Luo et al, EMBO Mol. Med. 4(6):453-461 (2012). 
     In one embodiment, an antigen binding domain against TRP-2 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Wang et al, J Exp Med. 184(6):2207-16 (1996). 
     In one embodiment, an antigen binding domain against CYP1B1 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Maecker et al, Blood 102 (9): 3287-3294 (2003). 
     In one embodiment, an antigen binding domain against RAGE-1 is an antigen binding portion, e.g., CDRs, of the antibody MAB5328 (EMD Millipore). 
     In one embodiment, an antigen binding domain against human telomerase reverse transcriptase is an antigen binding portion, e.g., CDRs, of the antibody cat no: LS-B95-100 (Lifespan Biosciences) 
     In one embodiment, an antigen binding domain against intestinal carboxyl esterase is an antigen binding portion, e.g., CDRs, of the antibody 4F12: cat no: LS-B6190-50 (Lifespan Biosciences). 
     In one embodiment, an antigen binding domain against mut hsp70-2 is an antigen binding portion, e.g., CDRs, of the antibody Lifespan Biosciences: monoclonal: cat no: LS-C133261-100 (Lifespan Biosciences). 
     In one embodiment, an antigen binding domain against CD79a is an antigen binding portion, e.g., CDRs, of the antibody Anti-CD79a antibody [HM47/A9] (ab3121), available from Abcam; antibody CD79A Antibody #3351 available from Cell Signalling Technology; or antibody HPA017748-Anti-CD79A antibody produced in rabbit, available from Sigma Aldrich. 
     In one embodiment, an antigen binding domain against CD79b is an antigen binding portion, e.g., CDRs, of the antibody polatuzumab vedotin, anti-CD79b described in Dornan et al., “Therapeutic potential of an anti-CD79b antibody-drug conjugate, anti-CD79b-vc-MMAE, for the treatment of non-Hodgkin lymphoma” Blood. 2009 Sep. 24; 114(13):2721-9. doi: 10.1182/blood-2009-02-205500. Epub 2009 Jul. 24, or the bispecific antibody Anti-CD79b/CD3 described in “4507 Pre-Clinical Characterization of T Cell-Dependent Bispecific Antibody Anti-CD79b/CD3 As a Potential Therapy for B Cell Malignancies” Abstracts of 56 th  ASH Annual Meeting and Exposition, San Francisco, Calif. Dec. 6-9, 2014. 
     In one embodiment, an antigen binding domain against CD72 is an antigen binding portion, e.g., CDRs, of the antibody J3-109 described in Myers, and Uckun, “An anti-CD72 immunotoxin against therapy-refractory B-lineage acute lymphoblastic leukemia.” Leuk Lymphoma. 1995 Jun.; 18(1-2):119-22, or anti-CD72 (10D6.8.1, mIgG1) described in Polson et al., “Antibody-Drug Conjugates for the Treatment of Non-Hodgkin&#39;s Lymphoma: Target and Linker-Drug Selection” Cancer Res Mar. 15, 2009 69; 2358. 
     In one embodiment, an antigen binding domain against LAIR1 is an antigen binding portion, e.g., CDRs, of the antibody ANT-301 LAIR1 antibody, available from ProSpec; or anti-human CD305 (LAIR1) Antibody, available from BioLegend. 
     In one embodiment, an antigen binding domain against FCAR is an antigen binding portion, e.g., CDRs, of the antibody CD89/FCARAntibody (Catalog #10414-H08H), available from Sino Biological Inc. 
     In one embodiment, an antigen binding domain against LILRA2 is an antigen binding portion, e.g., CDRs, of the antibody LILRA2 monoclonal antibody (M17), clone 3C7, available from Abnova, or Mouse Anti-LILRA2 antibody, Monoclonal (2D7), available from Lifespan Biosciences. 
     In one embodiment, an antigen binding domain against CD300LF is an antigen binding portion, e.g., CDRs, of the antibody Mouse Anti-CMRF35-like molecule 1 antibody, Monoclonal[UP-D2], available from BioLegend, or Rat Anti-CMRF35-like molecule 1 antibody, Monoclonal[234903], available from R&amp;D Systems. 
     In one embodiment, an antigen binding domain against CLEC12A is an antigen binding portion, e.g., CDRs, of the antibody Bispecific T cell Engager (BiTE) scFv-antibody and ADC described in Noordhuis et al., “Targeting of CLEC12A In Acute Myeloid Leukemia by Antibody-Drug-Conjugates and Bispecific CLL-1×CD3 BiTE Antibody” 53 rd  ASH Annual Meeting and Exposition, Dec. 10-13, 2011, and MCLA-117 (Merus). 
     In one embodiment, an antigen binding domain against BST2 (also called CD317) is an antigen binding portion, e.g., CDRs, of the antibody Mouse Anti-CD317 antibody, Monoclonal[3H4], available from Antibodies-Online or Mouse Anti-CD317 antibody, Monoclonal[696739], available from R&amp;D Systems. 
     In one embodiment, an antigen binding domain against EMR2 (also called CD312) is an antigen binding portion, e.g., CDRs, of the antibody Mouse Anti-CD312 antibody, Monoclonal[LS-B8033] available from Lifespan Biosciences, or Mouse Anti-CD312 antibody, Monoclonal[494025] available from R&amp;D Systems. 
     In one embodiment, an antigen binding domain against LY75 is an antigen binding portion, e.g., CDRs, of the antibody Mouse Anti-Lymphocyte antigen 75 antibody, Monoclonal[HD30] available from EMD Millipore or Mouse Anti-Lymphocyte antigen 75 antibody, Monoclonal[A15797] available from Life Technologies. 
     In one embodiment, an antigen binding domain against GPC3 is an antigen binding portion, e.g., CDRs, of the antibody hGC33 described in Nakano K, Ishiguro T, Konishi H, et al. Generation of a humanized anti-glypican 3 antibody by CDR grafting and stability optimization. Anticancer Drugs. 2010 Nov.; 21(10):907-916, or MDX-1414, HN3, or YP7, all three of which are described in Feng et al., “Glypican-3 antibodies: a new therapeutic target for liver cancer.” FEBS Lett. 2014 Jan. 21; 588(2):377-82. 
     In one embodiment, an antigen binding domain against FCRL5 is an antigen binding portion, e.g., CDRs, of the anti-FcRL5 antibody described in Elkins et al., “FcRL5 as a target of antibody-drug conjugates for the treatment of multiple myeloma” Mol Cancer Ther. 2012 Oct.; 11(10):2222-32. 
     In one embodiment, an antigen binding domain against IGLL1 is an antigen binding portion, e.g., CDRs, of the antibody Mouse Anti-Immunoglobulin lambda-like polypeptide 1 antibody, Monoclonal[A T1G4] available from Lifespan Biosciences, Mouse Anti-Immunoglobulin lambda-like polypeptide 1 antibody, Monoclonal[HSL11] available from BioLegend. 
     In one embodiment, the antigen binding domain comprises one, two three (e.g., all three) heavy chain CDRs, HC CDR1, HC CDR2 and HC CDR3, from an antibody listed above, and/or one, two, three (e.g., all three) light chain CDRs, LC CDR1, LC CDR2 and LC CDR3, from an antibody listed above. In one embodiment, the antigen binding domain comprises a heavy chain variable region and/or a variable light chain region of an antibody listed above. 
     In another aspect, the antigen binding domain comprises a humanized antibody or an antibody fragment. In some aspects, a non-human antibody is humanized, where specific sequences or regions of the antibody are modified to increase similarity to an antibody naturally produced in a human or fragment thereof. In one aspect, the antigen binding domain is humanized. 
     A humanized antibody can be produced using a variety of techniques known in the art, including but not limited to, CDR-grafting (see, e.g., European Patent No. EP 239,400; International Publication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089, each of which is incorporated herein in its entirety by reference), veneering or resurfacing (see, e.g., European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnicka et al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al., 1994, PNAS, 91:969-973, each of which is incorporated herein by its entirety by reference), chain shuffling (see, e.g., U.S. Pat. No. 5,565,332, which is incorporated herein in its entirety by reference), and techniques disclosed in, e.g., U.S. Patent Application Publication No. US2005/0042664, U.S. Patent Application Publication No. US2005/0048617, U.S. Pat. Nos. 6,407,213, 5,766,886, International Publication No. WO 9317105, Tan et al., J. Immunol., 169:1119-25 (2002), Caldas et al., Protein Eng., 13(5):353-60 (2000), Morea et al., Methods, 20(3):267-79 (2000), Baca et al., J. Biol. Chem., 272(16):10678-84 (1997), Roguska et al., Protein Eng., 9(10):895-904 (1996), Couto et al., Cancer Res., 55 (23 Supp):5973s-5977s (1995), Couto et al., Cancer Res., 55(8):1717-22 (1995), Sandhu J S, Gene, 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol., 235(3):959-73 (1994), each of which is incorporated herein in its entirety by reference. Often, framework residues in the framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, for example improve, antigen binding. These framework substitutions are identified by methods well-known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323, which are incorporated herein by reference in their entireties.) 
     A humanized antibody or antibody fragment has one or more amino acid residues remaining in it from a source which is nonhuman. These nonhuman amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. As provided herein, humanized antibodies or antibody fragments comprise one or more CDRs from nonhuman immunoglobulin molecules and framework regions wherein the amino acid residues comprising the framework are derived completely or mostly from human germline. Multiple techniques for humanization of antibodies or antibody fragments are well-known in the art and can essentially be performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody, i.e., CDR-grafting (EP 239,400; PCT Publication No. WO 91/09967; and U.S. Pat. Nos. 4,816,567; 6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640, the contents of which are incorporated herein by reference herein in their entirety). In such humanized antibodies and antibody fragments, substantially less than an intact human variable domain has been substituted by the corresponding sequence from a nonhuman species. Humanized antibodies are often human antibodies in which some CDR residues and possibly some framework (FR) residues are substituted by residues from analogous sites in rodent antibodies. Humanization of antibodies and antibody fragments can also be achieved by veneering or resurfacing (EP 592,106; EP 519,596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnicka et al., Protein Engineering, 7(6):805-814 (1994); and Roguska et al., PNAS, 91:969-973 (1994)) or chain shuffling (U.S. Pat. No. 5,565,332), the contents of which are incorporated herein by reference herein in their entirety. 
     The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is to reduce antigenicity. According to the so-called “best-fit” method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987), the contents of which are incorporated herein by reference herein in their entirety). Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (see, e.g., Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997); Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993), the contents of which are incorporated herein by reference herein in their entirety). In some embodiments, the framework region, e.g., all four framework regions, of the heavy chain variable region are derived from a VH4_4-59 germline sequence. In one embodiment, the framework region can comprise, one, two, three, four or five modifications, e.g., substitutions, e.g., from the amino acid at the corresponding murine sequence. In one embodiment, the framework region, e.g., all four framework regions of the light chain variable region are derived from a VK3_1.25 germline sequence. In one embodiment, the framework region can comprise, one, two, three, four or five modifications, e.g., substitutions, e.g., from the amino acid at the corresponding murine sequence. 
     In some aspects, the portion of a CAR composition of the invention that comprises an antibody fragment is humanized with retention of high affinity for the target antigen and other favorable biological properties. According to one aspect of the invention, humanized antibodies and antibody fragments are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, e.g., the analysis of residues that influence the ability of the candidate immunoglobulin to bind the target antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody or antibody fragment characteristic, such as increased affinity for the target antigen, is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding. 
     A humanized antibody or antibody fragment may retain a similar antigenic specificity as the original antibody, e.g., in the present invention, the ability to bind human a cancer associated antigen as described herein. In some embodiments, a humanized antibody or antibody fragment may have improved affinity and/or specificity of binding to human a cancer associated antigen as described herein. 
     In one aspect, the antigen binding domain of the invention is characterized by particular functional features or properties of an antibody or antibody fragment. For example, in one aspect, the portion of a CAR composition of the invention that comprises an antigen binding domain specifically binds a tumor antigen as described herein. 
     In one aspect, the anti-cancer associated antigen as described herein binding domain is a fragment, e.g., a single chain variable fragment (scFv). In one aspect, the anti-cancer associated antigen as described herein binding domain is a Fv, a Fab, a (Fab′)2, or a bi-functional (e.g. bi-specific) hybrid antibody (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)). In one aspect, the antibodies and fragments thereof of the invention binds a cancer associated antigen as described herein protein with wild-type or enhanced affinity. 
     In some instances, scFvs can be prepared according to method known in the art (see, for example, Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). ScFv molecules can be produced by linking VH and VL regions together using flexible polypeptide linkers. The scFv molecules comprise a linker (e.g., a Ser-Gly linker) with an optimized length and/or amino acid composition. The linker length can greatly affect how the variable regions of a scFv fold and interact. In fact, if a short polypeptide linker is employed (e.g., between 5-10 amino acids) intrachain folding is prevented. Interchain folding is also required to bring the two variable regions together to form a functional epitope binding site. For examples of linker orientation and size see, e.g., Hollinger et al. 1993 Proc Natl Acad. Sci. U.S.A. 90:6444-6448, U.S. Patent Application Publication Nos. 2005/0100543, 2005/0175606, 2007/0014794, and PCT publication Nos. WO2006/020258 and WO2007/024715, is incorporated herein by reference. 
     An scFv can comprise a linker of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more amino acid residues between its VL and VH regions. The linker sequence may comprise any naturally occurring amino acid. In some embodiments, the linker sequence comprises amino acids glycine and serine. In another embodiment, the linker sequence comprises sets of glycine and serine repeats such as (Gly 4 Ser)n, where n is a positive integer equal to or greater than 1 (SEQ ID NO:22). In one embodiment, the linker can be (Gly 4 Ser) 4  (SEQ ID NO:29) or (Gly 4 Ser) 3  (SEQ ID NO:30). Variation in the linker length may retain or enhance activity, giving rise to superior efficacy in activity studies. 
     In another aspect, the antigen binding domain is a T cell receptor (“TCR”), or a fragment thereof, for example, a single chain TCR (scTCR). Methods to make such TCRs are known in the art. See, e.g., Willemsen R A et al, Gene Therapy 7: 1369-1377 (2000); Zhang T et al, Cancer Gene Ther 11: 487-496 (2004); Aggen et al, Gene Ther. 19(4):365-74 (2012) (references are incorporated herein by its entirety). For example, scTCR can be engineered that contains the Vα and Vβ genes from a T cell clone linked by a linker (e.g., a flexible peptide). This approach is very useful to cancer associated target that itself is intracelluar, however, a fragment of such antigen (peptide) is presented on the surface of the cancer cells by MHC. 
     Bispecific CARs 
     In an embodiment a multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity for no more than two antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence which has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. In an embodiment the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In an embodiment the first and second epitopes overlap. In an embodiment the first and second epitopes do not overlap. In an embodiment the first and second epitopes are on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In an embodiment a bispecific antibody molecule comprises a heavy chain variable domain sequence and a light chain variable domain sequence which have binding specificity for a first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence which have binding specificity for a second epitope. In an embodiment a bispecific antibody molecule comprises a half antibody having binding specificity for a first epitope and a half antibody having binding specificity for a second epitope. In an embodiment a bispecific antibody molecule comprises a half antibody, or fragment thereof, having binding specificity for a first epitope and a half antibody, or fragment thereof, having binding specificity for a second epitope. In an embodiment a bispecific antibody molecule comprises a scFv, or fragment thereof, have binding specificity for a first epitope and a scFv, or fragment thereof, have binding specificity for a second epitope. 
     In certain embodiments, the antibody molecule is a multi-specific (e.g., a bispecific or a trispecific) antibody molecule. Protocols for generating bispecific or heterodimeric antibody molecules are known in the art; including but not limited to, for example, the “knob in a hole” approach described in, e.g., U.S. Pat. No. 5,731,168; the electrostatic steering Fc pairing as described in, e.g., WO 09/089004, WO 06/106905 and WO 2010/129304; Strand Exchange Engineered Domains (SEED) heterodimer formation as described in, e.g., WO 07/110205; Fab arm exchange as described in, e.g., WO 08/119353, WO 2011/131746, and WO 2013/060867; double antibody conjugate, e.g., by antibody cross-linking to generate a bi-specific structure using a heterobifunctional reagent having an amine-reactive group and a sulfhydryl reactive group as described in, e.g., U.S. Pat. No. 4,433,059; bispecific antibody determinants generated by recombining half antibodies (heavy-light chain pairs or Fabs) from different antibodies through cycle of reduction and oxidation of disulfide bonds between the two heavy chains, as described in, e.g., U.S. Pat. No. 4,444,878; trifunctional antibodies, e.g., three Fab′ fragments cross-linked through sulfhydryl reactive groups, as described in, e.g., U.S. Pat. No. 5,273,743; biosynthetic binding proteins, e.g., pair of scFvs cross-linked through C-terminal tails preferably through disulfide or amine-reactive chemical cross-linking, as described in, e.g., U.S. Pat. No. 5,534,254; bifunctional antibodies, e.g., Fab fragments with different binding specificities dimerized through leucine zippers (e.g., c-fos and c-jun) that have replaced the constant domain, as described in, e.g., U.S. Pat. No. 5,582,996; bispecific and oligospecific mono- and oligovalent receptors, e.g., VH-CH1 regions of two antibodies (two Fab fragments) linked through a polypeptide spacer between the CH1 region of one antibody and the VH region of the other antibody typically with associated light chains, as described in, e.g., U.S. Pat. No. 5,591,828; bispecific DNA-antibody conjugates, e.g., crosslinking of antibodies or Fab fragments through a double stranded piece of DNA, as described in, e.g., U.S. Pat. No. 5,635,602; bispecific fusion proteins, e.g., an expression construct containing two scFvs with a hydrophilic helical peptide linker between them and a full constant region, as described in, e.g., U.S. Pat. No. 5,637,481; multivalent and multispecific binding proteins, e.g., dimer of polypeptides having first domain with binding region of Ig heavy chain variable region, and second domain with binding region of Ig light chain variable region, generally termed diabodies (higher order structures are also encompassed creating for bispecifc, trispecific, or tetraspecific molecules, as described in, e.g., U.S. Pat. No. 5,837,242; minibody constructs with linked VL and VH chains further connected with peptide spacers to an antibody hinge region and CH3 region, which can be dimerized to form bispecific/multivalent molecules, as described in, e.g., U.S. Pat. No. 5,837,821; VH and VL domains linked with a short peptide linker (e.g., 5 or 10 amino acids) or no linker at all in either orientation, which can form dimers to form bispecific diabodies; trimers and tetramers, as described in, e.g., U.S. Pat. No. 5,844,094; String of VH domains (or VL domains in family members) connected by peptide linkages with crosslinkable groups at the C-terminus further associated with VL domains to form a series of FVs (or scFvs), as described in, e.g., U.S. Pat. No. 5,864,019; and single chain binding polypeptides with both a VH and a VL domain linked through a peptide linker are combined into multivalent structures through non-covalent or chemical crosslinking to form, e.g., homobivalent, heterobivalent, trivalent, and tetravalent structures using both scFV or diabody type format, as described in, e.g., U.S. Pat. No. 5,869,620. Additional exemplary multispecific and bispecific molecules and methods of making the same are found, for example, in U.S. Pat. Nos. 5,910,573, 5,932,448, 5,959,083, 5,989,830, 6,005,079, 6,239,259, 6,294,353, 6,333,396, 6,476,198, 6,511,663, 6,670,453, 6,743,896, 6,809,185, 6,833,441, 7,129,330, 7,183,076, 7,521,056, 7,527,787, 7,534,866, 7,612,181, US2002004587A1, US2002076406A1, US2002103345A1, US2003207346A1, US2003211078A1, US2004219643A1, US2004220388A1, US2004242847A1, US2005003403A1, US2005004352A1, US2005069552A1, US2005079170A1, US2005100543A1, US2005136049A1, US2005136051A1, US2005163782A1, US2005266425A1, US2006083747A1, US2006120960A1, US2006204493A1, US2006263367A1, US2007004909A1, US2007087381A1, US2007128150A1, US2007141049A1, US2007154901A1, US2007274985A1, US2008050370A1, US2008069820A1, US2008152645A1, US2008171855A1, US2008241884A1, US2008254512A1, US2008260738A1, US2009130106A1, US2009148905A1, US2009155275A1, US2009162359A1, US2009162360A1, US2009175851A1, US2009175867A1, US2009232811A1, US2009234105A1, US2009263392A1, US2009274649A1, EP346087A2, WO0006605A2, WO02072635A2, WO04081051A1, WO06020258A2, WO2007044887A2, WO2007095338A2, WO2007137760A2, WO2008119353A1, WO2009021754A2, WO2009068630A1, WO9103493A1, WO9323537A1, WO9409131A1, WO9412625A2, WO9509917A1, WO9637621A2, WO9964460A1. The contents of the above-referenced applications are incorporated herein by reference in their entireties. 
     Within each antibody or antibody fragment (e.g., scFv) of a bispecific antibody molecule, the VH can be upstream or downstream of the VL. In some embodiments, the upstream antibody or antibody fragment (e.g., scFv) is arranged with its VH (VH 1 ) upstream of its VL (VL 1 ) and the downstream antibody or antibody fragment (e.g., scFv) is arranged with its VL (VL 2 ) upstream of its VH (VH 2 ), such that the overall bispecific antibody molecule has the arrangement VH 1 -VL 1 -VL 2 -VH 2 . In other embodiments, the upstream antibody or antibody fragment (e.g., scFv) is arranged with its VL (VL 1 ) upstream of its VH (VH 1 ) and the downstream antibody or antibody fragment (e.g., scFv) is arranged with its VH (VH 2 ) upstream of its VL (VL 2 ), such that the overall bispecific antibody molecule has the arrangement VL 1 -VH 1 -VH 2 -VL 2 . Optionally, a linker is disposed between the two antibodies or antibody fragments (e.g., scFvs), e.g., between VL 1  and VL 2  if the construct is arranged as VH 1 -VL 1 -VL 2 -VH 2 , or between VH 1  and VH 2  if the construct is arranged as VL 1 -VH 1 -VH 2 -VL 2 . The linker may be a linker as described herein, e.g., a (Gly4-Ser)n linker, wherein n is 1, 2, 3, 4, 5, or 6, preferably 4 (SEQ ID NO: 72). In general, the linker between the two scFvs should be long enough to avoid mispairing between the domains of the two scFvs. Optionally, a linker is disposed between the VL and VH of the first scFv. Optionally, a linker is disposed between the VL and VH of the second scFv. In constructs that have multiple linkers, any two or more of the linkers can be the same or different. Accordingly, in some embodiments, a bispecific CAR comprises VLs, VHs, and optionally one or more linkers in an arrangement as described herein. 
     Stability and Mutations 
     The stability of an antigen binding domain to a cancer associated antigen as described herein, e.g., scFv molecules (e.g., soluble scFv), can be evaluated in reference to the biophysical properties (e.g., thermal stability) of a conventional control scFv molecule or a full length antibody. In one embodiment, the humanized scFv has a thermal stability that is greater than about 0.1, about 0.25, about 0.5, about 0.75, about 1, about 1.25, about 1.5, about 1.75, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10 degrees, about 11 degrees, about 12 degrees, about 13 degrees, about 14 degrees, or about 15 degrees Celsius than a control binding molecule (e.g. a conventional scFv molecule) in the described assays. 
     The improved thermal stability of the antigen binding domain to a cancer associated antigen described herein, e.g., scFv is subsequently conferred to the entire CAR construct, leading to improved therapeutic properties of the CAR construct. The thermal stability of the antigen binding domain of—a cancer associated antigen described herein, e.g., scFv, can be improved by at least about 2° C. or 3° C. as compared to a conventional antibody. In one embodiment, the antigen binding domain of—a cancer associated antigen described herein, e.g., scFv, has a 1° C. improved thermal stability as compared to a conventional antibody. In another embodiment, the antigen binding domain of a cancer associated antigen described herein, e.g., scFv, has a 2° C. improved thermal stability as compared to a conventional antibody. In another embodiment, the scFv has a 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15° C. improved thermal stability as compared to a conventional antibody. Comparisons can be made, for example, between the scFv molecules disclosed herein and scFv molecules or Fab fragments of an antibody from which the scFv VH and VL were derived. Thermal stability can be measured using methods known in the art. For example, in one embodiment, Tm can be measured. Methods for measuring Tm and other methods of determining protein stability are described in more detail below. 
     Mutations in scFv (arising through humanization or direct mutagenesis of the soluble scFv) can alter the stability of the scFv and improve the overall stability of the scFv and the CAR construct. Stability of the humanized scFv is compared against the murine scFv using measurements such as Tm, temperature denaturation and temperature aggregation. 
     The binding capacity of the mutant scFvs can be determined using assays know in the art and described herein. 
     In one embodiment, the antigen binding domain of—a cancer associated antigen described herein, e.g., scFv, comprises at least one mutation arising from the humanization process such that the mutated scFv confers improved stability to the CAR construct. In another embodiment, the antigen binding domain of—a cancer associated antigen described herein, e.g., scFv, comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mutations arising from the humanization process such that the mutated scFv confers improved stability to the CAR construct. 
     Methods of Evaluating Protein Stability 
     The stability of an antigen binding domain may be assessed using, e.g., the methods described below. Such methods allow for the determination of multiple thermal unfolding transitions where the least stable domain either unfolds first or limits the overall stability threshold of a multidomain unit that unfolds cooperatively (e.g., a multidomain protein which exhibits a single unfolding transition). The least stable domain can be identified in a number of additional ways. Mutagenesis can be performed to probe which domain limits the overall stability. Additionally, protease resistance of a multidomain protein can be performed under conditions where the least stable domain is known to be intrinsically unfolded via DSC or other spectroscopic methods (Fontana, et al., (1997) Fold. Des., 2: R17-26; Dimasi et al. (2009) J. Mol. Biol. 393: 672-692). Once the least stable domain is identified, the sequence encoding this domain (or a portion thereof) may be employed as a test sequence in the methods. 
     a) Thermal Stability 
     The thermal stability of the compositions may be analyzed using a number of non-limiting biophysical or biochemical techniques known in the art. In certain embodiments, thermal stability is evaluated by analytical spectroscopy. 
     An exemplary analytical spectroscopy method is Differential Scanning Calorimetry (DSC). DSC employs a calorimeter which is sensitive to the heat absorbances that accompany the unfolding of most proteins or protein domains (see, e.g. Sanchez-Ruiz, et al., Biochemistry, 27: 1648-52, 1988). To determine the thermal stability of a protein, a sample of the protein is inserted into the calorimeter and the temperature is raised until the Fab or scFv unfolds. The temperature at which the protein unfolds is indicative of overall protein stability. 
     Another exemplary analytical spectroscopy method is Circular Dichroism (CD) spectroscopy. CD spectrometry measures the optical activity of a composition as a function of increasing temperature. Circular dichroism (CD) spectroscopy measures differences in the absorption of left-handed polarized light versus right-handed polarized light which arise due to structural asymmetry. A disordered or unfolded structure results in a CD spectrum very different from that of an ordered or folded structure. The CD spectrum reflects the sensitivity of the proteins to the denaturing effects of increasing temperature and is therefore indicative of a protein&#39;s thermal stability (see van Mierlo and Steemsma, J. Biotechnol., 79(3):281-98, 2000). 
     Another exemplary analytical spectroscopy method for measuring thermal stability is Fluorescence Emission Spectroscopy (see van Mierlo and Steemsma, supra). Yet another exemplary analytical spectroscopy method for measuring thermal stability is Nuclear Magnetic Resonance (NMR) spectroscopy (see, e.g. van Mierlo and Steemsma, supra). 
     The thermal stability of a composition can be measured biochemically. An exemplary biochemical method for assessing thermal stability is a thermal challenge assay. In a “thermal challenge assay”, a composition is subjected to a range of elevated temperatures for a set period of time. For example, in one embodiment, test scFv molecules or molecules comprising scFv molecules are subject to a range of increasing temperatures, e.g., for 1-1.5 hours. The activity of the protein is then assayed by a relevant biochemical assay. For example, if the protein is a binding protein (e.g. an scFv or scFv-containing polypeptide) the binding activity of the binding protein may be determined by a functional or quantitative ELISA. 
     Such an assay may be done in a high-throughput format and those disclosed in the Examples using  E. coli  and high throughput screening. A library of antigen binding domains, e.g., that includes an antigen binding domain to—a cancer associated antigen described herein, e.g., scFv variants, may be created using methods known in the art. Antigen binding domain, e.g., to—a cancer associated antigen described herein, e.g., scFv, expression may be induced and the antigen binding domain, e.g., to—a cancer associated antigen described herein, e.g., scFv, may be subjected to thermal challenge. The challenged test samples may be assayed for binding and those antigen binding domains to—a cancer associated antigen described herein, e.g., scFvs, which are stable may be scaled up and further characterized. 
     Thermal stability is evaluated by measuring the melting temperature (Tm) of a composition using any of the above techniques (e.g. analytical spectroscopy techniques). The melting temperature is the temperature at the midpoint of a thermal transition curve wherein 50% of molecules of a composition are in a folded state (See e.g., Dimasi et al. (2009) J. Mol Biol. 393: 672-692). In one embodiment, Tm values for an antigen binding domain to—a cancer associated antigen described herein, e.g., scFv, are about 40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48° C., 49° C., 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C., 59° C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C., 67° C., 68° C., 69° C., 70° C., 71° C., 72° C., 73° C., 74° C., 75° C., 76° C., 77° C., 78° C., 79° C., 80° C., 81° C., 82° C., 83° C., 84° C., 85° C., 86° C., 87° C., 88° C., 89° C., 90° C., 91° C., 92° C., 93° C., 94° C., 95° C., 96° C., 97° C., 98° C., 99° C., 100° C. In one embodiment, Tm values for an IgG is about 40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48° C., 49° C., 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C., 59° C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C., 67° C., 68° C., 69° C., 70° C., 71° C., 72° C., 73° C., 74° C., 75° C., 76° C., 77° C., 78° C., 79° C., 80° C., 81° C., 82° C., 83° C., 84° C., 85° C., 86° C., 87° C., 88° C., 89° C., 90° C., 91° C., 92° C., 93° C., 94° C., 95° C., 96° C., 97° C., 98° C., 99° C., 100° C. In one embodiment, Tm values for an multivalent antibody is about 40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48° C., 49° C., 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C., 59° C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C., 67° C., 68° C., 69° C., 70° C., 71° C., 72° C., 73° C., 74° C., 75° C., 76° C., 77° C., 78° C., 79° C., 80° C., 81° C., 82° C., 83° C., 84° C., 85° C., 86° C., 87° C., 88° C., 89° C., 90° C., 91° C., 92° C., 93° C., 94° C., 95° C., 96° C., 97° C., 98° C., 99° C., 100° C. 
     Thermal stability is also evaluated by measuring the specific heat or heat capacity (Cp) of a composition using an analytical calorimetric technique (e.g. DSC). The specific heat of a composition is the energy (e.g. in kcal/mol) is required to rise by 1° C., the temperature of 1 mol of water. As large Cp is a hallmark of a denatured or inactive protein composition. The change in heat capacity (ΔCp) of a composition is measured by determining the specific heat of a composition before and after its thermal transition. Thermal stability may also be evaluated by measuring or determining other parameters of thermodynamic stability including Gibbs free energy of unfolding (ΔG), enthalpy of unfolding (ΔH), or entropy of unfolding (ΔS). One or more of the above biochemical assays (e.g. a thermal challenge assay) are used to determine the temperature (i.e. the Tc value) at which 50% of the composition retains its activity (e.g. binding activity). 
     In addition, mutations to the antigen binding domain of a cancer associated antigen described herein, e.g., scFv, can be made to alter the thermal stability of the antigen binding domain of a cancer associated antigen described herein, e.g., scFv, as compared with the unmutated antigen binding domain of a cancer associated antigen described herein, e.g., scFv. When the humanized antigen binding domain of a cancer associated antigen described herein, e.g., scFv, is incorporated into a CAR construct, the antigen binding domain of the cancer associated antigen described herein, e.g., humanized scFv, confers thermal stability to the overall CARs of the present invention. In one embodiment, the antigen binding domain to a cancer associated antigen described herein, e.g., scFv, comprises a single mutation that confers thermal stability to the antigen binding domain of the cancer associated antigen described herein, e.g., scFv. In another embodiment, the antigen binding domain to a cancer associated antigen described herein, e.g., scFv, comprises multiple mutations that confer thermal stability to the antigen binding domain to the cancer associated antigen described herein, e.g., scFv. In one embodiment, the multiple mutations in the antigen binding domain to a cancer associated antigen described herein, e.g., scFv, have an additive effect on thermal stability of the antigen binding domain to the cancer associated antigen described herein binding domain, e.g., scFv. 
     b) % Aggregation 
     The stability of a composition can be determined by measuring its propensity to aggregate. Aggregation can be measured by a number of non-limiting biochemical or biophysical techniques. For example, the aggregation of a composition may be evaluated using chromatography, e.g. Size-Exclusion Chromatography (SEC). SEC separates molecules on the basis of size. A column is filled with semi-solid beads of a polymeric gel that will admit ions and small molecules into their interior but not large ones. When a protein composition is applied to the top of the column, the compact folded proteins (i.e. non-aggregated proteins) are distributed through a larger volume of solvent than is available to the large protein aggregates. Consequently, the large aggregates move more rapidly through the column, and in this way the mixture can be separated or fractionated into its components. Each fraction can be separately quantified (e.g. by light scattering) as it elutes from the gel. Accordingly, the % aggregation of a composition can be determined by comparing the concentration of a fraction with the total concentration of protein applied to the gel. Stable compositions elute from the column as essentially a single fraction and appear as essentially a single peak in the elution profile or chromatogram. 
     c) Binding Affinity 
     The stability of a composition can be assessed by determining its target binding affinity. A wide variety of methods for determining binding affinity are known in the art. An exemplary method for determining binding affinity employs surface plasmon resonance. Surface plasmon resonance is an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). For further descriptions, see Jonsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26; Jonsson, U., i (1991) Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8:125-131; and Johnnson, B., et al. (1991) Anal. Biochem. 198:268-277. 
     In one aspect, the antigen binding domain of the CAR comprises an amino acid sequence that is homologous to an antigen binding domain amino acid sequence described herein, and the antigen binding domain retains the desired functional properties of the antigen binding domain described herein. 
     In one specific aspect, the CAR composition of the invention comprises an antibody fragment. In a further aspect, the antibody fragment comprises an scFv. 
     In various aspects, the antigen binding domain of the CAR is engineered by modifying one or more amino acids within one or both variable regions (e.g., VH and/or VL), for example within one or more CDR regions and/or within one or more framework regions. In one specific aspect, the CAR composition of the invention comprises an antibody fragment. In a further aspect, the antibody fragment comprises an scFv. 
     It will be understood by one of ordinary skill in the art that the antibody or antibody fragment of the invention may further be modified such that they vary in amino acid sequence (e.g., from wild-type), but not in desired activity. For example, additional nucleotide substitutions leading to amino acid substitutions at “non-essential” amino acid residues may be made to the protein For example, a nonessential amino acid residue in a molecule may be replaced with another amino acid residue from the same side chain family. In another embodiment, a string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side chain family members, e.g., a conservative substitution, in which an amino acid residue is replaced with an amino acid residue having a similar side chain, may be made. 
     Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). 
     Percent identity in the context of two or more nucleic acids or polypeptide sequences, refers to two or more sequences that are the same. Two sequences are “substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 60% identity, optionally 70%, 71%. 72%. 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length. 
     For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman, (1988) Proc. Nat′l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Brent et al., (2003) Current Protocols in Molecular Biology). 
     Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., (1977) Nuc. Acids Res. 25:3389-3402; and Altschul et al., (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. 
     The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, (1988) Comput. Appl. Biosci. 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. 
     In one aspect, the present invention contemplates modifications of the starting antibody or fragment (e.g., scFv) amino acid sequence that generate functionally equivalent molecules. For example, the VH or VL of an antigen binding domain to—a cancer associated antigen described herein, e.g., scFv, comprised in the CAR can be modified to retain at least about 70%, 71%. 72%. 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity of the starting VH or VL framework region of the antigen binding domain to the cancer associated antigen described herein, e.g., scFv. The present invention contemplates modifications of the entire CAR construct, e.g., modifications in one or more amino acid sequences of the various domains of the CAR construct in order to generate functionally equivalent molecules. The CAR construct can be modified to retain at least about 70%, 71%. 72%. 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%0, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity of the starting CAR construct. 
     Transmembrane Domain 
     With respect to the transmembrane domain, in various embodiments, a CAR can be designed to comprise a transmembrane domain that is attached to the extracellular domain of the CAR. A transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acid associated with the extracellular region of the protein from which the transmembrane was derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the extracellular region) and/or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids of the intracellular region). In one aspect, the transmembrane domain is one that is associated with one of the other domains of the CAR e.g., in one embodiment, the transmembrane domain may be from the same protein that the signaling domain, costimulatory domain or the hinge domain is derived from. In another aspect, the transmembrane domain is not derived from the same protein that any other domain of the CAR is derived from. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, e.g., to minimize interactions with other members of the receptor complex. In one aspect, the transmembrane domain is capable of homodimerization with another CAR on the cell surface of a CAR-expressing cell. In a different aspect, the amino acid sequence of the transmembrane domain may be modified or substituted so as to minimize interactions with the binding domains of the native binding partner present in the same CAR-expressing cell. 
     The transmembrane domain may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. In one aspect the transmembrane domain is capable of signaling to the intracellular domain(s) whenever the CAR has bound to a target. A transmembrane domain of particular use in this invention may include at least the transmembrane region(s) of e.g., the alpha, beta or zeta chain of the T-cell receptor, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In some embodiments, a transmembrane domain may include at least the transmembrane region(s) of, e.g., KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11 d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG/Cbp, NKG2D, NKG2C. 
     In some instances, the transmembrane domain can be attached to the extracellular region of the CAR, e.g., the antigen binding domain of the CAR, via a hinge, e.g., a hinge from a human protein. For example, in one embodiment, the hinge can be a human Ig (immunoglobulin) hinge (e.g., an IgG4 hinge, an IgD hinge), a GS linker (e.g., a GS linker described herein), a KIR2DS2 hinge or a CD8a hinge. In one embodiment, the hinge or spacer comprises (e.g., consists of) the amino acid sequence of SEQ ID NO:4. In one aspect, the transmembrane domain comprises (e.g., consists of) a transmembrane domain of SEQ ID NO: 12. 
     In one aspect, the hinge or spacer comprises an IgG4 hinge. For example, in one embodiment, the hinge or spacer comprises a hinge of the amino acid sequence 
                            (SEQ ID NO: 6)           ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMI                   SRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK                   TKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKV                   SNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEM                   TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT                   TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVM                   HEALHNHYTQKSLSLSLGKM.            
In some embodiments, the hinge or spacer comprises a hinge encoded by a nucleotide sequence of
 
     
       
         
           
               
            
               
                 (SEQ ID NO: 7) 
               
               
                 GAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCT 
               
               
                   
               
               
                 GGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGAGACCCTGA 
               
               
                   
               
               
                 TGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGACGTGTCCCAG 
               
               
                   
               
               
                 GAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCA 
               
               
                   
               
               
                 CAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTACCGGG 
               
               
                   
               
               
                 TGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAA 
               
               
                   
               
               
                 TACAAGTGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCGAGAAAAC 
               
               
                   
               
               
                 CATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTACACCCTGC 
               
               
                   
               
               
                 CCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTG 
               
               
                   
               
               
                 GTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGG 
               
               
                   
               
               
                 CCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACG 
               
               
                   
               
               
                 GCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAG 
               
               
                   
               
               
                 GAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCA 
               
               
                   
               
               
                 CTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGATG. 
               
            
           
         
       
     
     In one aspect, the hinge or spacer comprises an IgD hinge. For example, in one embodiment, the hinge or spacer comprises a hinge of the amino acid sequence 
                            (SEQ ID NO: 8)           RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTR                   NTGRGGEEKKKEKEKEEQEERETKTPECPSHTQPL                   GVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLT                   WEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPR                   SLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPV                   KLSLNLLASSDPPEAASWLLCEVSGFSPPNILLMW                   LEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVP                   APPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVT                   DH            
In some embodiments, the hinge or spacer comprises a hinge encoded by a nucleotide sequence of
 
     
       
         
           
               
            
               
                 (SEQ ID NO: 9) 
               
               
                 AGGTGGCCCGAAAGTCCCAAGGCCCAGGCATCTAGTGTTCCTACTGCACA 
               
               
                   
               
               
                 GCCCCAGGCAGAAGGCAGCCTAGCCAAAGCTACTACTGCACCTGCCACTA 
               
               
                   
               
               
                 CGCGCAATACTGGCCGTGGCGGGGAGGAGAAGAAAAAGGAGAAAGAGAAA 
               
               
                   
               
               
                 GAAGAACAGGAAGAGAGGGAGACCAAGACCCCTGAATGTCCATCCCATAC 
               
               
                   
               
               
                 CCAGCCGCTGGGCGTCTATCTCTTGACTCCCGCAGTACAGGACTTGTGGC 
               
               
                   
               
               
                 TTAGAGATAAGGCCACCTTTACATGTTTCGTCGTGGGCTCTGACCTGAAG 
               
               
                   
               
               
                 GATGCCCATTTGACTTGGGAGGTTGCCGGAAAGGTACCCACAGGGGGGGT 
               
               
                   
               
               
                 TGAGGAAGGGTTGCTGGAGCGCCATTCCAATGGCTCTCAGAGCCAGCACT 
               
               
                   
               
               
                 CAAGACTCACCCTTCCGAGATCCCTGTGGAACGCCGGGACCTCTGTCACA 
               
               
                   
               
               
                 TGTACTCTAAATCATCCTAGCCTGCCCCCACAGCGTCTGATGGCCCTTAG 
               
               
                   
               
               
                 AGAGCCAGCCGCCCAGGCACCAGTTAAGCTTAGCCTGAATCTGCTCGCCA 
               
               
                   
               
               
                 GTAGTGATCCCCCAGAGGCCGCCAGCTGGCTCTTATGCGAAGTGTCCGGC 
               
               
                   
               
               
                 TTTAGCCCGCCCAACATCTTGCTCATGTGGCTGGAGGACCAGCGAGAAGT 
               
               
                   
               
               
                 GAACACCAGCGGCTTCGCTCCAGCCCGGCCCCCACCCCAGCCGGGTTCTA 
               
               
                   
               
               
                 CCACATTCTGGGCCTGGAGTGTCTTAAGGGTCCCAGCACCACCTAGCCCC 
               
               
                   
               
               
                 CAGCCAGCCACATACACCTGTGTTGTGTCCCATGAAGATAGCAGGACCCT 
               
               
                   
               
               
                 GCTAAATGCTTCTAGGAGTCTGGAGGTTTCCTACGTGACTGACCATT. 
               
            
           
         
       
     
     In one aspect, the transmembrane domain may be recombinant, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In one aspect a triplet of phenylalanine, tryptophan and valine can be found at each end of a recombinant transmembrane domain. 
     Optionally, a short oligo- or polypeptide linker, between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the cytoplasmic region of the CAR. A glycine-serine doublet provides a particularly suitable linker. For example, in one aspect, the linker comprises the amino acid sequence of GGGGSGGGGS (SEQ ID NO: 10). In some embodiments, the linker is encoded by a nucleotide sequence of 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 11) 
               
               
                   
                 GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC. 
               
            
           
         
       
     
     In one aspect, the hinge or spacer comprises a KIR2DS2 hinge. 
     Cytoplasmic Domain 
     The cytoplasmic domain or region of the CAR includes an intracellular signaling domain. An intracellular signaling domain is generally responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR has been introduced. The term “effector function” refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. Thus the term “intracellular signaling domain” refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The term intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal. 
     Examples of intracellular signaling domains for use in the CAR of the invention include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any recombinant sequence that has the same functional capability. 
     It is known that signals generated through the TCR alone are insufficient for full activation of the T cell and that a secondary and/or costimulatory signal is also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary intracellular signaling domains) and those that act in an antigen-independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic domain, e.g., a costimulatory domain). 
     A primary signaling domain regulates primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary intracellular signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs. 
     Examples of ITAM containing primary intracellular signaling domains that are of particular use in the invention include those of CD3 zeta, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc Epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12. In one embodiment, a CAR of the invention comprises an intracellular signaling domain, e.g., a primary signaling domain of CD3-zeta. 
     In one embodiment, a primary signaling domain comprises a modified ITAM domain, e.g., a mutated ITAM domain which has altered (e.g., increased or decreased) activity as compared to the native ITAM domain. In one embodiment, a primary signaling domain comprises a modified ITAM-containing primary intracellular signaling domain, e.g., an optimized and/or truncated ITAM-containing primary intracellular signaling domain. In an embodiment, a primary signaling domain comprises one, two, three, four or more ITAM motifs. 
     The intracellular signalling domain of the CAR can comprise the CD3-zeta signaling domain by itself or it can be combined with any other desired intracellular signaling domain(s) useful in the context of a CAR of the invention. For example, the intracellular signaling domain of the CAR can comprise a CD3 zeta chain portion and a costimulatory signaling domain. The costimulatory signaling domain refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligands that is required for an efficient response of lymphocytes to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, and the like. For example, CD27 costimulation has been demonstrated to enhance expansion, effector function, and survival of human CART cells in vitro and augments human T cell persistence and antitumor activity in vivo (Song et al. Blood. 2012; 119(3):696-706). Further examples of such costimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), NKG2D, CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, and CD19a. 
     The intracellular signaling sequences within the cytoplasmic portion of the CAR of the invention may be linked to each other in a random or specified order. Optionally, a short oligo- or polypeptide linker, for example, between 2 and 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) in length may form the linkage between intracellular signaling sequence. In one embodiment, a glycine-serine doublet can be used as a suitable linker. In one embodiment, a single amino acid, e.g., an alanine, a glycine, can be used as a suitable linker. 
     In one aspect, the intracellular signaling domain is designed to comprise two or more, e.g., 2, 3, 4, 5, or more, costimulatory signaling domains. In an embodiment, the two or more, e.g., 2, 3, 4, 5, or more, costimulatory signaling domains, are separated by a linker molecule, e.g., a linker molecule described herein. In one embodiment, the intracellular signaling domain comprises two costimulatory signaling domains. In some embodiments, the linker molecule is a glycine residue. In some embodiments, the linker is an alanine residue. 
     In one aspect, the intracellular signaling domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD28. In one aspect, the intracellular signaling domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of 4-1BB. In one aspect, the signaling domain of 4-1BB is a signaling domain of SEQ ID NO: 14. In one aspect, the signaling domain of CD3-zeta is a signaling domain of SEQ ID NO: 18. 
     In one aspect, the intracellular signaling domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD27. In one aspect, the signaling domain of CD27 comprises an amino acid sequence of 
                    (SEQ ID NO: 16)       QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP.            
In one aspect, the signalling domain of CD27 is encoded by a nucleic acid sequence of
 
     
       
         
           
               
            
               
                 (SEQ ID NO: 17) 
               
               
                 AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCC 
               
               
                   
               
               
                 CCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCAC 
               
               
                   
               
               
                 GCGACTTCGCAGCCTATCGCTCC. 
               
            
           
         
       
     
     In one aspect, the CAR-expressing cell described herein can further comprise a second CAR, e.g., a second CAR that includes a different antigen binding domain, e.g., to the same target or a different target (e.g., a target other than a cancer associated antigen described herein or a different cancer associated antigen described herein). In one embodiment, the second CAR includes an antigen binding domain to a target expressed the same cancer cell type as the cancer associated antigen. In one embodiment, the CAR-expressing cell comprises a first CAR that targets a first antigen and includes an intracellular signaling domain having a costimulatory signaling domain but not a primary signaling domain, and a second CAR that targets a second, different, antigen and includes an intracellular signaling domain having a primary signaling domain but not a costimulatory signaling domain. While not wishing to be bound by theory, placement of a costimulatory signaling domain, e.g., 4-1BB, CD28, CD27 or OX-40, onto the first CAR, and the primary signaling domain, e.g., CD3 zeta, on the second CAR can limit the CAR activity to cells where both targets are expressed. In one embodiment, the CAR expressing cell comprises a first cancer associated antigen CAR that includes an antigen binding domain that binds a target antigen described herein, a transmembrane domain and a costimulatory domain and a second CAR that targets a different target antigen (e.g., an antigen expressed on that same cancer cell type as the first target antigen) and includes an antigen binding domain, a transmembrane domain and a primary signaling domain. In another embodiment, the CAR expressing cell comprises a first CAR that includes an antigen binding domain that binds a target antigen described herein, a transmembrane domain and a primary signaling domain and a second CAR that targets an antigen other than the first target antigen (e.g., an antigen expressed on the same cancer cell type as the first target antigen) and includes an antigen binding domain to the antigen, a transmembrane domain and a costimulatory signaling domain. 
     In one embodiment, the CAR-expressing cell comprises an XCAR described herein and an inhibitory CAR. In one embodiment, the inhibitory CAR comprises an antigen binding domain that binds an antigen found on normal cells but not cancer cells, e.g., normal cells that also express CLL. In one embodiment, the inhibitory CAR comprises the antigen binding domain, a transmembrane domain and an intracellular domain of an inhibitory molecule. For example, the intracellular domain of the inhibitory CAR can be an intracellular domain of PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGF beta. 
     In one embodiment, when the CAR-expressing cell comprises two or more different CARs, the antigen binding domains of the different CARs can be such that the antigen binding domains do not interact with one another. For example, a cell expressing a first and second CAR can have an antigen binding domain of the first CAR, e.g., as a fragment, e.g., an scFv, that does not form an association with the antigen binding domain of the second CAR, e.g., the antigen binding domain of the second CAR is a VHH. 
     In some embodiments, the antigen binding domain comprises a single domain antigen binding (SDAB) molecules include molecules whose complementary determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain variable domains, binding molecules naturally devoid of light chains, single domains derived from conventional 4-chain antibodies, engineered domains and single domain scaffolds other than those derived from antibodies. SDAB molecules may be any of the art, or any future single domain molecules. SDAB molecules may be derived from any species including, but not limited to mouse, human, camel, llama, lamprey, fish, shark, goat, rabbit, and bovine. This term also includes naturally occurring single domain antibody molecules from species other than Camelidae and sharks. 
     In one aspect, an SDAB molecule can be derived from a variable region of the immunoglobulin found in fish, such as, for example, that which is derived from the immunoglobulin isotype known as Novel Antigen Receptor (NAR) found in the serum of shark. Methods of producing single domain molecules derived from a variable region of NAR (“IgNARs”) are described in WO 03/014161 and Streltsov (2005) Protein Sci. 14:2901-2909. 
     According to another aspect, an SDAB molecule is a naturally occurring single domain antigen binding molecule known as heavy chain devoid of light chains. Such single domain molecules are disclosed in WO 9404678 and Hamers-Casterman, C. et al. (1993) Nature 363:446-448, for example. For clarity reasons, this variable domain derived from a heavy chain molecule naturally devoid of light chain is known herein as a VHH or nanobody to distinguish it from the conventional VH of four chain immunoglobulins. Such a VHH molecule can be derived from Camelidae species, for example in camel, llama, dromedary, alpaca and guanaco. Other species besides Camelidae may produce heavy chain molecules naturally devoid of light chain; such VHHs are within the scope of the invention. 
     The SDAB molecules can be recombinant, CDR-grafted, humanized, camelized, de-immunized and/or in vitro generated (e.g., selected by phage display). 
     It has also been discovered, that cells having a plurality of chimeric membrane embedded receptors comprising an antigen binding domain that interactions between the antigen binding domain of the receptors can be undesirable, e.g., because it inhibits the ability of one or more of the antigen binding domains to bind its cognate antigen. Accordingly, disclosed herein are cells having a first and a second non-naturally occurring chimeric membrane embedded receptor comprising antigen binding domains that minimize such interactions. Also disclosed herein are nucleic acids encoding a first and a second non-naturally occurring chimeric membrane embedded receptor comprising a antigen binding domains that minimize such interactions, as well as methods of making and using such cells and nucleic acids. In an embodiment the antigen binding domain of one of said first said second non-naturally occurring chimeric membrane embedded receptor, comprises an scFv, and the other comprises a single VH domain, e.g., a camelid, shark, or lamprey single VH domain, or a single VH domain derived from a human or mouse sequence. 
     In some embodiments, the claimed invention comprises a first and second CAR, wherein the antigen binding domain of one of said first CAR said second CAR does not comprise a variable light domain and a variable heavy domain. In some embodiments, the antigen binding domain of one of said first CAR said second CAR is an scFv, and the other is not an scFv. In some embodiments, the antigen binding domain of one of said first CAR said second CAR comprises a single VH domain, e.g., a camelid, shark, or lamprey single VH domain, or a single VH domain derived from a human or mouse sequence. In some embodiments, the antigen binding domain of one of said first CAR said second CAR comprises a nanobody. In some embodiments, the antigen binding domain of one of said first CAR said second CAR comprises a camelid VHH domain. 
     In some embodiments, the antigen binding domain of one of said first CAR said second CAR comprises an scFv, and the other comprises a single VH domain, e.g., a camelid, shark, or lamprey single VH domain, or a single VH domain derived from a human or mouse sequence. In some embodiments, the antigen binding domain of one of said first CAR said second CAR comprises an scFv, and the other comprises a nanobody. In some embodiments, the antigen binding domain of one of said first CAR said second CAR comprises an scFv, and the other comprises a camelid VHH domain. 
     In some embodiments, when present on the surface of a cell, binding of the antigen binding domain of said first CAR to its cognate antigen is not substantially reduced by the presence of said second CAR. In some embodiments, binding of the antigen binding domain of said first CAR to its cognate antigen in the presence of said second CAR is 85%, 90%, 95%, 96%, 97%, 98% or 99% of binding of the antigen binding domain of said first CAR to its cognate antigen in the absence of said second CAR. 
     In some embodiments, when present on the surface of a cell, the antigen binding domains of said first CAR said second CAR, associate with one another less than if both were scFv antigen binding domains. In some embodiments, the antigen binding domains of said first CAR said second CAR, associate with one another 85%, 90%, 95%, 96%, 97%, 98% or 99% less than if both were scFv antigen binding domains. 
     In another aspect, the CAR-expressing cell described herein can further express another agent, e.g., an agent which enhances the activity of a CAR-expressing cell. For example, in one embodiment, the agent can be an agent which inhibits an inhibitory molecule. Inhibitory molecules, e.g., PD1, can, in some embodiments, decrease the ability of a CAR-expressing cell to mount an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGF beta. In one embodiment, the agent which inhibits an inhibitory molecule, e.g., is a molecule described herein, e.g., an agent that comprises a first polypeptide, e.g., an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain described herein. In one embodiment, the agent comprises a first polypeptide, e.g., of an inhibitory molecule such as PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGF beta, or a fragment of any of these (e.g., at least a portion of an extracellular domain of any of these), and a second polypeptide which is an intracellular signaling domain described herein (e.g., comprising a costimulatory domain (e.g., 41BB, CD27 or CD28, e.g., as described herein) and/or a primary signaling domain (e.g., a CD3 zeta signaling domain described herein). In one embodiment, the agent comprises a first polypeptide of PD1 or a fragment thereof (e.g., at least a portion of an extracellular domain of PD1), and a second polypeptide of an intracellular signaling domain described herein (e.g., a CD28 signaling domain described herein and/or a CD3 zeta signaling domain described herein). PD1 is an inhibitory member of the CD28 family of receptors that also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed on activated B cells, T cells and myeloid cells (Agata et al. 1996 Int. Immunol 8:765-75). Two ligands for PD1, PD-L1 and PD-L2 have been shown to downregulate T cell activation upon binding to PD1 (Freeman et a. 2000 J Exp Med 192:1027-34; Latchman et al. 2001 Nat Immunol 2:261-8; Carter et al. 2002 Eur J Immunol 32:634-43). PD-L1 is abundant in human cancers (Dong et al. 2003 J Mol Med 81:281-7; Blank et al. 2005 Cancer Immunol. Immunother 54:307-314; Konishi et al. 2004 Clin Cancer Res 10:5094). Immune suppression can be reversed by inhibiting the local interaction of PD1 with PD-L1. 
     In one embodiment, the agent comprises the extracellular domain (ECD) of an inhibitory molecule, e.g., Programmed Death 1 (PD1), fused to a transmembrane domain and intracellular signaling domains such as 41BB and CD3 zeta (also referred to herein as a PD1 CAR). In one embodiment, the PD1 CAR, when used in combinations with a XCAR described herein, improves the persistence of the T cell. In one embodiment, the CAR is a PD1 CAR comprising the extracellular domain of PD1 indicated as underlined in SEQ ID NO: 26. In one embodiment, the PD1 CAR comprises the amino acid sequence of SEQ ID NO: 26. 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 26) 
               
               
                   
                 Malpvtalllplalllhaarp pgwfldspdrpwnp   
               
               
                   
               
               
                   
                 
                   ptfspallvvtegdnatftcsfsntsesfvlnwyr 
                 
               
               
                   
               
               
                   
                 
                   mspsngtdklaafpedrsapgqdcrfrvtalpngr 
                 
               
               
                   
               
               
                   
                 
                   dfhmsvvrarrndsgtylcgaislapkaqikeslr 
                 
               
               
                   
               
               
                   
                   aelrvterraevptahpspsprpagqfatlv tttp 
               
               
                   
               
               
                   
                 aprpptpaptiasqplslrpeacrpaaggavhtrg 
               
               
                   
               
               
                   
                 ldfacdiyiwaplagtcgvillslvitlyckrgrk 
               
               
                   
               
               
                   
                 kllyifkqpfmrpvqttqeedgcscrfpeeeeggc 
               
               
                   
               
               
                   
                 elrvkfsrsadapaykqgqnqlynelnlgrreeyd 
               
               
                   
               
               
                   
                 vldkrrgrdpemggkprrknpqeglynelqkdkma 
               
               
                   
               
               
                   
                 eayseigmkgerrrgkghdglyqglstatkdtyda 
               
               
                   
               
               
                   
                 lhmqalppr. 
               
            
           
         
       
     
     In one embodiment, the PD1 CAR comprises the amino acid sequence provided below (SEQ ID NO: 39). 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 39) 
               
               
                   
                 
                   pgwfldspdrpwnpptfspallvvtegdnatftcs 
                 
               
               
                   
               
               
                   
                 
                   fsntsesfvlnwyrmspsngtdklaafpedrsapg 
                 
               
               
                   
               
               
                   
                 
                   adcrfrvtalpngrdfhmsvvrarrndsgtylcga 
                 
               
               
                   
               
               
                   
                 
                   islapkaqikeslraelrvterraevptahpspsp 
                 
               
               
                   
               
               
                   
                   rpagqfqtlv tttpaprpptpaptiasqplslrpe 
               
               
                   
               
               
                   
                 acrpaaggavhtrgldfacdiyiwaplagtcgvil 
               
               
                   
               
               
                   
                 lslvitlyckrgrkkllyifkqpfmrpvqttqeed 
               
               
                   
               
               
                   
                 gcscrfpeeeeggcelrvkfsrsadapaykqgqnq 
               
               
                   
               
               
                   
                 lynelnlgrreeydvldkrrgrdpemggkprrknp 
               
               
                   
               
               
                   
                 qeglynelqkdkmaeayseigmkgerrrgkghdgl 
               
               
                   
               
               
                   
                 yqglstatkdtydalhmqalppr. 
               
            
           
         
       
     
     In one embodiment, the agent comprises a nucleic acid sequence encoding the PD1 CAR, e.g., the PD1 CAR described herein. In one embodiment, the nucleic acid sequence for the PD1 CAR is shown below, with the PD1 ECD underlined below in SEQ ID NO: 27. 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 27) 
               
               
                   
                 
                   atggccctccctgtcactgccctgcttctccccct 
                 
               
               
                   
               
               
                   
                 
                   cgcactcctgctccacgccgctagaccacccggat 
                 
               
               
                   
               
               
                   
                 
                   ggtttctggactctccggatcgcccgtggaatccc 
                 
               
               
                   
               
               
                   
                 
                   ccaaccttctcaccggcactcttggttgtgactga 
                 
               
               
                   
               
               
                   
                 
                   gggcgataatgcgaccttcacgtgctcgttctcca 
                 
               
               
                   
               
               
                   
                 
                   acacctccgaatcattcgtgctgaactggtaccgc 
                 
               
               
                   
               
               
                   
                 
                   atgagcccgtcaaaccagaccgacaagctcgccgc 
                 
               
               
                   
               
               
                   
                 
                   gtttccggaagatcggtcgcaaccgggacaggatt 
                 
               
               
                   
               
               
                   
                 
                   gtcggttccgcgtgactcaactgccgaatggcaga 
                 
               
               
                   
               
               
                   
                 
                   gacttccacatgagcgtggtccgcgctaggcgaaa 
                 
               
               
                   
               
               
                   
                 
                   cgactccgggacctacctgtgcggagccatctcgc 
                 
               
               
                   
               
               
                   
                 
                   tggcgcctaaggcccaaatcaaagagagcttgagg 
                 
               
               
                   
               
               
                   
                 
                   gccgaactgagagtgaccgagcgcagagctgaggt 
                 
               
               
                   
               
               
                   
                 
                   gccaactgcacatccatccccatcgcctcggcctg 
                 
               
               
                   
               
               
                   
                   cggggcagtttcagaccctggtc acgaccactccg 
               
               
                   
               
               
                   
                 gcgccgcgcccaccgactccggccccaactatcgc 
               
               
                   
               
               
                   
                 gagccagcccctgtcgctgaggccggaagcatgcc 
               
               
                   
               
               
                   
                 gccctgccgccggaggtgctgtgcatacccgggga 
               
               
                   
               
               
                   
                 ttggacttcgcatgcgacatctacatttgggctcc 
               
               
                   
               
               
                   
                 tctcgccggaacttgtggcgtgctccttctgtccc 
               
               
                   
               
               
                   
                 tggtcatcaccctgtactgcaagcggggtcggaaa 
               
               
                   
               
               
                   
                 aagcttctgtacattttcaagcagcccttcatgag 
               
               
                   
               
               
                   
                 gcccgtgcaaaccacccaggaggaggacggttgct 
               
               
                   
               
               
                   
                 cctgccggttccccgaagaggaagaaggaggttgc 
               
               
                   
               
               
                   
                 gagctgcgcgtgaagttctcccggagcgccgacgc 
               
               
                   
               
               
                   
                 ccccgcctataagcagggccagaaccagctgtaca 
               
               
                   
               
               
                   
                 acgaactgaacctgggacggcgggaagagtacgat 
               
               
                   
               
               
                   
                 gtgctggacaagcggcgcggccgggaccccgaaat 
               
               
                   
               
               
                   
                 gggcgggaagcctagaagaaagaaccctcaggaag 
               
               
                   
               
               
                   
                 gcctgtataacgagctgcagaaggacaagatggcc 
               
               
                   
               
               
                   
                 gaggcctactccgaaattgggatgaagggagagcg 
               
               
                   
               
               
                   
                 gcggaggggaaaggggcacgacggcctgtaccaag 
               
               
                   
               
               
                   
                 gactgtccaccgccaccaaggacacatacgatgcc 
               
               
                   
               
               
                   
                 ctgcacatgcaggcccttccccctcgc. 
               
            
           
         
       
     
     In another aspect, the present invention provides a population of CAR-expressing cells, e.g., CART cells. In some embodiments, the population of CAR-expressing cells comprises a mixture of cells expressing different CARs. For example, in one embodiment, the population of CART cells can include a first cell expressing a CAR having an antigen binding domain to a cancer associated antigen described herein, and a second cell expressing a CAR having a different antigen binding domain, e.g., an antigen binding domain to a different a cancer associated antigen described herein, e.g., an antigen binding domain to a cancer associated antigen described herein that differs from the cancer associated antigen bound by the antigen binding domain of the CAR expressed by the first cell. As another example, the population of CAR-expressing cells can include a first cell expressing a CAR that includes an antigen binding domain to a cancer associated antigen described herein, and a second cell expressing a CAR that includes an antigen binding domain to a target other than a cancer associated antigen as described herein. In one embodiment, the population of CAR-expressing cells includes, e.g., a first cell expressing a CAR that includes a primary intracellular signaling domain, and a second cell expressing a CAR that includes a secondary signaling domain. 
     In another aspect, the present invention provides a population of cells wherein at least one cell in the population expresses a CAR having an antigen binding domain to a cancer associated antigen described herein, and a second cell expressing another agent, e.g., an agent which enhances the activity of a CAR-expressing cell. For example, in one embodiment, the agent can be an agent which inhibits an inhibitory molecule. Inhibitory molecules, e.g., PD-1, can, in some embodiments, decrease the ability of a CAR-expressing cell to mount an immune effector response. Examples of inhibitory molecules include PD-1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGF beta. In one embodiment, the agent which inhibits an inhibitory molecule, e.g., is a molecule described herein, e.g., an agent that comprises a first polypeptide, e.g., an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain described herein. In one embodiment, the agent comprises a first polypeptide, e.g., of an inhibitory molecule such as PD-1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGF beta, or a fragment of any of these, and a second polypeptide which is an intracellular signaling domain described herein (e.g., comprising a costimulatory domain (e.g., 41BB, CD27, OX40 or CD28, e.g., as described herein) and/or a primary signaling domain (e.g., a CD3 zeta signaling domain described herein). In one embodiment, the agent comprises a first polypeptide of PD-1 or a fragment thereof, and a second polypeptide of an intracellular signaling domain described herein (e.g., a CD28 signaling domain described herein and/or a CD3 zeta signaling domain described herein). 
     In one aspect, the present invention provides methods comprising administering a population of CAR-expressing cells, e.g., CART cells, e.g., a mixture of cells expressing different CARs, in combination with another agent, e.g., a kinase inhibitor, such as a kinase inhibitor described herein. In another aspect, the present invention provides methods comprising administering a population of cells wherein at least one cell in the population expresses a CAR having an antigen binding domain of a cancer associated antigen described herein, and a second cell expressing another agent, e.g., an agent which enhances the activity of a CAR-expressing cell, in combination with another agent, e.g., a kinase inhibitor, such as a kinase inhibitor described herein. 
     Regulatable Chimeric Antigen Receptors 
     In some embodiments, a regulatable CAR (RCAR) where the CAR activity can be controlled is desirable to optimize the safety and efficacy of a CAR therapy. There are many ways CAR activities can be regulated. For example, inducible apoptosis using, e.g., a caspase fused to a dimerization domain (see, e.g., Di et al., N Egnl. J. Med. 2011 Nov. 3; 365(18):1673-1683), can be used as a safety switch in the CAR therapy of the instant invention. In an aspect, a RCAR comprises a set of polypeptides, typically two in the simplest embodiments, in which the components of a standard CAR described herein, e.g., an antigen binding domain and an intracellular signaling domain, are partitioned on separate polypeptides or members. In some embodiments, the set of polypeptides include a dimerization switch that, upon the presence of a dimerization molecule, can couple the polypeptides to one another, e.g., can couple an antigen binding domain to an intracellular signaling domain. 
     In an aspect, an RCAR comprises two polypeptides or members: 1) an intracellular signaling member comprising an intracellular signaling domain, e.g., a primary intracellular signaling domain described herein, and a first switch domain; 2) an antigen binding member comprising an antigen binding domain, e.g., that targets a tumor antigen described herein, as described herein and a second switch domain. Optionally, the RCAR comprises a transmembrane domain described herein. In an embodiment, a transmembrane domain can be disposed on the intracellular signaling member, on the antigen binding member, or on both. (Unless otherwise indicated, when members or elements of an RCAR are described herein, the order can be as provided, but other orders are included as well. In other words, in an embodiment, the order is as set out in the text, but in other embodiments, the order can be different. E.g., the order of elements on one side of a transmembrane region can be different from the example, e.g., the placement of a switch domain relative to a intracellular signaling domain can be different, e.g., reversed). 
     In an embodiment, the first and second switch domains can form an intracellular or an extracellular dimerization switch. In an embodiment, the dimerization switch can be a homodimerization switch, e.g., where the first and second switch domain are the same, or a heterodimerization switch, e.g., where the first and second switch domain are different from one another. 
     In embodiments, an RCAR can comprise a “multi switch.” A multi switch can comprise heterodimerization switch domains or homodimerization switch domains. A multi switch comprises a plurality of, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, switch domains, independently, on a first member, e.g., an antigen binding member, and a second member, e.g., an intracellular signaling member. In an embodiment, the first member can comprise a plurality of first switch domains, e.g., FKBP-based switch domains, and the second member can comprise a plurality of second switch domains, e.g., FRB-based switch domains. In an embodiment, the first member can comprise a first and a second switch domain, e.g., a FKBP-based switch domain and a FRB-based switch domain, and the second member can comprise a first and a second switch domain, e.g., a FKBP-based switch domain and a FRB-based switch domain. 
     In an embodiment, the intracellular signaling member comprises one or more intracellular signaling domains, e.g., a primary intracellular signaling domain and one or more costimulatory signaling domains. 
     In an embodiment, the antigen binding member may comprise one or more intracellular signaling domains, e.g., one or more costimulatory signaling domains. In an embodiment, the antigen binding member comprises a plurality, e.g., 2 or 3 costimulatory signaling domains described herein, e.g., selected from 41BB, CD28, CD27, ICOS, and OX40, and in embodiments, no primary intracellular signaling domain. In an embodiment, the antigen binding member comprises the following costimulatory signaling domains, from the extracellular to intracellular direction: 41BB-CD27; 41BB-CD27; CD27-41BB; 41BB-CD28; CD28-41BB; OX40-CD28; CD28-OX40; CD28-41BB; or 41BB-CD28. In such embodiments, the intracellular binding member comprises a CD3zeta domain. In one such embodiment the RCAR comprises (1) an antigen binding member comprising, an antigen binding domain, a transmembrane domain, and two costimulatory domains and a first switch domain; and (2) an intracellular signaling domain comprising a transmembrane domain or membrane tethering domain and at least one primary intracellular signaling domain, and a second switch domain. 
     An embodiment provides RCARs wherein the antigen binding member is not tethered to the surface of the CAR cell. This allows a cell having an intracellular signaling member to be conveniently paired with one or more antigen binding domains, without transforming the cell with a sequence that encodes the antigen binding member. In such embodiments, the RCAR comprises: 1) an intracellular signaling member comprising: a first switch domain, a transmembrane domain, an intracellular signaling domain, e.g., a primary intracellular signaling domain, and a first switch domain; and 2) an antigen binding member comprising: an antigen binding domain, and a second switch domain, wherein the antigen binding member does not comprise a transmembrane domain or membrane tethering domain, and, optionally, does not comprise an intracellular signaling domain. In some embodiments, the RCAR may further comprise 3) a second antigen binding member comprising: a second antigen binding domain, e.g., a second antigen binding domain that binds a different antigen than is bound by the antigen binding domain; and a second switch domain. 
     Also provided herein are RCARs wherein the antigen binding member comprises bispecific activation and targeting capacity. In this embodiment, the antigen binding member can comprise a plurality, e.g., 2, 3, 4, or 5 antigen binding domains, e.g., scFvs, wherein each antigen binding domain binds to a target antigen, e.g. different antigens or the same antigen, e.g., the same or different epitopes on the same antigen. In an embodiment, the plurality of antigen binding domains are in tandem, and optionally, a linker or hinge region is disposed between each of the antigen binding domains. Suitable linkers and hinge regions are described herein. 
     An embodiment provides RCARs having a configuration that allows switching of proliferation. In this embodiment, the RCAR comprises: 1) an intracellular signaling member comprising: optionally, a transmembrane domain or membrane tethering domain; one or more co-stimulatory signaling domain, e.g., selected from 41BB, CD28, CD27, ICOS, and OX40, and a switch domain; and 2) an antigen binding member comprising: an antigen binding domain, a transmembrane domain, and a primary intracellular signaling domain, e.g., a CD3zeta domain, wherein the antigen binding member does not comprise a switch domain, or does not comprise a switch domain that dimerizes with a switch domain on the intracellular signaling member. In an embodiment, the antigen binding member does not comprise a co-stimulatory signaling domain. In an embodiment, the intracellular signaling member comprises a switch domain from a homodimerization switch. In an embodiment, the intracellular signaling member comprises a first switch domain of a heterodimerization switch and the RCAR comprises a second intracellular signaling member which comprises a second switch domain of the heterodimerization switch. In such embodiments, the second intracellular signaling member comprises the same intracellular signaling domains as the intracellular signaling member. In an embodiment, the dimerization switch is intracellular. In an embodiment, the dimerization switch is extracellular. 
     In any of the RCAR configurations described here, the first and second switch domains comprise a FKBP-FRB based switch as described herein. 
     Also provided herein are cells comprising an RCAR described herein. Any cell that is engineered to express a RCAR can be used as a RCARX cell. In an embodiment the RCARX cell is a T cell, and is referred to as a RCART cell. In an embodiment the RCARX cell is an NK cell, and is referred to as a RCARN cell. 
     Also provided herein are nucleic acids and vectors comprising RCAR encoding sequences. Sequence encoding various elements of an RCAR can be disposed on the same nucleic acid molecule, e.g., the same plasmid or vector, e.g., viral vector, e.g., lentiviral vector. In an embodiment, (i) sequence encoding an antigen binding member and (ii) sequence encoding an intracellular signaling member, can be present on the same nucleic acid, e.g., vector. Production of the corresponding proteins can be achieved, e.g., by the use of separate promoters, or by the use of a bicistronic transcription product (which can result in the production of two proteins by cleavage of a single translation product or by the translation of two separate protein products). In an embodiment, a sequence encoding a cleavable peptide, e.g., a P2A or F2A sequence, is disposed between (i) and (ii). Examples of peptide cleavage sites include the following, wherein the GSG residues are optional: 
     
       
         
           
               
            
               
                 T2A: 
               
               
                 (SEQ ID NO: 68) 
               
               
                 (GSG) E G R G S L L T C G D V E E N P G P 
               
               
                   
               
               
                 P2A: 
               
               
                 (SEQ ID NO: 69) 
               
               
                 (GSG) A T N F S L L K Q A G D V E E N P G P 
               
               
                   
               
               
                 E2A: 
               
               
                 (SEQ ID NO: 70) 
               
               
                 (GSG)Q C T N Y A L L K L A G D V E S N P G P 
               
               
                   
               
               
                 F2A: 
               
               
                 (SEQ ID NO: 71) 
               
               
                 (GSG) V K Q T L N F D L L K L A G D V E S N P G P 
               
            
           
         
       
     
     In an embodiment, a sequence encoding an IRES, e.g., an EMCV or EV71 IRES, is disposed between (i) and (ii). In these embodiments, (i) and (ii) are transcribed as a single RNA. In an embodiment, a first promoter is operably linked to (i) and a second promoter is operably linked to (ii), such that (i) and (ii) are transcribed as separate mRNAs. 
     Alternatively, the sequence encoding various elements of an RCAR can be disposed on the different nucleic acid molecules, e.g., different plasmids or vectors, e.g., viral vector, e.g., lentiviral vector. E.g., the (i) sequence encoding an antigen binding member can be present on a first nucleic acid, e.g., a first vector, and the (ii) sequence encoding an intracellular signaling member can be present on the second nucleic acid, e.g., the second vector. 
     Dimerization Switches 
     Dimerization switches can be non-covalent or covalent. In a non-covalent dimerization switch, the dimerization molecule promotes a non-covalent interaction between the switch domains. In a covalent dimerization switch, the dimerization molecule promotes a covalent interaction between the switch domains. 
     In an embodiment, the RCAR comprises a FKBP/FRAP, or FKBP/FRB-based dimerization switch. FKBP12 (FKBP, or FK506 binding protein) is an abundant cytoplasmic protein that serves as the initial intracellular target for the natural product immunosuppressive drug, rapamycin. Rapamycin binds to FKBP and to the large PI3K homolog FRAP (RAFT, mTOR). FRB is a 93 amino acid portion of FRAP, that is sufficient for binding the FKBP-rapamycin complex (Chen, J., Zheng, X. F., Brown, E. J. &amp; Schreiber, S. L. (1995)  Identification of an  11- kDa FKBP 12- rapamycin - binding domain within the  289- kDa FKBP 12- rapamycin - associated protein and characterization of a critical serine residue . Proc Natl Acad Sci USA 92: 4947-51.) 
     In embodiments, an FKBP/FRAP, e.g., an FKBP/FRB, based switch can use a dimerization molecule, e.g., rapamycin or a rapamycin analog. 
     The amino acid sequence of FKBP is as follows: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 54) 
               
               
                   
                 D V P D Y A S L G G P S S P K K K 
               
               
                   
               
               
                   
                 R K V S R G V Q V E T I S P G D G 
               
               
                   
               
               
                   
                 R T F P K R G Q T C V V H Y T G M 
               
               
                   
               
               
                   
                 L E D G K K F D S S R D R N K P F 
               
               
                   
               
               
                   
                 K F M L G K Q E V I R G W E E G V 
               
               
                   
               
               
                   
                 A Q M S V G Q R A K L T I S P D Y 
               
               
                   
               
               
                   
                 A Y G A T G H P G I I P P H A T L 
               
               
                   
               
               
                   
                 V F D V E L L K L E T S Y 
               
            
           
         
       
     
     In embodiments, an FKBP switch domain can comprise a fragment of FKBP having the ability to bind with FRB, or a fragment or analog thereof, in the presence of rapamycin or a rapalog, e.g., the underlined portion of SEQ ID NO: 54, which is: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 55) 
               
               
                   
                 V Q V E T I S P G D G R T F P K R 
               
               
                   
               
               
                   
                 G Q T C V V H Y T G M L E D G K K 
               
               
                   
               
               
                   
                 F D S S R D R N K P F K F M L G K 
               
               
                   
               
               
                   
                 Q E V I R G W E E G V A Q M S V G 
               
               
                   
               
               
                   
                 Q R A K L T I S P D Y A Y G A T G 
               
               
                   
               
               
                   
                 H P G I I P P H A T L V F D V E L 
               
               
                   
               
               
                   
                 L K L E T S 
               
            
           
         
       
     
     The amino acid sequence of FRB is as follows: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 56) 
               
               
                   
                 ILWHEMWHEG LEEASRLYFG ERNVKGMFEV 
               
               
                   
               
               
                   
                 LEPLHAMMER GPQTLKETSF NQAYGRDLME 
               
               
                   
               
               
                   
                 AQEWCRKYMK SGNVKDLTQA WDLYYHVFRR 
               
               
                   
               
               
                   
                 ISK 
               
            
           
         
       
     
     “FKBP/FRAP, e.g., an FKBP/FRB, based switch” as that term is used herein, refers to a dimerization switch comprising: a first switch domain, which comprises an FKBP fragment or analog thereof having the ability to bind with FRB, or a fragment or analog thereof, in the presence of rapamycin or a rapalog, e.g., RAD001, and has at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identity with, or differs by no more than 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid residues from, the FKBP sequence of SEQ ID NO: 54 or 55; and a second switch domain, which comprises an FRB fragment or analog thereof having the ability to bind with FRB, or a fragment or analog thereof, in the presence of rapamycin or a rapalog, and has at least 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identity with, or differs by no more than 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 amino acid residues from, the FRB sequence of SEQ ID NO: 56. In an embodiment, a RCAR described herein comprises one switch domain comprises amino acid residues disclosed in SEQ ID NO: 54 (or SEQ ID NO: 55), and one switch domain comprises amino acid residues disclosed in SEQ ID NO: 56. 
     In embodiments, the FKBP/FRB dimerization switch comprises a modified FRB switch domain that exhibits altered, e.g., enhanced, complex formation between an FRB-based switch domain, e.g., the modified FRB switch domain, a FKBP-based switch domain, and the dimerization molecule, e.g., rapamycin or a rapalogue, e.g., RAD001. In an embodiment, the modified FRB switch domain comprises one or more mutations, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, selected from mutations at amino acid position(s) L2031, E2032, S2035, R2036, F2039, G2040, T2098, W2101, D2102, Y2105, and F2108, where the wild-type amino acid is mutated to any other naturally-occurring amino acid. In an embodiment, a mutant FRB comprises a mutation at E2032, where E2032 is mutated to phenylalanine (E2032F), methionine (E2032M), arginine (E2032R), valine (E2032V), tyrosine (E2032Y), isoleucine (E2032I), e.g., SEQ ID NO: 57, or leucine (E2032L), e.g., SEQ ID NO: 58. In an embodiment, a mutant FRB comprises a mutation at T2098, where T2098 is mutated to phenylalanine (T2098F) or leucine (T2098L), e.g., SEQ ID NO: 59. In an embodiment, a mutant FRB comprises a mutation at E2032 and at T2098, where E2032 is mutated to any amino acid, and where T2098 is mutated to any amino acid, e.g., SEQ ID NO: 60. In an embodiment, a mutant FRB comprises an E20321 and a T2098L mutation, e.g., SEQ ID NO: 61. In an embodiment, a mutant FRB comprises an E2032L and a T2098L mutation, e.g., SEQ ID NO: 62. 
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 Exemplary mutant FRB having increased affinity 
               
               
                 for a dimerization molecule 
               
            
           
           
               
               
               
            
               
                   
                   
                 SEQ ID 
               
               
                 FRB mutant 
                 Amino Acid Sequence 
                 NO: 
               
               
                   
               
               
                 E2032I 
                 ILWHEMWHEGLIEASRLYFGERN 
                 57 
               
               
                 mutant 
                 VKGMFEVLEPLHAMMERGPQTLK 
                   
               
               
                   
                 ETSFNQAYGRDLMEAQEWCRKYM 
                   
               
               
                   
                 KSGNVKDLTQAWDLYYHVFRRIS 
                   
               
               
                   
                 KTS 
                   
               
               
                   
               
               
                 E2032L 
                 ILWHEMWHEGLLEASRLYFGERN 
                 58 
               
               
                 mutant 
                 VKGMFEVLEPLHAMMERGPQTLK 
                   
               
               
                   
                 ETSFNQAYGRDLMEAQEWCRKYM 
                   
               
               
                   
                 KSGNVKDLTQAWDLYYHVFRRIS 
                   
               
               
                   
                 KTS 
                   
               
               
                   
               
               
                 T2098L 
                 ILWHEMWHEGLEEASRLYFGERN 
                 59 
               
               
                 mutant 
                 VKGMFEVLEPLHAMMERGPQTLK 
                   
               
               
                   
                 ETSFNQAYGRDLMEAQEWCRKYM 
                   
               
               
                   
                 KSGNVKDLLQAWDLYYHVFRRIS 
                   
               
               
                   
                 KTS 
                   
               
               
                   
               
               
                 E2032, 
                 ILWHEMWHEGL   X   EASRLYFGERN 
                 60 
               
               
                 T2098 
                 VKGMFEVLEPLHAMMERGPQTLK 
                   
               
               
                 mutant 
                   
                   
               
               
                   
                 ETSFNQAYGRDLMEAQEWCRKYM 
                   
               
               
                   
                 KSGNVKDL   X   QAWDLYYHVFRRIS 
                   
               
               
                   
                 KTS 
                   
               
               
                   
               
               
                 E2032I, 
                 ILWHEMWHEGLIEASRLYFGERN 
                 61 
               
               
                 T2098L 
                 VKGMFEVLEPLHAMMERGPQTLK 
                   
               
               
                 mutant 
                   
                   
               
               
                   
                 ETSFNQAYGRDLMEAQEWCRKYM 
                   
               
               
                   
                 KSGNVKDLLQAWDLYYHVFRRIS 
                   
               
               
                   
                 KTS 
                   
               
               
                   
               
               
                 E2032L, 
                 ILWHEMWHEGLLEASRLYFGERN 
                 62 
               
               
                 T2098L 
                 VKGMFEVLEPLHAMMERGPQTLK 
                   
               
               
                 mutant 
                   
                   
               
               
                   
                 ETSFNQAYGRDLMEAQEWCRKYM 
                   
               
               
                   
                 KSGNVKDLLQAWDLYYHVFRRIS 
                   
               
               
                   
                 KTS 
               
               
                   
               
            
           
         
       
     
     Other suitable dimerization switches include a GyrB-GyrB based dimerization switch, a Gibberellin-based dimerization switch, a tag/binder dimerization switch, and a halo-tag/snap-tag dimerization switch. Following the guidance provided herein, such switches and relevant dimerization molecules will be apparent to one of ordinary skill. 
     Dimerization Molecule 
     Association between the switch domains is promoted by the dimerization molecule. In the presence of dimerization molecule interaction or association between switch domains allows for signal transduction between a polypeptide associated with, e.g., fused to, a first switch domain, and a polypeptide associated with, e.g., fused to, a second switch domain. In the presence of non-limiting levels of dimerization molecule signal transduction is increased by 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 5, 10, 50, 100 fold, e.g., as measured in a system described herein. 
     Rapamycin and rapamycin analogs (sometimes referred to as rapalogues), e.g., RAD001, can be used as dimerization molecules in a FKBP/FRB-based dimerization switch described herein. In an embodiment the dimerization molecule can be selected from rapamycin (sirolimus), RAD001 (everolimus), zotarolimus, temsirolimus, AP-23573 (ridaforolimus), biolimus and AP21967. Additional rapamycin analogs suitable for use with FKBP/FRB-based dimerization switches are further described in the section entitled “Combination Therapies”, or in the subsection entitled “Exemplary mTOR inhibitors.” 
     Split CAR 
     In some embodiments, the CAR-expressing cell uses a split CAR. The split CAR approach is described in more detail in publications WO2014/055442 and WO2014/055657. Briefly, a split CAR system comprises a cell expressing a first CAR having a first antigen binding domain and a costimulatory domain (e.g., 41B), and the cell also expresses a second CAR having a second antigen binding domain and an intracellular signaling domain (e.g., CD3 zeta). When the cell encounters the first antigen, the costimulatory domain is activated, and the cell proliferates. When the cell encounters the second antigen, the intracellular signaling domain is activated and cell-killing activity begins. Thus, the CAR-expressing cell is only fully activated in the presence of both antigens. 
     RNA Transfection 
     Disclosed herein are methods for producing an in vitro transcribed RNA CAR. The present invention also includes a CAR encoding RNA construct that can be directly transfected into a cell. A method for generating mRNA for use in transfection can involve in vitro transcription (IVT) of a template with specially designed primers, followed by polyA addition, to produce a construct containing 3′ and 5′ untranslated sequence (“UTR”), a 5′ cap and/or Internal Ribosome Entry Site (IRES), the nucleic acid to be expressed, and a polyA tail, typically 50-2000 bases in length (SEQ ID NO:32). RNA so produced can efficiently transfect different kinds of cells. In one aspect, the template includes sequences for the CAR. 
     In one aspect, a CAR of the present invention is encoded by a messenger RNA (mRNA). In one aspect, the mRNA encoding a CAR described herein is introduced into an immune effector cell, e.g., a T cell or a NK cell, for production of a CAR-expressing cell, e.g., a CART cell or a CAR NK cell. 
     In one embodiment, the in vitro transcribed RNA CAR can be introduced to a cell as a form of transient transfection. The RNA is produced by in vitro transcription using a polymerase chain reaction (PCR)-generated template. DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase. The source of the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any other appropriate source of DNA. The desired temple for in vitro transcription is a CAR described herein. For example, the template for the RNA CAR comprises an extracellular region comprising a single chain variable domain of an antibody to a tumor associated antigen described herein; a hinge region (e.g., a hinge region described herein), a transmembrane domain (e.g., a transmembrane domain described herein such as a transmembrane domain of CD8a); and a cytoplasmic region that includes an intracellular signaling domain, e.g., an intracellular signaling domain described herein, e.g., comprising the signaling domain of CD3-zeta and the signaling domain of 4-1BB. 
     In one embodiment, the DNA to be used for PCR contains an open reading frame. The DNA can be from a naturally occurring DNA sequence from the genome of an organism. In one embodiment, the nucleic acid can include some or all of the 5′ and/or 3′ untranslated regions (UTRs). The nucleic acid can include exons and introns. In one embodiment, the DNA to be used for PCR is a human nucleic acid sequence. In another embodiment, the DNA to be used for PCR is a human nucleic acid sequence including the 5′ and 3′ UTRs. The DNA can alternatively be an artificial DNA sequence that is not normally expressed in a naturally occurring organism. An exemplary artificial DNA sequence is one that contains portions of genes that are ligated together to form an open reading frame that encodes a fusion protein. The portions of DNA that are ligated together can be from a single organism or from more than one organism. 
     PCR is used to generate a template for in vitro transcription of mRNA which is used for transfection. Methods for performing PCR are well known in the art. Primers for use in PCR are designed to have regions that are substantially complementary to regions of the DNA to be used as a template for the PCR. “Substantially complementary,” as used herein, refers to sequences of nucleotides where a majority or all of the bases in the primer sequence are complementary, or one or more bases are non-complementary, or mismatched. Substantially complementary sequences are able to anneal or hybridize with the intended DNA target under annealing conditions used for PCR. The primers can be designed to be substantially complementary to any portion of the DNA template. For example, the primers can be designed to amplify the portion of a nucleic acid that is normally transcribed in cells (the open reading frame), including 5′ and 3′ UTRs. The primers can also be designed to amplify a portion of a nucleic acid that encodes a particular domain of interest. In one embodiment, the primers are designed to amplify the coding region of a human cDNA, including all or portions of the 5′ and 3′ UTRs. Primers useful for PCR can be generated by synthetic methods that are well known in the art. “Forward primers” are primers that contain a region of nucleotides that are substantially complementary to nucleotides on the DNA template that are upstream of the DNA sequence that is to be amplified. “Upstream” is used herein to refer to a location 5, to the DNA sequence to be amplified relative to the coding strand. “Reverse primers” are primers that contain a region of nucleotides that are substantially complementary to a double-stranded DNA template that are downstream of the DNA sequence that is to be amplified. “Downstream” is used herein to refer to a location 3′ to the DNA sequence to be amplified relative to the coding strand. 
     Any DNA polymerase useful for PCR can be used in the methods disclosed herein. The reagents and polymerase are commercially available from a number of sources. 
     Chemical structures with the ability to promote stability and/or translation efficiency may also be used. The RNA preferably has 5′ and 3′ UTRs. In one embodiment, the 5′ UTR is between one and 3000 nucleotides in length. The length of 5′ and 3′ UTR sequences to be added to the coding region can be altered by different methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTRs. Using this approach, one of ordinary skill in the art can modify the 5′ and 3′ UTR lengths required to achieve optimal translation efficiency following transfection of the transcribed RNA. 
     The 5′ and 3′ UTRs can be the naturally occurring, endogenous 5′ and 3′ UTRs for the nucleic acid of interest. Alternatively, UTR sequences that are not endogenous to the nucleic acid of interest can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modifications of the template. The use of UTR sequences that are not endogenous to the nucleic acid of interest can be useful for modifying the stability and/or translation efficiency of the RNA. For example, it is known that AU-rich elements in 3′ UTR sequences can decrease the stability of mRNA. Therefore, 3′ UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art. 
     In one embodiment, the 5′ UTR can contain the Kozak sequence of the endogenous nucleic acid. Alternatively, when a 5′ UTR that is not endogenous to the nucleic acid of interest is being added by PCR as described above, a consensus Kozak sequence can be redesigned by adding the 5′ UTR sequence. Kozak sequences can increase the efficiency of translation of some RNA transcripts, but does not appear to be required for all RNAs to enable efficient translation. The requirement for Kozak sequences for many mRNAs is known in the art. In other embodiments the 5′ UTR can be 5′UTR of an RNA virus whose RNA genome is stable in cells. In other embodiments various nucleotide analogues can be used in the 3′ or 5′ UTR to impede exonuclease degradation of the mRNA. 
     To enable synthesis of RNA from a DNA template without the need for gene cloning, a promoter of transcription should be attached to the DNA template upstream of the sequence to be transcribed. When a sequence that functions as a promoter for an RNA polymerase is added to the 5′ end of the forward primer, the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame that is to be transcribed. In one preferred embodiment, the promoter is a T7 polymerase promoter, as described elsewhere herein. Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3 and SP6 promoters are known in the art. 
     In a preferred embodiment, the mRNA has both a cap on the 5′ end and a 3′ poly(A) tail which determine ribosome binding, initiation of translation and stability mRNA in the cell. On a circular DNA template, for instance, plasmid DNA, RNA polymerase produces a long concatameric product which is not suitable for expression in eukaryotic cells. The transcription of plasmid DNA linearized at the end of the 3′ UTR results in normal sized mRNA which is not effective in eukaryotic transfection even if it is polyadenylated after transcription. 
     On a linear DNA template, phage T7 RNA polymerase can extend the 3′ end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003). 
     The conventional method of integration of polyA/T stretches into a DNA template is molecular cloning. However polyA/T sequence integrated into plasmid DNA can cause plasmid instability, which is why plasmid DNA templates obtained from bacterial cells are often highly contaminated with deletions and other aberrations. This makes cloning procedures not only laborious and time consuming but often not reliable. That is why a method which allows construction of DNA templates with polyA/T 3′ stretch without cloning highly desirable. 
     The polyA/T segment of the transcriptional DNA template can be produced during PCR by using a reverse primer containing a polyT tail, such as 100T tail (SEQ ID NO: 35) (size can be 50-5000 T (SEQ ID NO: 36)), or after PCR by any other method, including, but not limited to, DNA ligation or in vitro recombination. Poly(A) tails also provide stability to RNAs and reduce their degradation. Generally, the length of a poly(A) tail positively correlates with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is between 100 and 5000 adenosines (SEQ ID NO: 37). 
     Poly(A) tails of RNAs can be further extended following in vitro transcription with the use of a poly(A) polymerase, such as  E. coli  polyA polymerase (E-PAP). In one embodiment, increasing the length of a poly(A) tail from 100 nucleotides to between 300 and 400 nucleotides (SEQ ID NO: 38) results in about a two-fold increase in the translation efficiency of the RNA. Additionally, the attachment of different chemical groups to the 3′ end can increase mRNA stability. Such attachment can contain modified/artificial nucleotides, aptamers and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase the stability of the RNA. 
     5′ caps on also provide stability to RNA molecules. In a preferred embodiment, RNAs produced by the methods disclosed herein include a 5′ cap. The 5′ cap is provided using techniques known in the art and described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)). 
     The RNAs produced by the methods disclosed herein can also contain an internal ribosome entry site (IRES) sequence. The IRES sequence may be any viral, chromosomal or artificially designed sequence which initiates cap-independent ribosome binding to mRNA and facilitates the initiation of translation. Any solutes suitable for cell electroporation, which can contain factors facilitating cellular permeability and viability such as sugars, peptides, lipids, proteins, antioxidants, and surfactants can be included. 
     RNA can be introduced into target cells using any of a number of different methods, for instance, commercially available methods which include, but are not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg Germany), cationic liposome mediated transfection using lipofection, polymer encapsulation, peptide mediated transfection, or biolistic particle delivery systems such as “gene guns” (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001). 
     Non-Viral Delivery Methods 
     In some aspects, non-viral methods can be used to deliver a nucleic acid encoding a CAR described herein into a cell or tissue or a subject. 
     In some embodiments, the non-viral method includes the use of a transposon (also called a transposable element). In some embodiments, a transposon is a piece of DNA that can insert itself at a location in a genome, for example, a piece of DNA that is capable of self-replicating and inserting its copy into a genome, or a piece of DNA that can be spliced out of a longer nucleic acid and inserted into another place in a genome. For example, a transposon comprises a DNA sequence made up of inverted repeats flanking genes for transposition. 
     Exemplary methods of nucleic acid delivery using a transposon include a Sleeping Beauty transposon system (SBTS) and a piggyBac (PB) transposon system. See, e.g., Aronovich et al. Hum. Mol. Genet. 20.R1(2011):R14-20; Singh et al. Cancer Res. 15(2008):2961-2971; Huang et al. Mol. Ther. 16(2008):580-589; Grabundzija et al. Mol. Ther. 18(2010):1200-1209; Kebriaei et al. Blood. 122.21(2013):166; Williams. Molecular Therapy 16.9(2008):1515-16; Bell et al. Nat. Protoc. 2.12(2007):3153-65; and Ding et al. Cell. 122.3(2005):473-83, all of which are incorporated herein by reference. 
     The SBTS includes two components: 1) a transposon containing a transgene and 2) a source of transposase enzyme. The transposase can transpose the transposon from a carrier plasmid (or other donor DNA) to a target DNA, such as a host cell chromosome/genome. For example, the transposase binds to the carrier plasmid/donor DNA, cuts the transposon (including transgene(s)) out of the plasmid, and inserts it into the genome of the host cell. See, e.g., Aronovich et al. supra. 
     Exemplary transposons include a pT2-based transposon. See, e.g., Grabundzija et al. Nucleic Acids Res. 41.3(2013):1829-47; and Singh et al. Cancer Res. 68.8(2008): 2961-2971, all of which are incorporated herein by reference. Exemplary transposases include a Tc1/mariner-type transposase, e.g., the SB10 transposase or the SB 11 transposase (a hyperactive transposase which can be expressed, e.g., from a cytomegalovirus promoter). See, e.g., Aronovich et al.; Kebriaei et al.; and Grabundzija et al., all of which are incorporated herein by reference. 
     Use of the SBTS permits efficient integration and expression of a transgene, e.g., a nucleic acid encoding a CAR described herein. Provided herein are methods of generating a cell, e.g., T cell or NK cell, that stably expresses a CAR described herein, e.g., using a transposon system such as SBTS. 
     In accordance with methods described herein, in some embodiments, one or more nucleic acids, e.g., plasmids, containing the SBTS components are delivered to a cell (e.g., T or NK cell). For example, the nucleic acid(s) are delivered by standard methods of nucleic acid (e.g., plasmid DNA) delivery, e.g., methods described herein, e.g., electroporation, transfection, or lipofection. In some embodiments, the nucleic acid contains a transposon comprising a transgene, e.g., a nucleic acid encoding a CAR described herein. In some embodiments, the nucleic acid contains a transposon comprising a transgene (e.g., a nucleic acid encoding a CAR described herein) as well as a nucleic acid sequence encoding a transposase enzyme. In other embodiments, a system with two nucleic acids is provided, e.g., a dual-plasmid system, e.g., where a first plasmid contains a transposon comprising a transgene, and a second plasmid contains a nucleic acid sequence encoding a transposase enzyme. For example, the first and the second nucleic acids are co-delivered into a host cell. 
     In some embodiments, cells, e.g., T or NK cells, are generated that express a CAR described herein by using a combination of gene insertion using the SBTS and genetic editing using a nuclease (e.g., Zinc finger nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), the CRISPR/Cas system, or engineered meganuclease re-engineered homing endonucleases). 
     In some embodiments, use of a non-viral method of delivery permits reprogramming of cells, e.g., T or NK cells, and direct infusion of the cells into a subject. Advantages of non-viral vectors include but are not limited to the ease and relatively low cost of producing sufficient amounts required to meet a patient population, stability during storage, and lack of immunogenicity. 
     Nucleic Acid Constructs Encoding a CAR 
     The present invention also provides nucleic acid molecules encoding one or more CAR constructs described herein. In one aspect, the nucleic acid molecule is provided as a messenger RNA transcript. In one aspect, the nucleic acid molecule is provided as a DNA construct. 
     Accordingly, in one aspect, the invention pertains to a nucleic acid molecule encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain that binds to a tumor antigen described herein, a transmembrane domain (e.g., a transmembrane domain described herein), and an intracellular signaling domain (e.g., an intracellular signaling domain described herein) comprising a stimulatory domain, e.g., a costimulatory signaling domain (e.g., a costimulatory signaling domain described herein) and/or a primary signaling domain (e.g., a primary signaling domain described herein, e.g., a zeta chain described herein). In one embodiment, the transmembrane domain is transmembrane domain of a protein selected from the group consisting of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. In some embodiments, a transmembrane domain may include at least the transmembrane region(s) of, e.g., KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG/Cbp. 
     In one embodiment, the transmembrane domain comprises a sequence of SEQ ID NO: 12, or a sequence with 95-99% identity thereof. In one embodiment, the antigen binding domain is connected to the transmembrane domain by a hinge region, e.g., a hinge described herein. In one embodiment, the hinge region comprises SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 or SEQ ID NO:10, or a sequence with 95-99% identity thereof. In one embodiment, the isolated nucleic acid molecule further comprises a sequence encoding a costimulatory domain. In one embodiment, the costimulatory domain is a functional signaling domain of a protein selected from the group consisting of OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), and 4-1BB (CD137). Further examples of such costimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, and PAG/Cbp. In one embodiment, the costimulatory domain comprises a sequence of SEQ ID NO:16, or a sequence with 95-99% identity thereof. In one embodiment, the intracellular signaling domain comprises a functional signaling domain of 4-1BB and a functional signaling domain of CD3 zeta. In one embodiment, the intracellular signaling domain comprises the sequence of SEQ ID NO: 14 or SEQ ID NO:16, or a sequence with 95-99% identity thereof, and the sequence of SEQ ID NO: 18 or SEQ ID NO:20, or a sequence with 95-99% identity thereof, wherein the sequences comprising the intracellular signaling domain are expressed in the same frame and as a single polypeptide chain. 
     In another aspect, the invention pertains to an isolated nucleic acid molecule encoding a CAR construct comprising a leader sequence of SEQ ID NO: 2, a scFv domain as described herein, a hinge region of SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 or SEQ ID NO:10 (or a sequence with 95-99% identity thereof), a transmembrane domain having a sequence of SEQ ID NO: 12 (or a sequence with 95-99% identity thereof), a 4-1BB costimulatory domain having a sequence of SEQ ID NO:14 or a CD27 costimulatory domain having a sequence of SEQ ID NO:16 (or a sequence with 95-99% identity thereof), and a CD3 zeta stimulatory domain having a sequence of SEQ ID NO:18 or SEQ ID NO:20 (or a sequence with 95-99% identity thereof). 
     In another aspect, the invention pertains to a nucleic acid molecule encoding a chimeric antigen receptor (CAR) molecule that comprises an antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a stimulatory domain, and wherein said antigen binding domain binds to a tumor antigen selected from a group consisting of: CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1 (CLECL1), CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-11Ra, PSCA, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, CD20, Folate receptor alpha, ERBB2 (Her2/neu), MUC1, EGFR, NCAM, Prostase, PRSS21, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, ber-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1/CD248, TEM7R, CLDN6, TSHR, GPRC5D, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1/Galectin 8, MelanA/MART1, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor, Cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, and IGLL1. 
     In one embodiment, the encoded CAR molecule further comprises a sequence encoding a costimulatory domain. In one embodiment, the costimulatory domain is a functional signaling domain of a protein selected from the group consisting of OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18) and 4-1BB (CD137). In one embodiment, the costimulatory domain comprises a sequence of SEQ ID NO: 14. In one embodiment, the transmembrane domain is a transmembrane domain of a protein selected from the group consisting of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. In one embodiment, the transmembrane domain comprises a sequence of SEQ ID NO:12. In one embodiment, the intracellular signaling domain comprises a functional signaling domain of 4-1BB and a functional signaling domain of zeta. In one embodiment, the intracellular signaling domain comprises the sequence of SEQ ID NO: 14 and the sequence of SEQ ID NO: 18, wherein the sequences comprising the intracellular signaling domain are expressed in the same frame and as a single polypeptide chain. In one embodiment, the anti-a cancer associated antigen as described herein binding domain is connected to the transmembrane domain by a hinge region. In one embodiment, the hinge region comprises SEQ ID NO:4. In one embodiment, the hinge region comprises SEQ ID NO:6 or SEQ ID NO:8 or SEQ ID NO:10. 
     The nucleic acid sequences coding for the desired molecules can be obtained using recombinant methods known in the art, such as, for example by screening libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques. Alternatively, the gene of interest can be produced synthetically, rather than cloned. 
     The present invention also provides vectors in which a DNA of the present invention is inserted. Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity. A retroviral vector may also be, e.g., a gammaretroviral vector. A gammaretroviral vector may include, e.g., a promoter, a packaging signal (W), a primer binding site (PBS), one or more (e.g., two) long terminal repeats (LTR), and a transgene of interest, e.g., a gene encoding a CAR. A gammaretroviral vector may lack viral structural gens such as gag, pol, and env. Exemplary gammaretroviral vectors include Murine Leukemia Virus (MLV), Spleen-Focus Forming Virus (SFFV), and Myeloproliferative Sarcoma Virus (MPSV), and vectors derived therefrom. Other gammaretroviral vectors are described, e.g., in Tobias Maetzig et al., “Gammaretroviral Vectors: Biology, Technology and Application” Viruses. 2011 June; 3(6): 677-713. 
     In another embodiment, the vector comprising the nucleic acid encoding the desired CAR of the invention is an adenoviral vector (A5/35). In another embodiment, the expression of nucleic acids encoding CARs can be accomplished using of transposons such as sleeping beauty, crisper, CAS9, and zinc finger nucleases. See below June et al. 2009 Nature Reviews Immunology  9.10: 704-716, is incorporated herein by reference. 
     In brief summary, the expression of natural or synthetic nucleic acids encoding CARs is typically achieved by operably linking a nucleic acid encoding the CAR polypeptide or portions thereof to a promoter, and incorporating the construct into an expression vector. The vectors can be suitable for replication and integration eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence. 
     The expression constructs of the present invention may also be used for nucleic acid immunization and gene therapy, using standard gene delivery protocols. Methods for gene delivery are known in the art. See, e.g., U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated by reference herein in their entireties. In another embodiment, the invention provides a gene therapy vector. 
     The nucleic acid can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. 
     Further, the expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193). 
     A number of viral based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. A number of adenovirus vectors are known in the art. In one embodiment, lentivirus vectors are used. 
     Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription. Exemplary promoters include the CMV IE gene, EF-1a, ubiquitin C, or phosphoglycerokinase (PGK) promoters. 
     An example of a promoter that is capable of expressing a CAR encoding nucleic acid molecule in a mammalian T cell is the EF1a promoter. The native EF1a promoter drives expression of the alpha subunit of the elongation factor-1 complex, which is responsible for the enzymatic delivery of aminoacyl tRNAs to the ribosome. The EF1a promoter has been extensively used in mammalian expression plasmids and has been shown to be effective in driving CAR expression from nucleic acid molecules cloned into a lentiviral vector. See, e.g., Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). In one aspect, the EF1a promoter comprises the sequence provided as SEQ ID NO: 1. 
     Another example of a promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the elongation factor-1α promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter. 
     A vector may also include, e.g., a signal sequence to facilitate secretion, a polyadenylation signal and transcription terminator (e.g., from Bovine Growth Hormone (BGH) gene), an element allowing episomal replication and replication in prokaryotes (e.g. SV40 origin and ColE1 or others known in the art) and/or elements to allow selection (e.g., ampicillin resistance gene and/or zeocin marker). 
     In order to assess the expression of a CAR polypeptide or portions thereof, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic-resistance genes, such as neo and the like. 
     Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5′ flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription. 
     Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means. 
     Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY). A preferred method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection 
     Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362. 
     Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). Other methods of state-of-the-art targeted delivery of nucleic acids are available, such as delivery of polynucleotides with targeted nanoparticles or other suitable sub-micron sized delivery system. 
     In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes. 
     Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K &amp; K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about −20° C. Chloroform is used as the only solvent since it is more readily evaporated than methanol. “Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine-nucleic acid complexes. 
     Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to the inhibitor of the present invention, in order to confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention. 
     The present invention further provides a vector comprising a CAR encoding nucleic acid molecule. In one aspect, a CAR vector can be directly transduced into a cell, e.g., a T cell or a NK cell. In one aspect, the vector is a cloning or expression vector, e.g., a vector including, but not limited to, one or more plasmids (e.g., expression plasmids, cloning vectors, minicircles, minivectors, double minute chromosomes), retroviral and lentiviral vector constructs. In one aspect, the vector is capable of expressing the CAR construct in mammalian immune effector cells (e.g., T cells, NK cells). In one aspect, the mammalian T cell is a human T cell. In one aspect, the mammalian NK cell is a human NK cell. 
     Sources of Cells 
     Prior to expansion and genetic modification or other modification, a source of cells, e.g., T cells or natural killer (NK) cells, can be obtained from a subject. The term “subject” is intended to include living organisms in which an immune response can be elicited (e.g., mammals). Examples of subjects include humans, monkeys, chimpanzees, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. 
     In certain aspects of the present disclosure, immune effector cells, e.g., T cells, can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll™ separation. In one preferred aspect, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one aspect, the cells collected by apheresis may be washed to remove the plasma fraction and, optionally, to place the cells in an appropriate buffer or media for subsequent processing steps. In one embodiment, the cells are washed with phosphate buffered saline (PBS). In an alternative embodiment, the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. 
     Initial activation steps in the absence of calcium can lead to magnified activation. As those of ordinary skill in the art would readily appreciate a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to the manufacturer&#39;s instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS, PlasmaLyte A, or other saline solution with or without buffer. Alternatively, the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media. 
     It is recognized that the methods of the application can utilize culture media conditions comprising 5% or less, for example 2%, human AB serum, and employ known culture media conditions and compositions, for example those described in Smith et al., “Ex vivo expansion of human T cells for adoptive immunotherapy using the novel Xeno-free CTS Immune Cell Serum Replacement”  Clinical  &amp;  Translational Immunology  (2015) 4, e31; doi:10.1038/cti.2014.31. 
     In one aspect, T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. 
     The methods described herein can include, e.g., selection of a specific subpopulation of immune effector cells, e.g., T cells, that are a T regulatory cell-depleted population, CD25+ depleted cells, using, e.g., a negative selection technique, e.g., described herein. Preferably, the population of T regulatory depleted cells contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% of CD25+ cells. 
     In one embodiment, T regulatory cells, e.g., CD25+ T cells, are removed from the population using an anti-CD25 antibody, or fragment thereof, or a CD25-binding ligand, IL-2. 
     In one embodiment, the anti-CD25 antibody, or fragment thereof, or CD25-binding ligand is conjugated to a substrate, e.g., a bead, or is otherwise coated on a substrate, e.g., a bead. In one embodiment, the anti-CD25 antibody, or fragment thereof, is conjugated to a substrate as described herein. 
     In one embodiment, the T regulatory cells, e.g., CD25+ T cells, are removed from the population using CD25 depletion reagent from Miltenyi™. In one embodiment, the ratio of cells to CD25 depletion reagent is 1e7 cells to 20 uL, or 1e7 cells to 15 uL, or 1e7 cells to 10 uL, or 1e7 cells to 5 uL, or 1e7 cells to 2.5 uL, or 1e7 cells to 1.25 uL. In one embodiment, e.g., for T regulatory cells, e.g., CD25+ depletion, greater than 500 million cells/ml is used. In a further aspect, a concentration of cells of 600, 700, 800, or 900 million cells/ml is used. 
     In one embodiment, the population of immune effector cells to be depleted includes about 6×10 9  CD25+ T cells. In other aspects, the population of immune effector cells to be depleted include about 1×10 9  to 1×10 10  CD25+ T cell, and any integer value in between. In one embodiment, the resulting population T regulatory depleted cells has 2×10 9  T regulatory cells, e.g., CD25+ cells, or less (e.g., 1×10 9 , 5×10 8 , 1×10 8 , 5×10 7 , 1×10 7 , or less CD25+ cells). 
     In one embodiment, the T regulatory cells, e.g., CD25+ cells, are removed from the population using the CliniMAC system with a depletion tubing set, such as, e.g., tubing 162-01. In one embodiment, the CliniMAC system is run on a depletion setting such as, e.g., DEPLETION2.1. 
     Without wishing to be bound by a particular theory, decreasing the level of negative regulators of immune cells (e.g., decreasing the number of unwanted immune cells, e.g., TREG cells), in a subject prior to apheresis or during manufacturing of a CAR-expressing cell product can reduce the risk of subject relapse. For example, methods of depleting TREG cells are known in the art. Methods of decreasing TREG cells include, but are not limited to, cyclophosphamide, anti-GITR antibody (an anti-GITR antibody described herein), CD25-depletion, and combinations thereof. 
     In some embodiments, the manufacturing methods comprise reducing the number of (e.g., depleting) TREG cells prior to manufacturing of the CAR-expressing cell. For example, manufacturing methods comprise contacting the sample, e.g., the apheresis sample, with an anti-GITR antibody and/or an anti-CD25 antibody (or fragment thereof, or a CD25-binding ligand), e.g., to deplete TREG cells prior to manufacturing of the CAR-expressing cell (e.g., T cell, NK cell) product. 
     In an embodiment, a subject is pre-treated with one or more therapies that reduce TREG cells prior to collection of cells for CAR-expressing cell product manufacturing, thereby reducing the risk of subject relapse to CAR-expressing cell treatment. In an embodiment, methods of decreasing TREG cells include, but are not limited to, administration to the subject of one or more of cyclophosphamide, anti-GITR antibody, CD25-depletion, or a combination thereof. Administration of one or more of cyclophosphamide, anti-GITR antibody, CD25-depletion, or a combination thereof, can occur before, during or after an infusion of the CAR-expressing cell product. 
     In an embodiment, a subject is pre-treated with cyclophosphamide prior to collection of cells for CAR-expressing cell product manufacturing, thereby reducing the risk of subject relapse to CAR-expressing cell treatment. In an embodiment, a subject is pre-treated with an anti-GITR antibody prior to collection of cells for CAR-expressing cell product manufacturing, thereby reducing the risk of subject relapse to CAR-expressing cell treatment. 
     In one embodiment, the population of cells to be removed are neither the regulatory T cells or tumor cells, but cells that otherwise negatively affect the expansion and/or function of CART cells, e.g. cells expressing CD14, CD11b, CD33, CD15, or other markers expressed by potentially immune suppressive cells. In one embodiment, such cells are envisioned to be removed concurrently with regulatory T cells and/or tumor cells, or following said depletion, or in another order. 
     The methods described herein can include more than one selection step, e.g., more than one depletion step. Enrichment of a T cell population by negative selection can be accomplished, e.g., with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail can include antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. 
     The methods described herein can further include removing cells from the population which express a tumor antigen, e.g., a tumor antigen that does not comprise CD25, e.g., CD19, CD30, CD38, CD123, CD20, CD14 or CD11b, to thereby provide a population of T regulatory depleted, e.g., CD25+ depleted, and tumor antigen depleted cells that are suitable for expression of a CAR, e.g., a CAR described herein. In one embodiment, tumor antigen expressing cells are removed simultaneously with the T regulatory, e.g., CD25+ cells. For example, an anti-CD25 antibody, or fragment thereof, and an anti-tumor antigen antibody, or fragment thereof, can be attached to the same substrate, e.g., bead, which can be used to remove the cells or an anti-CD25 antibody, or fragment thereof, or the anti-tumor antigen antibody, or fragment thereof, can be attached to separate beads, a mixture of which can be used to remove the cells. In other embodiments, the removal of T regulatory cells, e.g., CD25+ cells, and the removal of the tumor antigen expressing cells is sequential, and can occur, e.g., in either order. 
     Also provided are methods that include removing cells from the population which express a check point inhibitor, e.g., a check point inhibitor described herein, e.g., one or more of PD1+ cells, LAG3+ cells, and TIM3+ cells, to thereby provide a population of T regulatory depleted, e.g., CD25+ depleted cells, and check point inhibitor depleted cells, e.g., PD1+, LAG3+ and/or TIM3+ depleted cells. Exemplary check point inhibitors include B7-H1, B7-1, CD160, P1H, 2B4, PD1, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, TIGIT, CTLA-4, BTLA and LAIR1. In one embodiment, check point inhibitor expressing cells are removed simultaneously with the T regulatory, e.g., CD25+ cells. For example, an anti-CD25 antibody, or fragment thereof, and an anti-check point inhibitor antibody, or fragment thereof, can be attached to the same bead which can be used to remove the cells, or an anti-CD25 antibody, or fragment thereof, and the anti-check point inhibitor antibody, or fragment there, can be attached to separate beads, a mixture of which can be used to remove the cells. In other embodiments, the removal of T regulatory cells, e.g., CD25+ cells, and the removal of the check point inhibitor expressing cells is sequential, and can occur, e.g., in either order. 
     Methods described herein can include a positive selection step. For example, T cells can isolated by incubation with anti-CD3/anti-CD28 (e.g., 3×28)-conjugated beads, such as DYNABEADS® M-450 CD3/CD28 T, for a time period sufficient for positive selection of the desired T cells. In one embodiment, the time period is about 30 minutes. In a further embodiment, the time period ranges from 30 minutes to 36 hours or longer and all integer values there between. In a further embodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another embodiment, the time period is 10 to 24 hours, e.g., 24 hours. Longer incubation times may be used to isolate T cells in any situation where there are few T cells as compared to other cell types, such in isolating tumor infiltrating lymphocytes (TIL) from tumor tissue or from immunocompromised individuals. Further, use of longer incubation times can increase the efficiency of capture of CD8+ T cells. Thus, by simply shortening or lengthening the time T cells are allowed to bind to the CD3/CD28 beads and/or by increasing or decreasing the ratio of beads to T cells (as described further herein), subpopulations of T cells can be preferentially selected for or against at culture initiation or at other time points during the process. Additionally, by increasing or decreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on the beads or other surface, subpopulations of T cells can be preferentially selected for or against at culture initiation or at other desired time points. 
     In one embodiment, a T cell population can be selected that expresses one or more of IFN-γ, TNFα, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10, IL-13, granzyme B, and perforin, or other appropriate molecules, e.g., other cytokines. Methods for screening for cell expression can be determined, e.g., by the methods described in PCT Publication No.: WO 2013/126712. 
     For isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In certain aspects, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (e.g., increase the concentration of cells), to ensure maximum contact of cells and beads. For example, in one aspect, a concentration of 10 billion cells/ml, 9 billion/ml, 8 billion/ml, 7 billion/ml, 6 billion/ml, or 5 billion/ml is used. In one aspect, a concentration of 1 billion cells/ml is used. In yet one aspect, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/ml is used. In further aspects, concentrations of 125 or 150 million cells/ml can be used. 
     Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells, or from samples where there are many tumor cells present (e.g., leukemic blood, tumor tissue, etc.). Such populations of cells may have therapeutic value and would be desirable to obtain. For example, using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression. 
     In a related aspect, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and surface (e.g., particles such as beads), interactions between the particles and cells is minimized. This selects for cells that express high amounts of desired antigens to be bound to the particles. For example, CD4+ T cells express higher levels of CD28 and are more efficiently captured than CD8+ T cells in dilute concentrations. In one aspect, the concentration of cells used is 5×10 6 /ml. In other aspects, the concentration used can be from about 1×10 5 /ml to 1×10 6 /ml, and any integer value in between. 
     In other aspects, the cells may be incubated on a rotator for varying lengths of time at varying speeds at either 2-10° C. or at room temperature. 
     T cells for stimulation can also be frozen after a washing step. Wishing not to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or culture media containing 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitable cell freezing media containing for example, Hespan and PlasmaLyte A, the cells then are frozen to −80° C. at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at −20° C. or in liquid nitrogen. 
     In certain aspects, cryopreserved cells are thawed and washed as described herein and allowed to rest for one hour at room temperature prior to activation using the methods of the present invention. 
     Also contemplated in the context of the invention is the collection of blood samples or apheresis product from a subject at a time period prior to when the expanded cells as described herein might be needed. As such, the source of the cells to be expanded can be collected at any time point necessary, and desired cells, such as T cells, isolated and frozen for later use in immune effector cell therapy for any number of diseases or conditions that would benefit from immune effector cell therapy, such as those described herein. In one aspect a blood sample or an apheresis is taken from a generally healthy subject. In certain aspects, a blood sample or an apheresis is taken from a generally healthy subject who is at risk of developing a disease, but who has not yet developed a disease, and the cells of interest are isolated and frozen for later use. In certain aspects, the T cells may be expanded, frozen, and used at a later time. In certain aspects, samples are collected from a patient shortly after diagnosis of a particular disease as described herein but prior to any treatments. In a further aspect, the cells are isolated from a blood sample or an apheresis from a subject prior to any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and irradiation. 
     In a further aspect of the present invention, T cells are obtained from a patient directly following treatment that leaves the subject with functional T cells. In this regard, it has been observed that following certain cancer treatments, in particular treatments with drugs that damage the immune system, shortly after treatment during the period when patients would normally be recovering from the treatment, the quality of T cells obtained may be optimal or improved for their ability to expand ex vivo. Likewise, following ex vivo manipulation using the methods described herein, these cells may be in a preferred state for enhanced engraftment and in vivo expansion. Thus, it is contemplated within the context of the present invention to collect blood cells, including T cells, dendritic cells, or other cells of the hematopoietic lineage, during this recovery phase. Further, in certain aspects, mobilization (for example, mobilization with GM-CSF) and conditioning regimens can be used to create a condition in a subject wherein repopulation, recirculation, regeneration, and/or expansion of particular cell types is favored, especially during a defined window of time following therapy. Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system. 
     In one embodiment, the immune effector cells expressing a CAR molecule, e.g., a CAR molecule described herein, are obtained from a subject that has received a low, immune enhancing dose of an mTOR inhibitor. In an embodiment, the population of immune effector cells, e.g., T cells, to be engineered to express a CAR, are harvested after a sufficient time, or after sufficient dosing of the low, immune enhancing, dose of an mTOR inhibitor, such that the level of PD1 negative immune effector cells, e.g., T cells, or the ratio of PD1 negative immune effector cells, e.g., T cells/PD1 positive immune effector cells, e.g., T cells, in the subject or harvested from the subject has been, at least transiently, increased. 
     In other embodiments, population of immune effector cells, e.g., T cells, which have, or will be engineered to express a CAR, can be treated ex vivo by contact with an amount of an mTOR inhibitor that increases the number of PD1 negative immune effector cells, e.g., T cells or increases the ratio of PD1 negative immune effector cells, e.g., T cells/PD1 positive immune effector cells, e.g., T cells. 
     In one embodiment, a T cell population is diaglycerol kinase (DGK)-deficient. DGK-deficient cells include cells that do not express DGK RNA or protein, or have reduced or inhibited DGK activity. DGK-deficient cells can be generated by genetic approaches, e.g., administering RNA-interfering agents, e.g., siRNA, shRNA, miRNA, to reduce or prevent DGK expression. Alternatively, DGK-deficient cells can be generated by treatment with DGK inhibitors described herein. 
     In one embodiment, a T cell population is Ikaros-deficient. Ikaros-deficient cells include cells that do not express Ikaros RNA or protein, or have reduced or inhibited Ikaros activity, Ikaros-deficient cells can be generated by genetic approaches, e.g., administering RNA-interfering agents, e.g., siRNA, shRNA, miRNA, to reduce or prevent Ikaros expression. Alternatively, Ikaros-deficient cells can be generated by treatment with Ikaros inhibitors, e.g., lenalidomide. 
     In embodiments, a T cell population is DGK-deficient and Ikaros-deficient, e.g., does not express DGK and Ikaros, or has reduced or inhibited DGK and Ikaros activity. Such DGK and Ikaros-deficient cells can be generated by any of the methods described herein. 
     In an embodiment, the NK cells are obtained from the subject. In another embodiment, the NK cells are an NK cell line, e.g., NK-92 cell line (Conkwest). 
     Allogeneic CAR 
     In embodiments described herein, the immune effector cell can be an allogeneic immune effector cell, e.g., T cell or NK cell. For example, the cell can be an allogeneic T cell, e.g., an allogeneic T cell lacking expression of a functional T cell receptor (TCR) and/or human leukocyte antigen (HLA), e.g., HLA class I and/or HLA class II. 
     A T cell lacking a functional TCR can be, e.g., engineered such that it does not express any functional TCR on its surface, engineered such that it does not express one or more subunits that comprise a functional TCR or engineered such that it produces very little functional TCR on its surface. Alternatively, the T cell can express a substantially impaired TCR, e.g., by expression of mutated or truncated forms of one or more of the subunits of the TCR. The term “substantially impaired TCR” means that this TCR will not elicit an adverse immune reaction in a host. 
     A T cell described herein can be, e.g., engineered such that it does not express a functional HLA on its surface. For example, a T cell described herein, can be engineered such that cell surface expression HLA, e.g., HLA class 1 and/or HLA class II, is downregulated. 
     In some embodiments, the T cell can lack a functional TCR and a functional HLA, e.g., HLA class I and/or HLA class II. 
     Modified T cells that lack expression of a functional TCR and/or HLA can be obtained by any suitable means, including a knock out or knock down of one or more subunit of TCR or HLA. For example, the T cell can include a knock down of TCR and/or HLA using siRNA, shRNA, clustered regularly interspaced short palindromic repeats (CRISPR) transcription-activator like effector nuclease (TALEN), or zinc finger endonuclease (ZFN). 
     In some embodiments, the allogeneic cell can be a cell which does not express or expresses at low levels an inhibitory molecule, e.g. by any method described herein. For example, the cell can be a cell that does not express or expresses at low levels an inhibitory molecule, e.g., that can decrease the ability of a CAR-expressing cell to mount an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGF beta. Inhibition of an inhibitory molecule, e.g., by inhibition at the DNA, RNA or protein level, can optimize a CAR-expressing cell performance. In embodiments, an inhibitory nucleic acid, e.g., an inhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA or shRNA, a clustered regularly interspaced short palindromic repeats (CRISPR), a transcription-activator like effector nuclease (TALEN), or a zinc finger endonuclease (ZFN), e.g., as described herein, can be used. 
     siRNA and shRNA to Inhibit TCR or HLA 
     In some embodiments, TCR expression and/or HLA expression can be inhibited using siRNA or shRNA that targets a nucleic acid encoding a TCR and/or HLA in a T cell. 
     Expression of siRNA and shRNAs in T cells can be achieved using any conventional expression system, e.g., such as a lentiviral expression system. 
     Exemplary shRNAs that downregulate expression of components of the TCR are described, e.g., in US Publication No.: 2012/0321667. Exemplary siRNA and shRNA that downregulate expression of HLA class I and/or HLA class II genes are described, e.g., in U.S. publication No.: US 2007/0036773. 
     CRISPR to Inhibit TCR or HLA 
     “CRISPR” or “CRISPR to TCR and/or HLA” or “CRISPR to inhibit TCR and/or HLA” as used herein refers to a set of clustered regularly interspaced short palindromic repeats, or a system comprising such a set of repeats. “Cas”, as used herein, refers to a CRISPR-associated protein. A “CRISPR/Cas” system refers to a system derived from CRISPR and Cas which can be used to silence or mutate a TCR and/or HLA gene. 
     Naturally-occurring CRISPR/Cas systems are found in approximately 40% of sequenced eubacteria genomes and 90% of sequenced archaea. Grissa et al. (2007) BMC Bioinformatics 8: 172. This system is a type of prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages and provides a form of acquired immunity. Barrangou et al. (2007) Science 315: 1709-1712; Marragini et al. (2008) Science 322: 1843-1845. 
     The CRISPR/Cas system has been modified for use in gene editing (silencing, enhancing or changing specific genes) in eukaryotes such as mice or primates. Wiedenheft et al. (2012) Nature 482: 331-8. This is accomplished by introducing into the eukaryotic cell a plasmid containing a specifically designed CRISPR and one or more appropriate Cas. 
     The CRISPR sequence, sometimes called a CRISPR locus, comprises alternating repeats and spacers. In a naturally-occurring CRISPR, the spacers usually comprise sequences foreign to the bacterium such as a plasmid or phage sequence; in the TCR and/or HLA CRISPR/Cas system, the spacers are derived from the TCR or HLA gene sequence. 
     RNA from the CRISPR locus is constitutively expressed and processed by Cas proteins into small RNAs. These comprise a spacer flanked by a repeat sequence. The RNAs guide other Cas proteins to silence exogenous genetic elements at the RNA or DNA level. Horvath et al. (2010)  Science  327: 167-170; Makarova et al. (2006) Biology Direct 1: 7. The spacers thus serve as templates for RNA molecules, analogously to siRNAs. Pennisi (2013) Science 341: 833-836. 
     As these naturally occur in many different types of bacteria, the exact arrangements of the CRISPR and structure, function and number of Cas genes and their product differ somewhat from species to species. Haft et al. (2005) PLoS Comput. Biol. 1: e60; Kunin et al. (2007) Genome Biol. 8: R61; Mojica et al. (2005) J. Mol. Evol. 60: 174-182; Bolotin et al. (2005) Microbiol. 151: 2551-2561; Pourcel et al. (2005) Microbiol. 151: 653-663; and Stern et al. (2010) Trends. Genet. 28: 335-340. For example, the Cse (Cas subtype,  E. coli ) proteins (e.g., CasA) form a functional complex, Cascade, that processes CRISPR RNA transcripts into spacer-repeat units that Cascade retains. Brouns et al. (2008) Science 321: 960-964. In other prokaryotes, Cas6 processes the CRISPR transcript. The CRISPR-based phage inactivation in  E. coli  requires Cascade and Cas3, but not Cas1 or Cas2. The Cmr (Cas RAMP module) proteins in  Pyrococcus furiosus  and other prokaryotes form a functional complex with small CRISPR RNAs that recognizes and cleaves complementary target RNAs. A simpler CRISPR system relies on the protein Cas9, which is a nuclease with two active cutting sites, one for each strand of the double helix. Combining Cas9 and modified CRISPR locus RNA can be used in a system for gene editing. Pennisi (2013) Science 341: 833-836. 
     The CRISPR/Cas system can thus be used to edit a TCR and/or HLA gene (adding or deleting a basepair), or introducing a premature stop which thus decreases expression of a TCR and/or HLA. The CRISPR/Cas system can alternatively be used like RNA interference, turning off TCR and/or HLA gene in a reversible fashion. In a mammalian cell, for example, the RNA can guide the Cas protein to a TCR and/or HLA promoter, sterically blocking RNA polymerases. 
     Artificial CRISPR/Cas systems can be generated which inhibit TCR and/or HLA, using technology known in the art, e.g., that described in U.S. Publication No. 20140068797, and Cong (2013) Science 339: 819-823. Other artificial CRISPR/Cas systems that are known in the art may also be generated which inhibit TCR and/or HLA, e.g., that described in Tsai (2014) Nature Biotechnol., 32:6 569-576, U.S. Pat. Nos. 8,871,445; 8,865,406; 8,795,965; 8,771,945; and 8,697,359. 
     TALEN to Inhibit TCR and/or HLA 
     “TALEN” or “TALEN to HLA and/or TCR” or “TALEN to inhibit HLA and/or TCR” refers to a transcription activator-like effector nuclease, an artificial nuclease which can be used to edit the HLA and/or TCR gene. 
     TALENs are produced artificially by fusing a TAL effector DNA binding domain to a DNA cleavage domain. Transcription activator-like effects (TALEs) can be engineered to bind any desired DNA sequence, including a portion of the HLA or TCR gene. By combining an engineered TALE with a DNA cleavage domain, a restriction enzyme can be produced which is specific to any desired DNA sequence, including a HLA or TCR sequence. These can then be introduced into a cell, wherein they can be used for genome editing. Boch (2011)  Nature Biotech.  29: 135-6; and Boch et al. (2009)  Science  326: 1509-12; Moscou et al. (2009)  Science  326: 3501. 
     TALEs are proteins secreted by  Xanthomonas  bacteria. The DNA binding domain contains a repeated, highly conserved 33-34 amino acid sequence, with the exception of the 12th and 13th amino acids. These two positions are highly variable, showing a strong correlation with specific nucleotide recognition. They can thus be engineered to bind to a desired DNA sequence. 
     To produce a TALEN, a TALE protein is fused to a nuclease (N), which is a wild-type or mutated FokI endonuclease. Several mutations to FokI have been made for its use in TALENs; these, for example, improve cleavage specificity or activity. Cermak et al. (2011)  Nucl. Acids Res.  39: e82; Miller et al. (2011)  Nature Biotech.  29: 143-8; Hockemeyer et al. (2011)  Nature Biotech.  29: 731-734; Wood et al. (2011)  Science  333: 307; Doyon et al. (2010)  Nature Methods  8: 74-79; Szczepek et al. (2007)  Nature Biotech.  25: 786-793; and Guo et al. (2010)  J. Mol. Biol.  200: 96. 
     The FokI domain functions as a dimer, requiring two constructs with unique DNA binding domains for sites in the target genome with proper orientation and spacing. Both the number of amino acid residues between the TALE DNA binding domain and the FokI cleavage domain and the number of bases between the two individual TALEN binding sites appear to be important parameters for achieving high levels of activity. Miller et al. (2011) Nature Biotech. 29: 143-8. 
     A HLA or TCR TALEN can be used inside a cell to produce a double-stranded break (DSB). A mutation can be introduced at the break site if the repair mechanisms improperly repair the break via non-homologous end joining. For example, improper repair may introduce a frame shift mutation. Alternatively, foreign DNA can be introduced into the cell along with the TALEN; depending on the sequences of the foreign DNA and chromosomal sequence, this process can be used to correct a defect in the HLA or TCR gene or introduce such a defect into a wt HLA or TCR gene, thus decreasing expression of HLA or TCR. 
     TALENs specific to sequences in HLA or TCR can be constructed using any method known in the art, including various schemes using modular components. Zhang et al. (2011)  Nature Biotech.  29: 149-53; Geibler et al. (2011) PLoS ONE 6: e19509. 
     Zinc Finger Nuclease to Inhibit HLA and/or TCR 
     “ZFN” or “Zinc Finger Nuclease” or “ZFN to HLA and/or TCR” or “ZFN to inhibit HLA and/or TCR” refer to a zinc finger nuclease, an artificial nuclease which can be used to edit the HLA and/or TCR gene. 
     Like a TALEN, a ZFN comprises a FokI nuclease domain (or derivative thereof) fused to a DNA-binding domain. In the case of a ZFN, the DNA-binding domain comprises one or more zinc fingers. Carroll et al. (2011)  Genetics Society of America  188: 773-782; and Kim et al. (1996)  Proc. Natl. Acad. Sci. USA  93: 1156-1160. 
     A zinc finger is a small protein structural motif stabilized by one or more zinc ions. A zinc finger can comprise, for example, Cys2His2, and can recognize an approximately 3-bp sequence. Various zinc fingers of known specificity can be combined to produce multi-finger polypeptides which recognize about 6, 9, 12, 15 or 18-bp sequences. Various selection and modular assembly techniques are available to generate zinc fingers (and combinations thereof) recognizing specific sequences, including phage display, yeast one-hybrid systems, bacterial one-hybrid and two-hybrid systems, and mammalian cells. 
     Like a TALEN, a ZFN must dimerize to cleave DNA. Thus, a pair of ZFNs are required to target non-palindromic DNA sites. The two individual ZFNs must bind opposite strands of the DNA with their nucleases properly spaced apart. Bitinaite et al. (1998)  Proc. Natl. Acad. Sci. USA  95: 10570-5. 
     Also like a TALEN, a ZFN can create a double-stranded break in the DNA, which can create a frame-shift mutation if improperly repaired, leading to a decrease in the expression and amount of HLA and/or TCR in a cell. ZFNs can also be used with homologous recombination to mutate in the HLA or TCR gene. 
     ZFNs specific to sequences in HLA AND/OR TCR can be constructed using any method known in the art. See, e.g., Provasi (2011)  Nature Med.  18: 807-815; Torikai (2013)  Blood  122: 1341-1349; Cathomen et al. (2008)  Mol. Ther.  16: 1200-7; Guo et al. (2010)  J. Mol. Biol.  400: 96; U.S. Patent Publication 2011/0158957; and U.S. Patent Publication 2012/0060230. 
     Telomerase Expression 
     While not wishing to be bound by any particular theory, in some embodiments, a therapeutic T cell has short term persistence in a patient, due to shortened telomeres in the T cell; accordingly, transfection with a telomerase gene can lengthen the telomeres of the T cell and improve persistence of the T cell in the patient. See Carl June, “Adoptive T cell therapy for cancer in the clinic”, Journal of Clinical Investigation, 117:1466-1476 (2007). Thus, in an embodiment, an immune effector cell, e.g., a T cell, ectopically expresses a telomerase subunit, e.g., the catalytic subunit of telomerase, e.g., TERT, e.g., hTERT. In some aspects, this disclosure provides a method of producing a CAR-expressing cell, comprising contacting a cell with a nucleic acid encoding a telomerase subunit, e.g., the catalytic subunit of telomerase, e.g., TERT, e.g., hTERT. The cell may be contacted with the nucleic acid before, simultaneous with, or after being contacted with a construct encoding a CAR. 
     In one aspect, the disclosure features a method of making a population of immune effector cells (e.g., T cells, NK cells). In an embodiment, the method comprises: providing a population of immune effector cells (e.g., T cells or NK cells), contacting the population of immune effector cells with a nucleic acid encoding a CAR; and contacting the population of immune effector cells with a nucleic acid encoding a telomerase subunit, e.g., hTERT, under conditions that allow for CAR and telomerase expression. 
     In an embodiment, the nucleic acid encoding the telomerase subunit is DNA. In an embodiment, the nucleic acid encoding the telomerase subunit comprises a promoter capable of driving expression of the telomerase subunit. 
     In an embodiment, hTERT has the amino acid sequence of GenBank Protein ID AAC51724.1 (Meyerson et al., “hEST2, the Putative Human Telomerase Catalytic Subunit Gene, Is Up-Regulated in Tumor Cells and during Immortalization” Cell Volume 90, Issue 4, 22 Aug. 1997, Pages 785-795) as follows: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 63) 
               
               
                   
                 MPRAPRCRAVRSLLRSHYREVLPLATFVRRLGPQG 
               
               
                   
               
               
                   
                 WRLVQRGDPAAFRALVAQCLVCVPWDARPPPAAPS 
               
               
                   
               
               
                   
                 FRQVSCLKELVARVLQRLCERGAKNVLAFGFALLD 
               
               
                   
               
               
                   
                 GARGGPPEAFTTSVRSYLPNTVTDALRGSGAWGLL 
               
               
                   
               
               
                   
                 LRRVGDDVLVHLLARCALFVLVAPSCAYQVCGPPL 
               
               
                   
               
               
                   
                 YQLGAATQARPPPHASGPRRRLGCERAWNHSVREA 
               
               
                   
               
               
                   
                 GVPLGLPAPGARRRGGSASRSLPLPKRPRRGAAPE 
               
               
                   
               
               
                   
                 PERTPVGQGSWAHPGRTRGPSDRGFCVVSPARPAE 
               
               
                   
               
               
                   
                 EATSLEGALSGTRHSHPSVGRQHHAGPPSTSRPPR 
               
               
                   
               
               
                   
                 PWDTPCPPVYAETKHFLYSSGDKEQLRPSFLLSSL 
               
               
                   
               
               
                   
                 RPSLTGARRLVETIFLGSRPWMPGTPRRLPRLPQR 
               
               
                   
               
               
                   
                 YWQMRPLFLELLGNHAQCPYGVLLKTHCPLRAAVT 
               
               
                   
               
               
                   
                 PAAGVCAREKPQGSVAAPEEEDTDPRRLVQLLRQH 
               
               
                   
               
               
                   
                 SSPWQVYGFVRACLRRLVPPGLWGSRHNERRFLRN 
               
               
                   
               
               
                   
                 TKKFISLGKHAKLSLQELTWKMSVRGCAWLRRSPG 
               
               
                   
               
               
                   
                 VGCVPAAEHRLREEILAKFLHWLMSVYVVELLRSF 
               
               
                   
               
               
                   
                 FYVTETTFQKNRLFFYRKSVWSKLQSIGIRQHLKR 
               
               
                   
               
               
                   
                 VQLRELSEAEVRQHREARPALLTSRLRFIPKPDGL 
               
               
                   
               
               
                   
                 RPIVNMDYVVGARTFRREKRAERLTSRVKALFSVL 
               
               
                   
               
               
                   
                 NYERARRPGLLGASVLGLDDIHRAWRTFVLRVRAQ 
               
               
                   
               
               
                   
                 DPPPELYFVKVDVTGAYDTIPQDRLTEVIASIIKP 
               
               
                   
               
               
                   
                 QNTYCVRRYAVVQKAAHGHVRKAFKSHVSTLTDLQ 
               
               
                   
               
               
                   
                 PYMRQFVAHLQETSPLRDAVVIEQSSSLNEASSGL 
               
               
                   
               
               
                   
                 FDVFLRFMCHHAVRIRGKSYVQCQGIPQGSILSTL 
               
               
                   
               
               
                   
                 LCSLCYGDMENKLFAGIRRDGLLLRLVDDFLLVTP 
               
               
                   
               
               
                   
                 HLTHAKTFLRTLVRGVPEYGCVVNLRKTVVNFPVE 
               
               
                   
               
               
                   
                 DEALGGTAFVQMPAHGLFPWCGLLLDTRTLEVQSD 
               
               
                   
               
               
                   
                 YSSYARTSIRASLTFNRGFKAGRNMRRKLFGVLRL 
               
               
                   
               
               
                   
                 KCHSLFLDLQVNSLQTVCTNIYKILLLQAYRFHAC 
               
               
                   
               
               
                   
                 VLQLPFHQQVWKNPTFFLRVISDTASLCYSILKAK 
               
               
                   
               
               
                   
                 NAGMSLGAKGAAGPLPSEAVQWLCHQAFLLKLTRH 
               
               
                   
               
               
                   
                 RVTYVPLLGSLRTAQTQLSRKLPGTTLTALEAAAN 
               
               
                   
               
               
                   
                 PALPSDFKTILD 
               
            
           
         
       
     
     In an embodiment, the hTERT has a sequence at least 80%, 85%, 90%, 95%, 96{circumflex over ( )}, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 63. In an embodiment, the hTERT has a sequence of SEQ ID NO: 63. In an embodiment, the hTERT comprises a deletion (e.g., of no more than 5, 10, 15, 20, or 30 amino acids) at the N-terminus, the C-terminus, or both. In an embodiment, the hTERT comprises a transgenic amino acid sequence (e.g., of no more than 5, 10, 15, 20, or 30 amino acids) at the N-terminus, the C-terminus, or both. 
     In an embodiment, the hTERT is encoded by the nucleic acid sequence of GenBank Accession No. AF018167 (Meyerson et al., “hEST2, the Putative Human Telomerase Catalytic Subunit Gene, Is Up-Regulated in Tumor Cells and during Immortalization” Cell Volume 90, Issue 4, 22 Aug. 1997, Pages 785-795): 
     
       
         
           
               
               
            
               
                 (SEQ ID NO: 64) 
                   
               
            
           
           
               
               
               
            
               
                 1 
                 caggcagcgt ggtcctgctg cgcacgtggg aagccctggc cccggccacc cccgcgatgc 
                   
               
               
                   
               
               
                 61 
                 cgcgcgctcc ccgctgccga gccgtgcgct ccctgctgcg cagccactac cgcgaggtgc 
               
               
                   
               
               
                 121 
                 tgccgctggc cacgttcgtg cggcgcctgg ggccccaggg ctggcggctg gtgcagcgcg 
               
               
                   
               
               
                 181 
                 gggacccggc ggctttccgc gcgctggtgg cccagtgcct ggtgtgcgtg ccctgggacg 
               
               
                   
               
               
                 241 
                 cacggccgcc ccccgccgcc ccctccttcc gccaggtgtc ctgcctgaag gagctggtgg 
               
               
                   
               
               
                 301 
                 cccgagtgct gcagaggctg tgcgagcgcg gcgcgaagaa cgtgctggcc ttcggcttcg 
               
               
                   
               
               
                 361 
                 cgctgctgga cggggcccgc gggggccccc ccgaggcctt caccaccagc gtgcgcagct 
               
               
                   
               
               
                 421 
                 acctgcccaa cacggtgacc gacgcactgc gggggagcgg ggcgtggggg ctgctgttgc 
               
               
                   
               
               
                 481 
                 gccgcgtggg cgacgacgtg ctggttcacc tgctggcacg ctgcgcgctc tttgtgctgg 
               
               
                   
               
               
                 541 
                 tggctcccag ctgcgcctac caggtgtgcg ggccgccgct gtaccagctc ggcgctgcca 
               
               
                   
               
               
                 601 
                 ctcaggcccg gcccccgcca cacgctagtg gaccccgaag gcgtctggga tgcgaacggg 
               
               
                   
               
               
                 661 
                 cctggaacca tagcgtcagg gaggccgggg tccccctggg cctgccagcc ccgggtgcga 
               
               
                   
               
               
                 721 
                 ggaggcgcgg gggcagtgcc agccgaagtc tgccgttgcc caagaggccc aggcgtggcg 
               
               
                   
               
               
                 781 
                 ctgcccctga gccggagcgg acgcccgttg ggcaggggtc ctgggcccac ccgggcagga 
               
               
                   
               
               
                 841 
                 cgcgtggacc gagtgaccgt ggtttctgtg tggtgtcacc tgccagaccc gccgaagaag 
               
               
                   
               
               
                 901 
                 ccacctcttt ggagggtgcg ctctctggca cgcgccactc ccacccatcc gtgggccgcc 
               
               
                   
               
               
                 961 
                 agcaccacgc gggcccccca tccacatcgc ggccaccacg tccctgggac acgccttgtc 
               
               
                   
               
               
                 1021 
                 ccccggtgta cgccgagacc aagcacttcc tctactcctc aggcgacaag gagcagctgc 
               
               
                   
               
               
                 1081 
                 ggccctcctt cctactcagc tctctgaggc ccagcctgac tggcgctcgg aggctcgtgg 
               
               
                   
               
               
                 1141 
                 agaccatctt tctgggttcc aggccctgga tgccagggac tccccgcagg ttgccccgcc 
               
               
                   
               
               
                 1201 
                 tgccccagcg ctactggcaa atgcggcccc tgtttctgga gctgcttggg aaccacgcgc 
               
               
                   
               
               
                 1261 
                 agtgccccta cggggtgctc ctcaagacgc actgcccgct gcgagctgcg gtcaccccag 
               
               
                   
               
               
                 1321 
                 cagccggtgt ctgtgcccgg gagaagcccc agggctctgt ggcggccccc gaggaggagg 
               
               
                   
               
               
                 1381 
                 acacagaccc ccgtcgcctg gtgcagctgc tccgccagca cagcagcccc tggcaggtgt 
               
               
                   
               
               
                 1441 
                 acggcttcgt gcgggcctgc ctgcgccggc tggtgccccc aggcctctgg ggctccaggc 
               
               
                   
               
               
                 1501 
                 acaacgaacg ccgcttcctc aggaacacca agaagttcat ctccctgggg aagcatgcca 
               
               
                   
               
               
                 1561 
                 agctctcgct gcaggagctg acgtggaaga tgagcgtgcg gggctgcgct tggctgcgca 
               
               
                   
               
               
                 1621 
                 ggagcccagg ggttggctgt gttccggccg cagagcaccg tctgcgtgag gagatcctgg 
               
               
                   
               
               
                 1681 
                 ccaagttcct gcactggctg atgagtgtgt acgtcgtcga gctgctcagg tctttctttt 
               
               
                   
               
               
                 1741 
                 atgtcacgga gaccacgttt caaaagaaca ggctcttttt ctaccggaag agtgtctgga 
               
               
                   
               
               
                 1801 
                 gcaagttgca aagcattgga atcagacagc acttgaagag ggtgcagctg cgggagctgt 
               
               
                   
               
               
                 1861 
                 cggaagcaga ggtcaggcag catcgggaag ccaggcccgc cctgctgacg tccagactcc 
               
               
                   
               
               
                 1921 
                 gcttcatccc caagcctgac gggctgcggc cgattgtgaa catggactac gtcgtgggag 
               
               
                   
               
               
                 1981 
                 ccagaacgtt ccgcagagaa aagagggccg agcgtctcac ctcgagggtg aaggcactgt 
               
               
                   
               
               
                 2041 
                 tcagcgtgct caactacgag cgggcgcggc gccccggcct cctgggcgcc tctgtgctgg 
               
               
                   
               
               
                 2101 
                 gcctggacga tatccacagg gcctggcgca ccttcgtgct gcgtgtgcgg gcccaggacc 
               
               
                   
               
               
                 2161 
                 cgccgcctga gctgtacttt gtcaaggtgg atgtgacggg cgcgtacgac accatccccc 
               
               
                   
               
               
                 2221 
                 aggacaggct cacggaggtc atcgccagca tcatcaaacc ccagaacacg tactgcgtgc 
               
               
                   
               
               
                 2281 
                 gtcggtatgc cgtggtccag aaggccgccc atgggcacgt ccgcaaggcc ttcaagagcc 
               
               
                   
               
               
                 2341 
                 acgtctctac cttgacagac ctccagccgt acatgcgaca gttcgtggct cacctgcagg 
               
               
                   
               
               
                 2401 
                 agaccagccc gctgagggat gccgtcgtca tcgagcagag ctcctccctg aatgaggcca 
               
               
                   
               
               
                 2461 
                 gcagtggcct cttcgacgtc ttcctacgct tcatgtgcca ccacgccgtg cgcatcaggg 
               
               
                   
               
               
                 2521 
                 gcaagtccta cgtccagtgc caggggatcc cgcagggctc catcctctcc acgctgctct 
               
               
                   
               
               
                 2581 
                 gcagcctgtg ctacggcgac atggagaaca agctgtttgc ggggattcgg cgggacgggc 
               
               
                   
               
               
                 2641 
                 tgctcctgcg tttggtggat gatttcttgt tggtgacacc tcacctcacc cacgcgaaaa 
               
               
                   
               
               
                 2701 
                 ccttcctcag gaccctggtc cgaggtgtcc ctgagtatgg ctgcgtggtg aacttgcgga 
               
               
                   
               
               
                 2761 
                 agacagtggt gaacttccct gtagaagacg aggccctggg tggcacggct tttgttcaga 
               
               
                   
               
               
                 2821 
                 tgccggccca cggcctattc ccctggtgcg gcctgctgct ggatacccgg accctggagg 
               
               
                   
               
               
                 2881 
                 tgcagagcga ctactccagc tatgcccgga cctccatcag agccagtctc accttcaacc 
               
               
                   
               
               
                 2941 
                 gcggcttcaa ggctgggagg aacatgcgtc gcaaactctt tggggtcttg cggctgaagt 
               
               
                   
               
               
                 3001 
                 gtcacagcct gtttctggat ttgcaggtga acagcctcca gacggtgtgc accaacatct 
               
               
                   
               
               
                 3061 
                 acaagatcct cctgctgcag gcgtacaggt ttcacgcatg tgtgctgcag ctcccatttc 
               
               
                   
               
               
                 3121 
                 atcagcaagt ttggaagaac cccacatttt tcctgcgcgt catctctgac acggcctccc 
               
               
                   
               
               
                 3181 
                 tctgctactc catcctgaaa gccaagaacg cagggatgtc gctgggggcc aagggcgccg 
               
               
                   
               
               
                 3241 
                 ccggccctct gccctccgag gccgtgcagt ggctgtgcca ccaagcattc ctgctcaagc 
               
               
                   
               
               
                 3301 
                 tgactcgaca ccgtgtcacc tacgtgccac tcctggggtc actcaggaca gcccagacgc 
               
               
                   
               
               
                 3361 
                 agctgagtcg gaagctcccg gggacgacgc tgactgccct ggaggccgca gccaacccgg 
               
               
                   
               
               
                 3421 
                 cactgccctc agacttcaag accatcctgg actgatggcc acccgcccac agccaggccg 
               
               
                   
               
               
                 3481 
                 agagcagaca ccagcagccc tgtcacgccg ggctctacgt cccagggagg gaggggcggc 
               
               
                   
               
               
                 3541 
                 ccacacccag gcccgcaccg ctgggagtct gaggcctgag tgagtgtttg gccgaggcct 
               
               
                   
               
               
                 3601 
                 gcatgtccgg ctgaaggctg agtgtccggc tgaggcctga gcgagtgtcc agccaagggc 
               
               
                   
               
               
                 3661 
                 tgagtgtcca gcacacctgc cgtcttcact tccccacagg ctggcgctcg gctccacccc 
               
               
                   
               
               
                 3721 
                 agggccagct tttcctcacc aggagcccgg cttccactcc ccacatagga atagtccatc 
               
               
                   
               
               
                 3781 
                 cccagattcg ccattgttca cccctcgccc tgccctcctt tgccttccac ccccaccatc 
               
               
                   
               
               
                 3841 
                 caggtggaga ccctgagaag gaccctggga gctctgggaa tttggagtga ccaaaggtgt 
               
               
                   
               
               
                 3901 
                 gccctgtaca caggcgagga ccctgcacct ggatgggggt ccctgtgggt caaattgggg 
               
               
                   
               
               
                 3961 
                 ggaggtgctg tgggagtaaa atactgaata tatgagtttt tcagttttga aaaaaaaaaa 
               
               
                   
               
               
                 4021 
                 aaaaaaa 
               
            
           
         
       
     
     In an embodiment, the hTERT is encoded by a nucleic acid having a sequence at least 80%, 85%, 90%, 95%, 96, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 64. In an embodiment, the hTERT is encoded by a nucleic acid of SEQ ID NO: 64. 
     Activation and Expansion of Immune Effector Cells (e.g., T Cells) 
     Immune effector cells such as T cells may be activated and expanded generally using methods as described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005. 
     Generally, a population of immune effector cells e.g., T regulatory cell depleted cells, may be expanded by contact with a surface having attached thereto an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a costimulatory molecule on the surface of the T cells. In particular, T cell populations may be stimulated as described herein, such as by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For co-stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. To stimulate proliferation of either CD4+ T cells or CD8+ T cells, an anti-CD3 antibody and an anti-CD28 antibody can be used. Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besangon, France) can be used as can other methods commonly known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):13191328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999). 
     In certain aspects, the primary stimulatory signal and the costimulatory signal for the T cell may be provided by different protocols. For example, the agents providing each signal may be in solution or coupled to a surface. When coupled to a surface, the agents may be coupled to the same surface (i.e., in “cis” formation) or to separate surfaces (i.e., in “trans” formation). Alternatively, one agent may be coupled to a surface and the other agent in solution. In one aspect, the agent providing the costimulatory signal is bound to a cell surface and the agent providing the primary activation signal is in solution or coupled to a surface. In certain aspects, both agents can be in solution. In one aspect, the agents may be in soluble form, and then cross-linked to a surface, such as a cell expressing Fc receptors or an antibody or other binding agent which will bind to the agents. In this regard, see for example, U.S. Patent Application Publication Nos. 20040101519 and 20060034810 for artificial antigen presenting cells (aAPCs) that are contemplated for use in activating and expanding T cells in the present invention. 
     In one aspect, the two agents are immobilized on beads, either on the same bead, i.e., “cis,” or to separate beads, i.e., “trans.” By way of example, the agent providing the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof and the agent providing the costimulatory signal is an anti-CD28 antibody or antigen-binding fragment thereof, and both agents are co-immobilized to the same bead in equivalent molecular amounts. In one aspect, a 1:1 ratio of each antibody bound to the beads for CD4+ T cell expansion and T cell growth is used. In certain aspects of the present invention, a ratio of anti CD3:CD28 antibodies bound to the beads is used such that an increase in T cell expansion is observed as compared to the expansion observed using a ratio of 1:1. In one particular aspect an increase of from about 1 to about 3 fold is observed as compared to the expansion observed using a ratio of 1:1. In one aspect, the ratio of CD3:CD28 antibody bound to the beads ranges from 100:1 to 1:100 and all integer values there between. In one aspect, more anti-CD28 antibody is bound to the particles than anti-CD3 antibody, i.e., the ratio of CD3:CD28 is less than one. In certain aspects, the ratio of anti CD28 antibody to anti CD3 antibody bound to the beads is greater than 2:1. In one particular aspect, a 1:100 CD3:CD28 ratio of antibody bound to beads is used. In one aspect, a 1:75 CD3:CD28 ratio of antibody bound to beads is used. In a further aspect, a 1:50 CD3:CD28 ratio of antibody bound to beads is used. In one aspect, a 1:30 CD3:CD28 ratio of antibody bound to beads is used. In one preferred aspect, a 1:10 CD3:CD28 ratio of antibody bound to beads is used. In one aspect, a 1:3 CD3:CD28 ratio of antibody bound to the beads is used. In yet one aspect, a 3:1 CD3:CD28 ratio of antibody bound to the beads is used. 
     Ratios of particles to cells from 1:500 to 500:1 and any integer values in between may be used to stimulate T cells or other target cells. As those of ordinary skill in the art can readily appreciate, the ratio of particles to cells may depend on particle size relative to the target cell. For example, small sized beads could only bind a few cells, while larger beads could bind many. In certain aspects the ratio of cells to particles ranges from 1:100 to 100:1 and any integer values in-between and in further aspects the ratio comprises 1:9 to 9:1 and any integer values in between, can also be used to stimulate T cells. The ratio of anti-CD3- and anti-CD28-coupled particles to T cells that result in T cell stimulation can vary as noted above, however certain preferred values include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, and 15:1 with one preferred ratio being at least 1:1 particles per T cell. In one aspect, a ratio of particles to cells of 1:1 or less is used. In one particular aspect, a preferred particle: cell ratio is 1:5. In further aspects, the ratio of particles to cells can be varied depending on the day of stimulation. For example, in one aspect, the ratio of particles to cells is from 1:1 to 10:1 on the first day and additional particles are added to the cells every day or every other day thereafter for up to 10 days, at final ratios of from 1:1 to 1:10 (based on cell counts on the day of addition). In one particular aspect, the ratio of particles to cells is 1:1 on the first day of stimulation and adjusted to 1:5 on the third and fifth days of stimulation. In one aspect, particles are added on a daily or every other day basis to a final ratio of 1:1 on the first day, and 1:5 on the third and fifth days of stimulation. In one aspect, the ratio of particles to cells is 2:1 on the first day of stimulation and adjusted to 1:10 on the third and fifth days of stimulation. In one aspect, particles are added on a daily or every other day basis to a final ratio of 1:1 on the first day, and 1:10 on the third and fifth days of stimulation. One of skill in the art will appreciate that a variety of other ratios may be suitable for use in the present invention. In particular, ratios will vary depending on particle size and on cell size and type. In one aspect, the most typical ratios for use are in the neighborhood of 1:1, 2:1 and 3:1 on the first day. 
     In further aspects, the cells, such as T cells, are combined with agent-coated beads, the beads and the cells are subsequently separated, and then the cells are cultured. In an alternative aspect, prior to culture, the agent-coated beads and cells are not separated but are cultured together. In a further aspect, the beads and cells are first concentrated by application of a force, such as a magnetic force, resulting in increased ligation of cell surface markers, thereby inducing cell stimulation. 
     By way of example, cell surface proteins may be ligated by allowing paramagnetic beads to which anti-CD3 and anti-CD28 are attached (3×28 beads) to contact the T cells. In one aspect the cells (for example, 10 4  to 10 9  T cells) and beads (for example, DYNABEADS® M-450 CD3/CD28 T paramagnetic beads at a ratio of 1:1) are combined in a buffer, for example PBS (without divalent cations such as, calcium and magnesium). Again, those of ordinary skill in the art can readily appreciate any cell concentration may be used. For example, the target cell may be very rare in the sample and comprise only 0.01% of the sample or the entire sample (i.e., 100%) may comprise the target cell of interest. Accordingly, any cell number is within the context of the present invention. In certain aspects, it may be desirable to significantly decrease the volume in which particles and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and particles. For example, in one aspect, a concentration of about 10 billion cells/ml, 9 billion/ml, 8 billion/ml, 7 billion/ml, 6 billion/ml, 5 billion/ml, or 2 billion cells/ml is used. In one aspect, greater than 100 million cells/ml is used. In a further aspect, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used. In yet one aspect, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/ml is used. In further aspects, concentrations of 125 or 150 million cells/ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells. Such populations of cells may have therapeutic value and would be desirable to obtain in certain aspects. For example, using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression. 
     In one embodiment, cells transduced with a nucleic acid encoding a CAR, e.g., a CAR described herein, are expanded, e.g., by a method described herein. In one embodiment, the cells are expanded in culture for a period of several hours (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18, 21 hours) to about 14 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days). In one embodiment, the cells are expanded for a period of 4 to 9 days. In one embodiment, the cells are expanded for a period of 8 days or less, e.g., 7, 6 or 5 days. In one embodiment, the cells, e.g., a CD19 CAR cell described herein, are expanded in culture for 5 days, and the resulting cells are more potent than the same cells expanded in culture for 9 days under the same culture conditions. Potency can be defined, e.g., by various T cell functions, e.g. proliferation, target cell killing, cytokine production, activation, migration, or combinations thereof. In one embodiment, the cells, e.g., a CD19 CAR cell described herein, expanded for 5 days show at least a one, two, three or four fold increase in cells doublings upon antigen stimulation as compared to the same cells expanded in culture for 9 days under the same culture conditions. In one embodiment, the cells, e.g., the cells expressing a CD19 CAR described herein, are expanded in culture for 5 days, and the resulting cells exhibit higher proinflammatory cytokine production, e.g., IFN-7 and/or GM-CSF levels, as compared to the same cells expanded in culture for 9 days under the same culture conditions. In one embodiment, the cells, e.g., a CD19 CAR cell described herein, expanded for 5 days show at least a one, two, three, four, five, ten fold or more increase in pg/ml of proinflammatory cytokine production, e.g., IFN-7 and/or GM-CSF levels, as compared to the same cells expanded in culture for 9 days under the same culture conditions. 
     Several cycles of stimulation may also be desired such that culture time of T cells can be 60 days or more. Conditions appropriate for T cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-7, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-α or any other additives for the growth of cells known to the skilled artisan. Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. Media can include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and/or an amount of cytokine(s) sufficient for the growth and expansion of T cells. Antibiotics, e.g., penicillin and streptomycin, are included only in experimental cultures, not in cultures of cells that are to be infused into a subject. The target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5% CO 2 ). 
     In one embodiment, the cells are expanded in an appropriate media (e.g., media described herein) that includes one or more interleukin that result in at least a 200-fold (e.g., 200-fold, 250-fold, 300-fold, 350-fold) increase in cells over a 14 day expansion period, e.g., as measured by a method described herein such as flow cytometry. In one embodiment, the cells are expanded in the presence of IL-15 and/or IL-7 (e.g., IL-15 and IL-7). 
     In embodiments, methods described herein, e.g., CAR-expressing cell manufacturing methods, comprise removing T regulatory cells, e.g., CD25+ T cells, from a cell population, e.g., using an anti-CD25 antibody, or fragment thereof, or a CD25-binding ligand, IL-2. Methods of removing T regulatory cells, e.g., CD25+ T cells, from a cell population are described herein. In embodiments, the methods, e.g., manufacturing methods, further comprise contacting a cell population (e.g., a cell population in which T regulatory cells, such as CD25+ T cells, have been depleted; or a cell population that has previously contacted an anti-CD25 antibody, fragment thereof, or CD25-binding ligand) with IL-15 and/or IL-7. For example, the cell population (e.g., that has previously contacted an anti-CD25 antibody, fragment thereof, or CD25-binding ligand) is expanded in the presence of IL-15 and/or IL-7. 
     In some embodiments a CAR-expressing cell described herein is contacted with a composition comprising a interleukin-15 (IL-15) polypeptide, a interleukin-15 receptor alpha (IL-15Ra) polypeptide, or a combination of both a IL-15 polypeptide and a IL-15Ra polypeptide e.g., hetIL-15, during the manufacturing of the CAR-expressing cell, e.g., ex vivo. In embodiments, a CAR-expressing cell described herein is contacted with a composition comprising a IL-15 polypeptide during the manufacturing of the CAR-expressing cell, e.g., ex vivo. In embodiments, a CAR-expressing cell described herein is contacted with a composition comprising a combination of both a IL-15 polypeptide and a IL-15 Ra polypeptide during the manufacturing of the CAR-expressing cell, e.g., ex vivo. In embodiments, a CAR-expressing cell described herein is contacted with a composition comprising hetIL-15 during the manufacturing of the CAR-expressing cell, e.g., ex vivo. 
     In one embodiment the CAR-expressing cell described herein is contacted with a composition comprising hetIL-15 during ex vivo expansion. In an embodiment, the CAR-expressing cell described herein is contacted with a composition comprising an IL-15 polypeptide during ex vivo expansion. In an embodiment, the CAR-expressing cell described herein is contacted with a composition comprising both an IL-15 polypeptide and an IL-15Ra polypeptide during ex vivo expansion. In one embodiment the contacting results in the survival and proliferation of a lymphocyte subpopulation, e.g., CD8+ T cells. 
     T cells that have been exposed to varied stimulation times may exhibit different characteristics. For example, typical blood or apheresed peripheral blood mononuclear cell products have a helper T cell population (TH, CD4+) that is greater than the cytotoxic or suppressor T cell population (TC, CD8+). Ex vivo expansion of T cells by stimulating CD3 and CD28 receptors produces a population of T cells that prior to about days 8-9 consists predominately of TH cells, while after about days 8-9, the population of T cells comprises an increasingly greater population of TC cells. Accordingly, depending on the purpose of treatment, infusing a subject with a T cell population comprising predominately of TH cells may be advantageous. Similarly, if an antigen-specific subset of TC cells has been isolated it may be beneficial to expand this subset to a greater degree. 
     Further, in addition to CD4 and CD8 markers, other phenotypic markers vary significantly, but in large part, reproducibly during the course of the cell expansion process. Thus, such reproducibility enables the ability to tailor an activated T cell product for specific purposes. 
     Once a CAR described herein is constructed, various assays can be used to evaluate the activity of the molecule, such as but not limited to, the ability to expand T cells following antigen stimulation, sustain T cell expansion in the absence of re-stimulation, and anti-cancer activities in appropriate in vitro and animal models. Assays to evaluate the effects of a cars of the present invention are described in further detail below 
     Western blot analysis of CAR expression in primary T cells can be used to detect the presence of monomers and dimers. See, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). Very briefly, T cells (1:1 mixture of CD4 +  and CD8 +  T cells) expressing the CARs are expanded in vitro for more than 10 days followed by lysis and SDS-PAGE under reducing conditions. CARs containing the full length TCR-ζ cytoplasmic domain and the endogenous TCR-ζ chain are detected by western blotting using an antibody to the TCR-ζ chain. The same T cell subsets are used for SDS-PAGE analysis under non-reducing conditions to permit evaluation of covalent dimer formation. 
     In vitro expansion of CAR +  T cells following antigen stimulation can be measured by flow cytometry. For example, a mixture of CD4 +  and CD8 +  T cells are stimulated with αCD3/αCD28 aAPCs followed by transduction with lentiviral vectors expressing GFP under the control of the promoters to be analyzed. Exemplary promoters include the CMV IE gene, EF-1α, ubiquitin C, or phosphoglycerokinase (PGK) promoters. GFP fluorescence is evaluated on day 6 of culture in the CD4 +  and/or CD8 +  T cell subsets by flow cytometry. See, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). Alternatively, a mixture of CD4 +  and CD8 +  T cells are stimulated with αCD3/αCD28 coated magnetic beads on day 0, and transduced with CAR on day 1 using a bicistronic lentiviral vector expressing CAR along with eGFP using a 2A ribosomal skipping sequence. Cultures are re-stimulated with either a cancer associated antigen as described herein +  K562 cells (K562 expressing a cancer associated antigen as described herein), wild-type K562 cells (K562 wild type) or K562 cells expressing hCD32 and 4-1BBL in the presence of antiCD3 and anti-CD28 antibody (K562-BBL-3/28) following washing. Exogenous TL-2 is added to the cultures every other day at 100 IU/ml. GFP +  T cells are enumerated by flow cytometry using bead-based counting. See, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). 
     Sustained CAR +  T cell expansion in the absence of re-stimulation can also be measured. See, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). Briefly, mean T cell volume (fl) is measured on day 8 of culture using a Coulter Multisizer III particle counter, a Nexcelom Cellometer Vision or Millipore Scepter, following stimulation with αCD3/αCD28 coated magnetic beads on day 0, and transduction with the indicated CAR on day 1. 
     Animal models can also be used to measure a CART activity. For example, xenograft model using human a cancer associated antigen described herein-specific CAR +  T cells to treat a primary human pre-B ALL in immunodeficient mice can be used. See, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). Very briefly, after establishment of ALL, mice are randomized as to treatment groups. Different numbers of a cancer associated antigen-specific CAR engineered T cells are coinjected at a 1:1 ratio into NOD-SCID-T mice bearing B-ALL. The number of copies of a cancer associated antigen-specific CAR vector in spleen DNA from mice is evaluated at various times following T cell injection. Animals are assessed for leukemia at weekly intervals. Peripheral blood a cancer associate antigen as described herein +  B-ALL blast cell counts are measured in mice that are injected with a cancer associated antigen described herein-ζ CAR +  T cells or mock-transduced T cells. Survival curves for the groups are compared using the log-rank test. In addition, absolute peripheral blood CD4 +  and CD8 +  T cell counts 4 weeks following T cell injection in NOD-SCID-γ −/−  mice can also be analyzed. Mice are injected with leukemic cells and 3 weeks later are injected with T cells engineered to express CAR by a bicistronic lentiviral vector that encodes the CAR linked to eGFP. T cells are normalized to 45-50% input GFP +  T cells by mixing with mock-transduced cells prior to injection, and confirmed by flow cytometry. Animals are assessed for leukemia at 1-week intervals. Survival curves for the CAR +  T cell groups are compared using the log-rank test. 
     Dose dependent CAR treatment response can be evaluated. See, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). For example, peripheral blood is obtained 35-70 days after establishing leukemia in mice injected on day 21 with CAR T cells, an equivalent number of mock-transduced T cells, or no T cells. Mice from each group are randomly bled for determination of peripheral blood a cancer associate antigen as described herein +  ALL blast counts and then killed on days 35 and 49. The remaining animals are evaluated on days 57 and 70. 
     Assessment of cell proliferation and cytokine production has been previously described, e.g., at Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). Briefly, assessment of CAR-mediated proliferation is performed in microtiter plates by mixing washed T cells with K562 cells expressing a cancer associated antigen described herein (K19) or CD32 and CD137 (KT32-BBL) for a final T-cell:K562 ratio of 2:1. K562 cells are irradiated with gamma-radiation prior to use. Anti-CD3 (clone OKT3) and anti-CD28 (clone 9.3) monoclonal antibodies are added to cultures with KT32-BBL cells to serve as a positive control for stimulating T-cell proliferation since these signals support long-term CD8+ T cell expansion ex vivo. T cells are enumerated in cultures using CountBright™ fluorescent beads (Invitrogen, Carlsbad, Calif.) and flow cytometry as described by the manufacturer. CAR +  T cells are identified by GFP expression using T cells that are engineered with eGFP-2A linked CAR-expressing lentiviral vectors. For CAR+ T cells not expressing GFP, the CAR+ T cells are detected with biotinylated recombinant a cancer associate antigen as described herein protein and a secondary avidin-PE conjugate. CD4+ and CD8 +  expression on T cells are also simultaneously detected with specific monoclonal antibodies (BD Biosciences). Cytokine measurements are performed on supernatants collected 24 hours following re-stimulation using the human TH1/TH2 cytokine cytometric bead array kit (BD Biosciences, San Diego, Calif.) according the manufacturer&#39;s instructions. Fluorescence is assessed using a FACScalibur flow cytometer, and data is analyzed according to the manufacturer&#39;s instructions. 
     Cytotoxicity can be assessed by a standard 51Cr-release assay. See, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). Briefly, target cells (K562 lines and primary pro-B-ALL cells) are loaded with 51Cr (as NaCrO4, New England Nuclear, Boston, Mass.) at 37° C. for 2 hours with frequent agitation, washed twice in complete RPMI and plated into microtiter plates. Effector T cells are mixed with target cells in the wells in complete RPMI at varying ratios of effector cell:target cell (E:T). Additional wells containing media only (spontaneous release, SR) or a 1% solution of triton-X 100 detergent (total release, TR) are also prepared. After 4 hours of incubation at 37° C., supernatant from each well is harvested. Released 51Cr is then measured using a gamma particle counter (Packard Instrument Co., Waltham, Mass.). Each condition is performed in at least triplicate, and the percentage of lysis is calculated using the formula: % Lysis=(ER-SR)/(TR-SR), where ER represents the average 51Cr released for each experimental condition. 
     Imaging technologies can be used to evaluate specific trafficking and proliferation of CARs in tumor-bearing animal models. Such assays have been described, for example, in Barrett et al., Human Gene Therapy 22:1575-1586 (2011). Briefly, NOD/SCID/γc −/−  (NSG) mice are injected IV with Nalm-6 cells followed 7 days later with T cells 4 hour after electroporation with the CAR constructs. The T cells are stably transfected with a lentiviral construct to express firefly luciferase, and mice are imaged for bioluminescence. Alternatively, therapeutic efficacy and specificity of a single injection of CAR +  T cells in Nalm-6 xenograft model can be measured as the following: NSG mice are injected with Nalm-6 transduced to stably express firefly luciferase, followed by a single tail-vein injection of T cells electroporated with cars of the present invention 7 days later. Animals are imaged at various time points post injection. For example, photon-density heat maps of firefly luciferasepositive leukemia in representative mice at day 5 (2 days before treatment) and day 8 (24 hr post CAR +  PBLs) can be generated. 
     Other assays, including those described in the Example section herein as well as those that are known in the art can also be used to evaluate the CARs described herein. 
     Therapeutic Application 
     In one aspect, the invention provides methods for treating a disease associated with expression of a cancer associated antigen described herein. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express an XCAR, wherein X represents a tumor antigen as described herein, and wherein the cancer cells express said X tumor antigen. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a XCAR described herein, wherein the cancer cells express X. In one embodiment, X is expressed on both normal cells and cancers cells, but is expressed at lower levels on normal cells. In one embodiment, the method further comprises selecting a CAR that binds X with an affinity that allows the XCAR to bind and kill the cancer cells expressing X but less than 30%, 25%, 20%, 15%, 10%, 5% or less of the normal cells expressing X are killed, e.g., as determined by an assay described herein. For example, the assay described in  FIGS.  13 A and  13 B  can be used or a killing assay such as flow cytometry based on Cr51 CTL. In one embodiment, the selected CAR has an antigen binding domain that has a binding affinity KD of 10 −4  M to 10 −1  M, e.g., 10 −5  M to 10 −7  M, e.g., 10 −6  M or 10 −7  M, for the target antigen. In one embodiment, the selected antigen binding domain has a binding affinity that is at least five-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold or 1,000-fold less than a reference antibody, e.g., an antibody described herein. 
     In one embodiment, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express CD19 CAR, wherein the cancer cells express CD19. In one embodiment, the cancer to be treated is ALL (acute lymphoblastic leukemia), CLL (chronic lymphocytic leukemia), DLBCL (diffuse large B-cell lymphoma), MCL (Mantle cell lymphoma, or MM (multiple myeloma). 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express an EGFRvIIICAR, wherein the cancer cells express EGFRvIII. In one embodiment, the cancer to be treated is glioblastoma. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a mesothelinCAR, wherein the cancer cells express mesothelin. In one embodiment, the cancer to be treated is mesothelioma, pancreatic cancer, or ovarian cancer. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a CD123CAR, wherein the cancer cells express CD123. In one embodiment, the cancer to be treated is AML. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a CD22CAR, wherein the cancer cells express CD22. In one embodiment, the cancer to be treated is B cell malignancies. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a CS-1CAR, wherein the cancer cells express CS-1. In one embodiment, the cancer to be treated is multiple myeloma. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a CLL-1CAR, wherein the cancer cells express CLL-1. In one embodiment, the cancer to be treated is AML. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a CD33CAR, wherein the cancer cells express CD33. In one embodiment, the cancer to be treated is AML. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a GD2CAR, wherein the cancer cells express GD2. In one embodiment, the cancer to be treated is neuroblastoma. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a BCMACAR, wherein the cancer cells express BCMA. In one embodiment, the cancer to be treated is multiple myeloma. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a TnCAR, wherein the cancer cells express Tn antigen. In one embodiment, the cancer to be treated is ovarian cancer. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a PSMACAR, wherein the cancer cells express PSMA. In one embodiment, the cancer to be treated is prostate cancer. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a ROR1CAR, wherein the cancer cells express ROR1. In one embodiment, the cancer to be treated is B cell malignancies. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a FLT3 CAR, wherein the cancer cells express FLT3. In one embodiment, the cancer to be treated is AML. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a TAG72CAR, wherein the cancer cells express TAG72. In one embodiment, the cancer to be treated is gastrointestinal cancer. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a CD38CAR, wherein the cancer cells express CD38. In one embodiment, the cancer to be treated is multiple myeloma. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a CD44v6CAR, wherein the cancer cells express CD44v6. In one embodiment, the cancer to be treated is cervical cancer, AML, or MM. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a CEACAR, wherein the cancer cells express CEA. In one embodiment, the cancer to be treated is pastrointestinal cancer, or pancreatic cancer. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express an EPCAMCAR, wherein the cancer cells express EPCAM. In one embodiment, the cancer to be treated is gastrointestinal cancer. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a B7H3CAR, wherein the cancer cells express B7H3. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a KITCAR, wherein the cancer cells express KIT. In one embodiment, the cancer to be treated is gastrointestinal cancer. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express an IL-13Ra2CAR, wherein the cancer cells express IL-13Ra2. In one embodiment, the cancer to be treated is glioblastoma. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a PRSS21CAR, wherein the cancer cells express PRSS21. In one embodiment, the cancer to be treated is selected from ovarian, pancreatic, lung and breast cancer. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a CD30CAR, wherein the cancer cells express CD30. In one embodiment, the cancer to be treated is lymphomas, or leukemias. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a GD3CAR, wherein the cancer cells express GD3. In one embodiment, the cancer to be treated is melanoma. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a CD171CAR, wherein the cancer cells express CD171. In one embodiment, the cancer to be treated is neuroblastoma, ovarian cancer, melanoma, breast cancer, pancreatic cancer, colon cancers, or NSCLC (non-small cell lung cancer). 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express an IL-11RaCAR, wherein the cancer cells express IL-11Ra. In one embodiment, the cancer to be treated is osteosarcoma. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a PSCACAR, wherein the cancer cells express PSCA. In one embodiment, the cancer to be treated is prostate cancer. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a VEGFR2CAR, wherein the cancer cells express VEGFR2. In one embodiment, the cancer to be treated is a solid tumor. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a LewisYCAR, wherein the cancer cells express LewisY. In one embodiment, the cancer to be treated is ovarian cancer, or AML. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a CD24CAR, wherein the cancer cells express CD24. In one embodiment, the cancer to be treated is pancreatic cancer. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a PDGFR-betaCAR, wherein the cancer cells express PDGFR-beta. In one embodiment, the cancer to be treated is breast cancer, prostate cancer, GIST (gastrointestinal stromal tumor), CML, DFSP (dermatofibrosarcoma protuberans), or glioma. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a SSEA-4CAR, wherein the cancer cells express SSEA-4. In one embodiment, the cancer to be treated is glioblastoma, breast cancer, lung cancer, or stem cell cancer. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a CD20CAR, wherein the cancer cells express CD20. In one embodiment, the cancer to be treated is B cell malignancies. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a Folate receptor alphaCAR, wherein the cancer cells express folate receptor alpha. In one embodiment, the cancer to be treated is ovarian cancer, NSCLC, endometrial cancer, renal cancer, or other solid tumors. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express an ERBB2CAR, wherein the cancer cells express ERBB2 (Her2/neu). In one embodiment, the cancer to be treated is breast cancer, gastric cancer, colorectal cancer, lung cancer, or other solid tumors. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a MUC1CAR, wherein the cancer cells express MUC1. In one embodiment, the cancer to be treated is breast cancer, lung cancer, or other solid tumors. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express an EGFRCAR, wherein the cancer cells express EGFR. In one embodiment, the cancer to be treated is glioblastoma, SCLC (small cell lung cancer), SCCHN (squamous cell carcinoma of the head and neck), NSCLC, or other solid tumors. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a NCAMCAR, wherein the cancer cells express NCAM. In one embodiment, the cancer to be treated is neuroblastoma, or other solid tumors. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a CAIXCAR, wherein the cancer cells express CAIX. In one embodiment, the cancer to be treated is renal cancer, CRC, cervical cancer, or other solid tumors. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express an EphA2CAR, wherein the cancer cells express EphA2. In one embodiment, the cancer to be treated is GBM. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a GD3CAR, wherein the cancer cells express GD3. In one embodiment, the cancer to be treated is melanoma. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a Fucosyl GM1CAR, wherein the cancer cells express Fucosyl GM In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a sLeCAR, wherein the cancer cells express sLe. In one embodiment, the cancer to be treated is NSCLC, or AML. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a GM3CAR, wherein the cancer cells express GM3. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a TGS5CAR, wherein the cancer cells express TGS5. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a HMWMAACAR, wherein the cancer cells express HMWMAA. In one embodiment, the cancer to be treated is melanoma, glioblastoma, or breast cancer. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express an o-acetyl-GD2CAR, wherein the cancer cells express o-acetyl-GD2. In one embodiment, the cancer to be treated is neuroblastoma, or melanoma. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a CD19CAR, wherein the cancer cells express CD19. In one embodiment, the cancer to be treated isFolate receptor beta AML, myeloma 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a TEM1/CD248CAR, wherein the cancer cells express TEM1/CD248. In one embodiment, the cancer to be treated is a solid tumor. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a TEM7RCAR, wherein the cancer cells express TEM7R. In one embodiment, the cancer to be treated is solid tumor. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a CLDN6CAR, wherein the cancer cells express CLDN6. In one embodiment, the cancer to be treated is ovarian cancer, lung cancer, or breast cancer. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a TSHRCAR, wherein the cancer cells express TSHR. In one embodiment, the cancer to be treated is thyroid cancer, or multiple myeloma. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a GPRC5DCAR, wherein the cancer cells express GPRC5D. In one embodiment, the cancer to be treated is multiple myeloma. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a CXORF61CAR, wherein the cancer cells express CXORF61. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a CD97CAR, wherein the cancer cells express CD97. In one embodiment, the cancer to be treated is B cell malignancies, gastric cancer, pancreatic cancer, esophageal cancer, glioblastoma, breast cancer, or colorectal cancer. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a CD179aCAR, wherein the cancer cells express CD179a. In one embodiment, the cancer to be treated is B cell malignancies. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express an ALK CAR, wherein the cancer cells express ALK. In one embodiment, the cancer to be treated is NSCLC, ALCL (anaplastic large cell lymphoma), IMT (inflammatory myofibroblastic tumor), or neuroblastoma. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a Polysialic acid CAR, wherein the cancer cells express Polysialic acid. In one embodiment, the cancer to be treated is small cell lung cancer. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a PLAC1CAR, wherein the cancer cells express PLAC1. In one embodiment, the cancer to be treated is HCC (hepatocellular carcinoma). 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a GloboHCAR, wherein the cancer cells express GloboH. In one embodiment, the cancer to be treated is ovarian cancer, gastric cancer, prostate cancer, lung cancer, breast cancer, or pancreatic cancer. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a NY-BR-1CAR, wherein the cancer cells express NY-BR-1. In one embodiment, the cancer to be treated is breast cancer. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a UPK2CAR, wherein the cancer cells express UPK2. In one embodiment, the cancer to be treated is bladder cancer. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a HAVCR1CAR, wherein the cancer cells express HAVCR1. In one embodiment, the cancer to be treated is renal cancer. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a ADRB3CAR, wherein the cancer cells express ADRB3. In one embodiment, the cancer to be treated is Ewing sarcoma. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a PANX3CAR, wherein the cancer cells express PANX3. In one embodiment, the cancer to be treated is osteosarcoma. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a GPR20CAR, wherein the cancer cells express GPR20. In one embodiment, the cancer to be treated is GIST. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a LY6KCAR, wherein the cancer cells express LY6K. In one embodiment, the cancer to be treated is breast cancer, lung cancer, ovary caner, or cervix cancer. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a OR51E2CAR, wherein the cancer cells express OR51E2. In one embodiment, the cancer to be treated is prostate cancer. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a TARPCAR, wherein the cancer cells express TARP. In one embodiment, the cancer to be treated is prostate cancer. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a WT1CAR, wherein the cancer cells express WT1. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a NY-ESO-1CAR, wherein the cancer cells express NY-ESO-1. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a LAGE-1a CAR, wherein the cancer cells express LAGE-1a. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a MAGE-A1CAR, wherein the cancer cells express MAGE-A1. In one embodiment, the cancer to be treated is melanoma. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a MAGE A1CAR, wherein the cancer cells express MAGE A1. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a ETV6-AML CAR, wherein the cancer cells express ETV6-AML. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a sperm protein 17 CAR, wherein the cancer cells express sperm protein 17. In one embodiment, the cancer to be treated is ovarian cancer, HCC, or NSCLC. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a XAGE1CAR, wherein the cancer cells express XAGE1. In one embodiment, the cancer to be treated is Ewings, or rhabdo cancer. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a Tie 2 CAR, wherein the cancer cells express Tie 2. In one embodiment, the cancer to be treated is a solid tumor. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a MAD-CT-1CAR, wherein the cancer cells express MAD-CT-1. In one embodiment, the cancer to be treated is prostate cancer, or melanoma. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a MAD-CT-2CAR, wherein the cancer cells express MAD-CT-2. In one embodiment, the cancer to be treated is prostate cancer, melanoma. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a Fos-related antigen 1 CAR, wherein the cancer cells express Fos-related antigen 1. In one embodiment, the cancer to be treated is glioma, squamous cell cancer, or pancreatic cancer. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a p53CAR, wherein the cancer cells express p53. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a prostein CAR, wherein the cancer cells express prostein. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a survivin and telomerase CAR, wherein the cancer cells express survivin and telomerase. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a PCTA-1/Galectin 8 CAR, wherein the cancer cells express PCTA-1/Galectin 8. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a MelanA/MART1CAR, wherein the cancer cells express MelanA/MART1. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a Ras mutant CAR, wherein the cancer cells express Ras mutant. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a p53 mutant CAR, wherein the cancer cells express p53 mutant. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a hTERT CAR, wherein the cancer cells express hTERT. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a sarcoma translocation breakpoints CAR, wherein the cancer cells express sarcoma translocation breakpoints. In one embodiment, the cancer to be treated is sarcoma. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a ML-IAP CAR, wherein the cancer cells express ML-IAP. In one embodiment, the cancer to be treated is melanoma. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express an ERGCAR, wherein the cancer cells express ERG (TMPRSS2 ETS fusion gene). 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a NA17CAR, wherein the cancer cells express NA17. In one embodiment, the cancer to be treated is melanoma. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a PAX3CAR, wherein the cancer cells express PAX3. In one embodiment, the cancer to be treated is alveolar rhabdomyosarcoma. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express an androgen receptor CAR, wherein the cancer cells express androgen receptor. In one embodiment, the cancer to be treated is metastatic prostate cancer. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a Cyclin B1CAR, wherein the cancer cells express Cyclin B1. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a MYCNCAR, wherein the cancer cells express MYCN. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a RhoC CAR, wherein the cancer cells express RhoC. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a TRP-2CAR, wherein the cancer cells express TRP-2. In one embodiment, the cancer to be treated is melanoma. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a CYP1B1CAR, wherein the cancer cells express CYP1B1. In one embodiment, the cancer to be treated is breast cancer, colon cancer, lung cancer, esophagus cancer, skin cancer, lymph node cancer, brain cancer, or testis cancer. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a BORIS CAR, wherein the cancer cells express BORIS. In one embodiment, the cancer to be treated is lung cancer. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a SART3CAR, wherein the cancer cells express SART3 In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a PAX5CAR, wherein the cancer cells express PAX5. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a OY-TES1CAR, wherein the cancer cells express OY-TES1. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a LCK CAR, wherein the cancer cells express LCK. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a AKAP-4CAR, wherein the cancer cells express AKAP-4. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a SSX2CAR, wherein the cancer cells express SSX2. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a RAGE-1CAR, wherein the cancer cells express RAGE-1. In one embodiment, the cancer to be treated is RCC (renal cell cancer), or other solid tumors In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a human telomerase reverse transcriptase CAR, wherein the cancer cells express human telomerase reverse transcriptase. In one embodiment, the cancer to be treated is solid tumors. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a RU1CAR, wherein the cancer cells express RU1. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a RU2CAR, wherein the cancer cells express RU2. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express an intestinal carboxyl esterase CAR, wherein the cancer cells express intestinal carboxyl esterase. In one embodiment, the cancer to be treated is thyroid cancer, RCC, CRC (colorectal cancer), breast cancer, or other solid tumors. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a Prostase CAR, wherein the cancer cells express Prostase. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a PAPCAR, wherein the cancer cells express PAP. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express an IGF-I receptor CAR, wherein the cancer cells express IGF-I receptor. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a gp100 CAR, wherein the cancer cells express gp100. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a bcr-abl CAR, wherein the cancer cells express bcr-abl. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a tyrosinase CAR, wherein the cancer cells express tyrosinase. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a Fucosyl GM1CAR, wherein the cancer cells express Fucosyl GM1. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a mut hsp70-2CAR, wherein the cancer cells express mut hsp70-2. In one embodiment, the cancer to be treated is melanoma. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a CD79a CAR, wherein the cancer cells express CD79a. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a CD79b CAR, wherein the cancer cells express CD79b. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a CD72 CAR, wherein the cancer cells express CD72. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a LAIR1 CAR, wherein the cancer cells express LAIR1. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a FCAR CAR, wherein the cancer cells express FCAR. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a LILRA2 CAR, wherein the cancer cells express LILRA2. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a CD300LF CAR, wherein the cancer cells express CD300LF. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a CLEC12A CAR, wherein the cancer cells express CLEC12A. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a BST2 CAR, wherein the cancer cells express BST2. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express an EIR2 CAR, wherein the cancer cells express EMR2. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a LY75 CAR, wherein the cancer cells express LY75. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a GPC3 CAR, wherein the cancer cells express GPC3. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express a FCRL5 CAR, wherein the cancer cells express FCRL5. 
     In one aspect, the present invention provides methods of treating cancer by providing to the subject in need thereof immune effector cells (e.g., T cells, NK cells) that are engineered to express an IGLL1 CAR, wherein the cancer cells express IGLL1. 
     In one aspect, the present invention relates to treatment of a subject in vivo using an PD1 CAR such that growth of cancerous tumors is inhibited. A PD1 CAR may be used alone to inhibit the growth of cancerous tumors. Alternatively, PD1 CAR may be used in conjunction with other CARs, immunogenic agents, standard cancer treatments, or other antibodies. In one embodiment, the subject is treated with a PD1 CAR and an XCAR described herein. In an embodiment, a PD1 CAR is used in conjunction with another CAR, e.g., a CAR described herein, and a kinase inhibitor, e.g., a kinase inhibitor described herein. 
     In another aspect, a method of treating a subject, e.g., reducing or ameliorating, a hyperproliferative condition or disorder (e.g., a cancer), e.g., solid tumor, a soft tissue tumor, or a metastatic lesion, in a subject is provided. As used herein, the term “cancer” is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. Examples of solid tumors include malignancies, e.g., sarcomas, adenocarcinomas, and carcinomas, of the various organ systems, such as those affecting liver, lung, breast, lymphoid, gastrointestinal (e.g., colon), genitourinary tract (e.g., renal, urothelial cells), prostate and pharynx. Adenocarcinomas include malignancies such as most colon cancers, rectal cancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus. In one embodiment, the cancer is a melanoma, e.g., an advanced stage melanoma. Metastatic lesions of the aforementioned cancers can also be treated or prevented using the methods and compositions of the invention. Examples of other cancers that can be treated include bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin Disease, non-Hodgkin lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemias including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi&#39;s sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, and combinations of said cancers. Treatment of metastatic cancers, e.g., metastatic cancers that express PD-L1 (Iwai et al. (2005) Int. Immunol. 17:133-144) can be effected using the antibody molecules described herein. 
     Exemplary cancers whose growth can be inhibited include cancers typically responsive to immunotherapy. Non-limiting examples of cancers for treatment include melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g. clear cell carcinoma), prostate cancer (e.g. hormone refractory prostate adenocarcinoma), breast cancer, colon cancer and lung cancer (e.g. non-small cell lung cancer). Additionally, refractory or recurrent malignancies can be treated using the molecules described herein. 
     In one aspect, the invention pertains to a vector comprising a CAR operably linked to promoter for expression in mammalian immune effector cells (e.g., T cells, NK cells). In one aspect, the invention provides a recombinant immune effector cell expressing a CAR of the present invention for use in treating cancer expressing a cancer associate antigen as described herein. In one aspect, CAR-expressing cells of the invention is capable of contacting a tumor cell with at least one cancer associated antigen expressed on its surface such that the CAR-expressing cell targets the cancer cell and growth of the cancer is inhibited. 
     In one aspect, the invention pertains to a method of inhibiting growth of a cancer, comprising contacting the cancer cell with a CAR-expressing cell of the present invention such that the CART is activated in response to the antigen and targets the cancer cell, wherein the growth of the tumor is inhibited. 
     In one aspect, the invention pertains to a method of treating cancer in a subject. The method comprises administering to the subject CAR-expressing cell of the present invention such that the cancer is treated in the subject. In one aspect, the cancer associated with expression of a cancer associate antigen as described herein is a hematological cancer. In one aspect, the hematological cancer is a leukemia or a lymphoma. In one aspect, a cancer associated with expression of a cancer associate antigen as described herein includes cancers and malignancies including, but not limited to, e.g., one or more acute leukemias including but not limited to, e.g., B-cell acute Lymphoid Leukemia (“BALL”), T-cell acute Lymphoid Leukemia (“TALL”), acute lymphoid leukemia (ALL); one or more chronic leukemias including but not limited to, e.g., chronic myelogenous leukemia (CML), Chronic Lymphoid Leukemia (CLL). Additional cancers or hematologic conditions associated with expression of a cancer associate antigen as described herein include, but are not limited to, e.g., B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt&#39;s lymphoma, diffuse large B cell lymphoma, Follicular lymphoma, Hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, and “preleukemia” which are a diverse collection of hematological conditions united by ineffective production (or dysplasia) of myeloid blood cells, and the like. Further a disease associated with a cancer associate antigen as described herein expression include, but not limited to, e.g., atypical and/or non-classical cancers, malignancies, precancerous conditions or proliferative diseases associated with expression of a cancer associate antigen as described herein. 
     In some embodiments, a cancer that can be treated with CAR-expressing cell of the present invention is multiple myeloma. Multiple myeloma is a cancer of the blood, characterized by accumulation of a plasma cell clone in the bone marrow. Current therapies for multiple myeloma include, but are not limited to, treatment with lenalidomide, which is an analog of thalidomide. Lenalidomide has activities which include anti-tumor activity, angiogenesis inhibition, and immunomodulation. Generally, myeloma cells are thought to be negative for a cancer associate antigen as described herein expression by flow cytometry. Thus, in some embodiments, a CD19 CAR, e.g., as described herein, may be used to target myeloma cells. In some embodiments, cars of the present invention therapy can be used in combination with one or more additional therapies, e.g., lenalidomide treatment. 
     The invention includes a type of cellular therapy where immune effector cells (e.g., T cells, NK cells) are genetically modified to express a chimeric antigen receptor (CAR) and the CAR-expressing T cell or NK cell is infused to a recipient in need thereof. The infused cell is able to kill tumor cells in the recipient. Unlike antibody therapies, CAR-modified immune effector cells (e.g., T cells, NK cells) are able to replicate in vivo resulting in long-term persistence that can lead to sustained tumor control. In various aspects, the immune effector cells (e.g., T cells, NK cells) administered to the patient, or their progeny, persist in the patient for at least four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, thirteen months, fourteen month, fifteen months, sixteen months, seventeen months, eighteen months, nineteen months, twenty months, twenty-one months, twenty-two months, twenty-three months, two years, three years, four years, or five years after administration of the T cell or NK cell to the patient. 
     The invention also includes a type of cellular therapy where immune effector cells (e.g., T cells, NK cells) are modified, e.g., by in vitro transcribed RNA, to transiently express a chimeric antigen receptor (CAR) and the CAR T cell or NK cell is infused to a recipient in need thereof. The infused cell is able to kill tumor cells in the recipient. Thus, in various aspects, the immune effector cells (e.g., T cells, NK cells) administered to the patient, is present for less than one month, e.g., three weeks, two weeks, one week, after administration of the T cell or NK cell to the patient. 
     Without wishing to be bound by any particular theory, the anti-tumor immunity response elicited by the CAR-modified immune effector cells (e.g., T cells, NK cells) may be an active or a passive immune response, or alternatively may be due to a direct vs indirect immune response. In one aspect, the CAR transduced immune effector cells (e.g., T cells, NK cells) exhibit specific proinflammatory cytokine secretion and potent cytolytic activity in response to human cancer cells expressing the a cancer associate antigen as described herein, resist soluble a cancer associate antigen as described herein inhibition, mediate bystander killing and mediate regression of an established human tumor. For example, antigen-less tumor cells within a heterogeneous field of a cancer associate antigen as described herein-expressing tumor may be susceptible to indirect destruction by a cancer associate antigen as described herein-redirected immune effector cells (e.g., T cells, NK cells) that has previously reacted against adjacent antigen-positive cancer cells. 
     In one aspect, the fully-human CAR-modified immune effector cells (e.g., T cells, NK cells) of the invention may be a type of vaccine for ex vivo immunization and/or in vivo therapy in a mammal. In one aspect, the mammal is a human. 
     With respect to ex vivo immunization, at least one of the following occurs in vitro prior to administering the cell into a mammal: i) expansion of the cells, ii) introducing a nucleic acid encoding a CAR to the cells or iii) cryopreservation of the cells. 
     Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from a mammal (e.g., a human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing a CAR disclosed herein. The CAR-modified cell can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient may be a human and the CAR-modified cell can be autologous with respect to the recipient. Alternatively, the cells can be allogeneic, syngeneic or xenogeneic with respect to the recipient. 
     The procedure for ex vivo expansion of hematopoietic stem and progenitor cells is described in U.S. Pat. No. 5,199,942, incorporated herein by reference, can be applied to the cells of the present invention. Other suitable methods are known in the art, therefore the present invention is not limited to any particular method of ex vivo expansion of the cells. Briefly, ex vivo culture and expansion of immune effector cells (e.g., T cells, NK cells) comprises: (1) collecting CD34+ hematopoietic stem and progenitor cells from a mammal from peripheral blood harvest or bone marrow explants; and (2) expanding such cells ex vivo. In addition to the cellular growth factors described in U.S. Pat. No. 5,199,942, other factors such as flt3-L, IL-1, IL-3 and c-kit ligand, can be used for culturing and expansion of the cells. 
     In addition to using a cell-based vaccine in terms of ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response directed against an antigen in a patient. 
     Generally, the cells activated and expanded as described herein may be utilized in the treatment and prevention of diseases that arise in individuals who are immunocompromised. In particular, the CAR-modified immune effector cells (e.g., T cells, NK cells) of the invention are used in the treatment of diseases, disorders and conditions associated with expression of a cancer associate antigen as described herein. In certain aspects, the cells of the invention are used in the treatment of patients at risk for developing diseases, disorders and conditions associated with expression of a cancer associate antigen as described herein. Thus, the present invention provides methods for the treatment or prevention of diseases, disorders and conditions associated with expression of a cancer associate antigen as described herein comprising administering to a subject in need thereof, a therapeutically effective amount of the CAR-modified immune effector cells (e.g., T cells, NK cells) of the invention. 
     In one aspect the CAR-expressing cells of the inventions may be used to treat a proliferative disease such as a cancer or malignancy or is a precancerous condition such as a myelodysplasia, a myelodysplastic syndrome or a preleukemia. Further a disease associated with a cancer associate antigen as described herein expression include, but not limited to, e.g., atypical and/or non-classical cancers, malignancies, precancerous conditions or proliferative diseases expressing a cancer associated antigen as described herein. Non-cancer related indications associated with expression of a cancer associate antigen as described herein include, but are not limited to, e.g., autoimmune disease, (e.g., lupus), inflammatory disorders (allergy and asthma) and transplantation. 
     The CAR-modified immune effector cells (e.g., T cells, NK cells) of the present invention may be administered either alone, or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2 or other cytokines or cell populations. 
     Hematologic Cancer 
     Hematological cancer conditions are the types of cancer such as leukemia, lymphoma, and malignant lymphoproliferative conditions that affect blood, bone marrow and the lymphatic system. 
     Leukemia can be classified as acute leukemia and chronic leukemia. Acute leukemia can be further classified as acute myelogenous leukemia (AML) and acute lymphoid leukemia (ALL). Chronic leukemia includes chronic myelogenous leukemia (CML) and chronic lymphoid leukemia (CLL). Other related conditions include myelodysplastic syndromes (MDS, formerly known as “preleukemia”) which are a diverse collection of hematological conditions united by ineffective production (or dysplasia) of myeloid blood cells and risk of transformation to AML. 
     Lymphoma is a group of blood cell tumors that develop from lymphocytes. Exemplary lymphomas include non-Hodgkin lymphoma and Hodgkin lymphoma. 
     The present invention provides for compositions and methods for treating cancer. In one aspect, the cancer is a hematologic cancer including but is not limited to hematolical cancer is a leukemia or a lymphoma. In one aspect, the CAR-expressing cells of the invention may be used to treat cancers and malignancies such as, but not limited to, e.g., acute leukemias including but not limited to, e.g., B-cell acute lymphoid leukemia (“BALL”), T-cell acute lymphoid leukemia (“TALL”), acute lymphoid leukemia (ALL); one or more chronic leukemias including but not limited to, e.g., chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL); additional hematologic cancers or hematologic conditions including, but not limited to, e.g., B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt&#39;s lymphoma, diffuse large B cell lymphoma, Follicular lymphoma, Hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, and “preleukemia” which are a diverse collection of hematological conditions united by ineffective production (or dysplasia) of myeloid blood cells, and the like. Further a disease associated with a cancer associate antigen as described herein expression includes, but not limited to, e.g., atypical and/or non-classical cancers, malignancies, precancerous conditions or proliferative diseases expressing a cancer associate antigen as described herein. 
     The present invention also provides methods for inhibiting the proliferation or reducing a cancer associated antigen as described herein-expressing cell population, the methods comprising contacting a population of cells comprising a cancer associated antigen as described herein-expressing cell with a CAR-expressing T cell or NK cell of the invention that binds to the a cancer associate antigen as described herein-expressing cell. In a specific aspect, the present invention provides methods for inhibiting the proliferation or reducing the population of cancer cells expressing a cancer associated antigen as described herein, the methods comprising contacting a cancer associate antigen as described herein-expressing cancer cell population with a CAR-expressing T cell or NK cell of the invention that binds to a cancer associated antigen as described herein-expressing cell. In one aspect, the present invention provides methods for inhibiting the proliferation or reducing the population of cancer cells expressing a cancer associated antigen as described herein, the methods comprising contacting a cancer associated antigen as described herein-expressing cancer cell population with a CAR-expressing T cell or NK cell of the invention that binds to a cancer associated antigen as described herein-expressing cell. In certain aspects, a CAR-expressing T cell or NK cell of the invention reduces the quantity, number, amount or percentage of cells and/or cancer cells by at least 25%, at least 30%, at least 40%, at least 50%, at least 65%, at least 75%, at least 85%, at least 95%, or at least 99% in a subject with or animal model for myeloid leukemia or another cancer associated with a cancer associated antigen as described herein-expressing cells relative to a negative control. In one aspect, the subject is a human. 
     The present invention also provides methods for preventing, treating and/or managing a disease associated with a cancer associated antigen as described herein-expressing cells (e.g., a hematologic cancer or atypical cancer expressing a cancer associated antigen as described herein), the methods comprising administering to a subject in need a CAR T cell or NK cell of the invention that binds to a cancer associated antigen as described herein-expressing cell. In one aspect, the subject is a human. Non-limiting examples of disorders associated with a cancer associated antigen as described herein-expressing cells include autoimmune disorders (such as lupus), inflammatory disorders (such as allergies and asthma) and cancers (such as hematological cancers or atypical cancers expressing a cancer associated antigen as described herein). 
     The present invention also provides methods for preventing, treating and/or managing a disease associated with a cancer associated antigen as described herein-expressing cells, the methods comprising administering to a subject in need a CAR T cell or NK cell of the invention that binds to a cancer associated antigen as described herein-expressing cell. In one aspect, the subject is a human. 
     The present invention provides methods for preventing relapse of cancer associated with a cancer associated antigen as described herein-expressing cells, the methods comprising administering to a subject in need thereof aCAR T cell or NK cell of the invention that binds to a cancer associated antigen as described herein-expressing cell. In one aspect, the methods comprise administering to the subject in need thereof an effective amount of a CAR-expressingT cell or NK cell described herein that binds to a cancer associated antigen as described herein-expressing cell in combination with an effective amount of another therapy. 
     Combination Therapies 
     A CAR-expressing cell described herein may be used in combination with other known agents and therapies. Administered “in combination”, as used herein, means that two (or more) different treatments are delivered to the subject during the course of the subject&#39;s affliction with the disorder, e.g., the two or more treatments are delivered after the subject has been diagnosed with the disorder and before the disorder has been cured or eliminated or treatment has ceased for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery”. In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In some embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered. 
     A CAR-expressing cell described herein and the at least one additional therapeutic agent can be administered simultaneously, in the same or in separate compositions, or sequentially. For sequential administration, the CAR-expressing cell described herein can be administered first, and the additional agent can be administered second, or the order of administration can be reversed. 
     The CAR therapy and/or other therapeutic agents, procedures or modalities can be administered during periods of active disorder, or during a period of remission or less active disease. The CAR therapy can be administered before the other treatment, concurrently with the treatment, post-treatment, or during remission of the disorder. 
     When administered in combination, the CAR therapy and the additional agent (e.g., second or third agent), or all, can be administered in an amount or dose that is higher, lower or the same than the amount or dosage of each agent used individually, e.g., as a monotherapy. In certain embodiments, the administered amount or dosage of the CAR therapy, the additional agent (e.g., second or third agent), or all, is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dosage of each agent used individually, e.g., as a monotherapy. In other embodiments, the amount or dosage of the CAR therapy, the additional agent (e.g., second or third agent), or all, that results in a desired effect (e.g., treatment of cancer) is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50% lower) than the amount or dosage of each agent used individually, e.g., as a monotherapy, required to achieve the same therapeutic effect. 
     In further aspects, a CAR-expressing cell described herein may be used in a treatment regimen in combination with surgery, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation. peptide vaccine, such as that described in Izumoto et al. 2008 J Neurosurg 108:963-971. 
     In one embodiment, a CAR-expressing cell described herein can be used in combination with a chemotherapeutic agent. Exemplary chemotherapeutic agents include an anthracycline (e.g., doxorubicin (e.g., liposomal doxorubicin)). a  vinca  alkaloid (e.g., vinblastine, vincristine, vindesine, vinorelbine), an alkylating agent (e.g., cyclophosphamide, decarbazine, melphalan, ifosfamide, temozolomide), an immune cell antibody (e.g., alemtuzamab, gemtuzumab, rituximab, ofatumumab, tositumomab, brentuximab), an antimetabolite (including, e.g., folic acid antagonists, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors (e.g., fludarabine)), an mTOR inhibitor, a TNFR glucocorticoid induced TNFR related protein (GITR) agonist, a proteasome inhibitor (e.g., aclacinomycin A, gliotoxin or bortezomib), an immunomodulator such as thalidomide or a thalidomide derivative (e.g., lenalidomide). 
     General Chemotherapeutic agents considered for use in combination therapies include anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU@), chlorambucil (Leukeran®), cisplatin (Platinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin (Actinomycin D, Cosmegan), daunorubicin hydrochloride (Cerubidine®), daunorubicin citrate liposome injection (DaunoXome®), dexamethasone, docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5-fluorouracil (Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, Gemcitabine (difluorodeoxycitidine), hydroxyurea (Hydrea®), Idarubicin (Idamycin®), ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®), leucovorin calcium, melphalan (Alkeran®), 6-mercaptopurine (Purinethol®), methotrexate (Folex®), mitoxantrone (Novantrone®), mylotarg, paclitaxel (Taxol®), phoenix (Yttrium90/MX-DTPA), pentostatin, polifeprosan 20 with carmustine implant (Gliadel®), tamoxifen citrate (Nolvadex®), teniposide (Vumon®), 6-thioguanine, thiotepa, tirapazamine (Tirazone®), topotecan hydrochloride for injection (Hycamptin®), vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine (Navelbine®). 
     Exemplary alkylating agents include, without limitation, nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas and triazenes): uracil mustard (Aminouracil Mustard®, Chlorethaminacil®, Demethyldopan®, Desmethyldopan®, Haemanthamine®, Nordopan®, Uracil nitrogen Mustard®, Uracillost®, Uracilmostaza®, Uramustin®, Uramustine®), chlormethine (Mustargen®), cyclophosphamide (Cytoxan®, Neosar®, Clafen®, Endoxan®, Procytox®, Revimmune™), ifosfamide (Mitoxana®), melphalan (Alkeran®), Chlorambucil (Leukeran®), pipobroman (Amedel®, Vercyte®), triethylenemelamine (Hemel®, Hexalen®, Hexastat®), triethylenethiophosphoramine, Temozolomide (Temodar®), thiotepa (Thioplex®), busulfan (Busilvex®, Myleran®), carmustine (BiCNU@), lomustine (CeeNU®), streptozocin (Zanosar®), and Dacarbazine (DTIC-Dome®). Additional exemplary alkylating agents include, without limitation, Oxaliplatin (Eloxatin®); Temozolomide (Temodar® and Temodal®); Dactinomycin (also known as actinomycin-D, Cosmegen®); Melphalan (also known as L-PAM, L-sarcolysin, and phenylalanine mustard, Alkeran®); Altretamine (also known as hexamethylmelamine (HMM), Hexalen®); Carmustine (BiCNU@); Bendamustine (Treanda®); Busulfan (Busulfex® and Myleran®); Carboplatin (Paraplatin®); Lomustine (also known as CCNU, CeeNU®); Cisplatin (also known as CDDP, Platinol® and Platinol®-AQ); Chlorambucil (Leukeran®); Cyclophosphamide (Cytoxan® and Neosar®); Dacarbazine (also known as DTIC, DIC and imidazole carboxamide, DTIC-Dome®); Altretamine (also known as hexamethylmelamine (HMM), Hexalen®); Ifosfamide (Ifex®); Prednumustine; Procarbazine (Matulane®); Mechlorethamine (also known as nitrogen mustard, mustine and mechloroethamine hydrochloride, Mustargen®); Streptozocin (Zanosar®); Thiotepa (also known as thiophosphoamide, TESPA and TSPA, Thioplex®); Cyclophosphamide (Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune®); and Bendamustine HCl (Treanda®). 
     In embodiments, a CAR-expressing cell described herein is administered to a subject in combination with fludarabine, cyclophosphamide, and/or rituximab. In embodiments, a CAR-expressing cell described herein is administered to a subject in combination with fludarabine, cyclophosphamide, and rituximab (FCR). In embodiments, the subject has CLL. For example, the subject has a deletion in the short arm of chromosome 17 (del(17p), e.g., in a leukemic cell). In other examples, the subject does not have a del(17p). In embodiments, the subject comprises a leukemic cell comprising a mutation in the immunoglobulin heavy-chain variable-region (IgV H ) gene. In other embodiments, the subject does not comprise a leukemic cell comprising a mutation in the immunoglobulin heavy-chain variable-region (IgV H ) gene. In embodiments, the fludarabine is administered at a dosage of about 10-50 mg/m 2  (e.g., about 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, or 45-50 mg/m 2 ), e.g., intravenously. In embodiments, the cyclophosphamide is administered at a dosage of about 200-300 mg/m 2  (e.g., about 200-225, 225-250, 250-275, or 275-300 mg/m 2 ), e.g., intravenously. In embodiments, the rituximab is administered at a dosage of about 400-600 mg/m2 (e.g., 400-450, 450-500, 500-550, or 550-600 mg/m 2 ), e.g., intravenously. 
     In embodiments, a CAR-expressing cell described herein is administered to a subject in combination with bendamustine and rituximab. In embodiments, the subject has CLL. For example, the subject has a deletion in the short arm of chromosome 17 (del(17p), e.g., in a leukemic cell). In other examples, the subject does not have a del(17p). In embodiments, the subject comprises a leukemic cell comprising a mutation in the immunoglobulin heavy-chain variable-region (IgV H ) gene. In other embodiments, the subject does not comprise a leukemic cell comprising a mutation in the immunoglobulin heavy-chain variable-region (IgV H ) gene. In embodiments, the bendamustine is administered at a dosage of about 70-110 mg/m2 (e.g., 70-80, 80-90, 90-100, or 100-110 mg/m2), e.g., intravenously. In embodiments, the rituximab is administered at a dosage of about 400-600 mg/m2 (e.g., 400-450, 450-500, 500-550, or 550-600 mg/m 2 ), e.g., intravenously. 
     In embodiments, a CAR-expressing cell described herein is administered to a subject in combination with rituximab, cyclophosphamide, doxorubicine, vincristine, and/or a corticosteroid (e.g., prednisone). In embodiments, a CAR-expressing cell described herein is administered to a subject in combination with rituximab, cyclophosphamide, doxorubicine, vincristine, and prednisone (R-CHOP). In embodiments, the subject has diffuse large B-cell lymphoma (DLBCL). In embodiments, the subject has nonbulky limited-stage DLBCL (e.g., comprises a tumor having a size/diameter of less than 7 cm). In embodiments, the subject is treated with radiation in combination with the R-CHOP. For example, the subject is administered R-CHOP (e.g., 1-6 cycles, e.g., 1, 2, 3, 4, 5, or 6 cycles of R-CHOP), followed by radiation. In some cases, the subject is administered R-CHOP (e.g., 1-6 cycles, e.g., 1, 2, 3, 4, 5, or 6 cycles of R-CHOP) following radiation. 
     In embodiments, a CAR-expressing cell described herein is administered to a subject in combination with etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and/or rituximab. In embodiments, a CAR-expressing cell described herein is administered to a subject in combination with etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab (EPOCH-R). In embodiments, a CAR-expressing cell described herein is administered to a subject in combination with dose-adjusted EPOCH-R (DA-EPOCH-R). In embodiments, the subject has a B cell lymphoma, e.g., a Myc-rearranged aggressive B cell lymphoma. 
     In embodiments, a CAR-expressing cell described herein is administered to a subject in combination with rituximab and/or lenalidomide. Lenalidomide ((RS)-3-(4-Amino-1-oxo 1,3-dihydro-2H-isoindol-2-yl)piperidine-2,6-dione) is an immunomodulator. In embodiments, a CAR-expressing cell described herein is administered to a subject in combination with rituximab and lenalidomide. In embodiments, the subject has follicular lymphoma (FL) or mantle cell lymphoma (MCL). In embodiments, the subject has FL and has not previously been treated with a cancer therapy. In embodiments, lenalidomide is administered at a dosage of about 10-20 mg (e.g., 10-15 or 15-20 mg), e.g., daily. In embodiments, rituximab is administered at a dosage of about 350-550 mg/m 2  (e.g., 350-375, 375-400, 400-425, 425-450, 450-475, or 475-500 mg/m 2 ), e.g., intravenously. 
     Exemplary mTOR inhibitors include, e.g., temsirolimus; ridaforolimus (formally known as deferolimus, (1R,2R,4S)-4-[(2R)-2 [(1R,9S,12S,15R,16E,18R,19R,21R, 23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23, 29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.0 4,9 ] hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyl dimethylphosphinate, also known as AP23573 and MK8669, and described in PCT Publication No. WO 03/064383); everolimus (Afinitor® or RAD001); rapamycin (AY22989, Sirolimus®); simapimod (CAS 164301-51-3); emsirolimus, (5-{2,4-Bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl}-2-methoxyphenyl)methanol (AZD8055); 2-Amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one (PF04691502, CAS 1013101-36-4); and N 2 -[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-L-arginylglycyl-L-α-aspartylL-serine-, inner salt (SF1126, CAS 936487-67-1) (SEQ ID NO: 1262), and XL765. 
     Exemplary immunomodulators include, e.g., afutuzumab (available from Roche®); pegfilgrastim (Neulasta®); lenalidomide (CC-5013, Revlimid®); thalidomide (Thalomid®), actimid (CC4047); and IRX-2 (mixture of human cytokines including interleukin 1, interleukin 2, and interferon 7, CAS 951209-71-5, available from IRX Therapeutics). 
     Exemplary anthracyclines include, e.g., doxorubicin (Adriamycin® and Rubex®); bleomycin (Lenoxane®); daunorubicin (dauorubicin hydrochloride, daunomycin, and rubidomycin hydrochloride, Cerubidine®); daunorubicin liposomal (daunorubicin citrate liposome, DaunoXome®); mitoxantrone (DHAD, Novantrone®); epirubicin (Ellence™); idarubicin (Idamycin®, Idamycin PFS®); mitomycin C (Mutamycin®); geldanamycin; herbimycin; ravidomycin; and desacetylravidomycin. 
     Exemplary  vinca  alkaloids include, e.g., vinorelbine tartrate (Navelbine®), Vincristine (Oncovin®), and Vindesine (Eldisine®)); vinblastine (also known as vinblastine sulfate, vincaleukoblastine and VLB, Alkaban-AQ® and Velban®); and vinorelbine (Navelbine®). 
     Exemplary proteosome inhibitors include bortezomib (Velcade®); carfilzomib (PX-171-007, (S)-4-Methyl-N-((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)-pentanamide); marizomib (NPI-0052); ixazomib citrate (MLN-9708); delanzomib (CEP-18770); and O-Methyl-N-[(2-methyl-5-thiazolyl)carbonyl]-L-seryl-O-methyl-N-[(1S)-2-[(2R)-2-methyl-2-oxiranyl]-2-oxo-1-(phenylmethyl)ethyl]-L-serinamide (ONX-0912). 
     In embodiments, a CAR-expressing cell described herein is administered to a subject in combination with brentuximab. Brentuximab is an antibody-drug conjugate of anti-CD30 antibody and monomethyl auristatin E. In embodiments, the subject has Hodgkin&#39;s lymphoma (HL), e.g., relapsed or refractory HL. In embodiments, the subject comprises CD30+HL. In embodiments, the subject has undergone an autologous stem cell transplant (ΔSCT). In embodiments, the subject has not undergone an ΔSCT. In embodiments, brentuximab is administered at a dosage of about 1-3 mg/kg (e.g., about 1-1.5, 1.5-2, 2-2.5, or 2.5-3 mg/kg), e.g., intravenously, e.g., every 3 weeks. 
     In embodiments, a CAR-expressing cell described herein is administered to a subject in combination with brentuximab and dacarbazine or in combination with brentuximab and bendamustine. Dacarbazine is an alkylating agent with a chemical name of 5-(3,3-Dimethyl-1-triazenyl)imidazole-4-carboxamide. Bendamustine is an alkylating agent with a chemical name of 4-[5-[Bis(2-chloroethyl)amino]-1-methylbenzimidazol-2-yl]butanoic acid. In embodiments, the subject has Hodgkin&#39;s lymphoma (HL). In embodiments, the subject has not previously been treated with a cancer therapy. In embodiments, the subject is at least 60 years of age, e.g., 60, 65, 70, 75, 80, 85, or older. In embodiments, dacarbazine is administered at a dosage of about 300-450 mg/m 2  (e.g., about 300-325, 325-350, 350-375, 375-400, 400-425, or 425-450 mg/m 2 ), e.g., intravenously. In embodiments, bendamustine is administered at a dosage of about 75-125 mg/m2 (e.g., 75-100 or 100-125 mg/m 2 , e.g., about 90 mg/m 2 ), e.g., intravenously. In embodiments, brentuximab is administered at a dosage of about 1-3 mg/kg (e.g., about 1-1.5, 1.5-2, 2-2.5, or 2.5-3 mg/kg), e.g., intravenously, e.g., every 3 weeks. 
     In some embodiments, a CAR-expressing cell described herein is administered to a subject in combination with a CD20 inhibitor, e.g., an anti-CD20 antibody (e.g., an anti-CD20 mono- or bispecific antibody) or a fragment thereof. Exemplary anti-CD20 antibodies include but are not limited to rituximab, ofatumumab, ocrelizumab, veltuzumab, obinutuzumab, TRU-015 (Trubion Pharmaceuticals), ocaratuzumab, and Pro131921 (Genentech). See, e.g., Lim et al. Haematologica. 95.1(2010):135-43. 
     In some embodiments, the anti-CD20 antibody comprises rituximab. Rituximab is a chimeric mouse/human monoclonal antibody IgGa1 kappa that binds to CD20 and causes cytolysis of a CD20 expressing cell, e.g., as described in www.accessdata.fda.gov/drugsatfda_docs/label/2010/103705s53111bl.pdf. In embodiments, a CAR-expressing cell described herein is administered to a subject in combination with rituximab. In embodiments, the subject has CLL or SLL. 
     In some embodiments, rituximab is administered intravenously, e.g., as an intravenous infusion. For example, each infusion provides about 500-2000 mg (e.g., about 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900-950, 950-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400, 1400-1500, 1500-1600, 1600-1700, 1700-1800, 1800-1900, or 1900-2000 mg) of rituximab. In some embodiments, rituximab is administered at a dose of 150 mg/m 2  to 750 mg/m 2 , e.g., about 150-175 mg/m 2 , 175-200 mg/m 2 , 200-225 mg/m 2 , 225-250 mg/m 2 , 250-300 mg/m 2 , 300-325 mg/m 2 , 325-350 mg/m 2 , 350-375 mg/m 2 , 375-400 mg/m 2 , 400-425 mg/m 2 , 425-450 mg/m 2 , 450-475 mg/m 2 , 475-500 mg/m 2 , 500-525 mg/m 2 , 525-550 mg/m 2 , 550-575 mg/m 2 , 575-600 mg/m 2 , 600-625 mg/m 2 , 625-650 mg/m 2 , 650-675 mg/m 2 , or 675-700 mg/m 2 , where m 2  indicates the body surface area of the subject. In some embodiments, rituximab is administered at a dosing interval of at least 4 days, e.g., 4, 7, 14, 21, 28, 35 days, or more. For example, rituximab is administered at a dosing interval of at least 0.5 weeks, e.g., 0.5, 1, 2, 3, 4, 5, 6, 7, 8 weeks, or more. In some embodiments, rituximab is administered at a dose and dosing interval described herein for a period of time, e.g., at least 2 weeks, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 weeks, or greater. For example, rituximab is administered at a dose and dosing interval described herein for a total of at least 4 doses per treatment cycle (e.g., at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more doses per treatment cycle). 
     In some embodiments, the anti-CD20 antibody comprises ofatumumab. Ofatumumab is an anti-CD20 IgG1κ human monoclonal antibody with a molecular weight of approximately 149 kDa. For example, ofatumumab is generated using transgenic mouse and hybridoma technology and is expressed and purified from a recombinant murine cell line (NSO). See, e.g., www.accessdata.fda.gov/drugsatfda_docs/label/2009/1253261bl.pdf; and Clinical Trial Identifier number NCT01363128, NCT01515176, NCT01626352, and NCT01397591. In embodiments, a CAR-expressing cell described herein is administered to a subject in combination with ofatumumab. In embodiments, the subject has CLL or SLL. 
     In some embodiments, ofatumumab is administered as an intravenous infusion. For example, each infusion provides about 150-3000 mg (e.g., about 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900-950, 950-1000, 1000-1200, 1200-1400, 1400-1600, 1600-1800, 1800-2000, 2000-2200, 2200-2400, 2400-2600, 2600-2800, or 2800-3000 mg) of ofatumumab. In embodiments, ofatumumab is administered at a starting dosage of about 300 mg, followed by 2000 mg, e.g., for about 11 doses, e.g., for 24 weeks. In some embodiments, ofatumumab is administered at a dosing interval of at least 4 days, e.g., 4, 7, 14, 21, 28, 35 days, or more. For example, ofatumumab is administered at a dosing interval of at least 1 week, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 26, 28, 20, 22, 24, 26, 28, 30 weeks, or more. In some embodiments, ofatumumab is administered at a dose and dosing interval described herein for a period of time, e.g., at least 1 week, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 40, 50, 60 weeks or greater, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or greater, or 1, 2, 3, 4, 5 years or greater. For example, ofatumumab is administered at a dose and dosing interval described herein for a total of at least 2 doses per treatment cycle (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, or more doses per treatment cycle). 
     In some cases, the anti-CD20 antibody comprises ocrelizumab. Ocrelizumab is a humanized anti-CD20 monoclonal antibody, e.g., as described in Clinical Trials Identifier Nos. NCT00077870, NCT01412333, NCT00779220, NCT00673920, NCT01194570, and Kappos et al. Lancet. 19.378(2011):1779-87. 
     In some cases, the anti-CD20 antibody comprises veltuzumab. Veltuzumab is a humanized monoclonal antibody against CD20. See, e.g., Clinical Trial Identifier No. NCT00547066, NCT00546793, NCT01101581, and Goldenberg et al. Leuk Lymphoma. 51(5)(2010):747-55. 
     In some cases, the anti-CD20 antibody comprises GA101. GA101 (also called obinutuzumab or R05072759) is a humanized and glyco-engineered anti-CD20 monoclonal antibody. See, e.g., Robak. Curr. Opin. Investig. Drugs. 10.6(2009):588-96; Clinical Trial Identifier Numbers: NCT01995669, NCT01889797, NCT02229422, and NCT01414205; and www.accessdata.fda.gov/drugsatfda_docs/label/2013/125486s0001bl.pdf. 
     In some cases, the anti-CD20 antibody comprises AME-133v. AME-133v (also called LY2469298 or ocaratuzumab) is a humanized IgG1 monoclonal antibody against CD20 with increased affinity for the FcTRIIIa receptor and an enhanced antibody dependent cellular cytotoxicity (ADCC) activity compared with rituximab. See, e.g., Robak et al. BioDrugs 25.1(2011):13-25; and Forero-Torres et al. Clin Cancer Res. 18.5(2012):1395-403. 
     In some cases, the anti-CD20 antibody comprises PRO131921. PRO131921 is a humanized anti-CD20 monoclonal antibody engineered to have better binding to FcγRIIIa and enhanced ADCC compared with rituximab. See, e.g., Robak et al. BioDrugs 25.1(2011):13-25; and Casulo et al. Clin Immunol. 154.1(2014):37-46; and Clinical Trial Identifier No. NCT00452127. 
     In some cases, the anti-CD20 antibody comprises TRU-015. TRU-015 is an anti-CD20 fusion protein derived from domains of an antibody against CD20. TRU-015 is smaller than monoclonal antibodies, but retains Fc-mediated effector functions. See, e.g., Robak et al. BioDrugs 25.1(2011):13-25. TRU-015 contains an anti-CD20 single-chain variable fragment (scFv) linked to human IgG1 hinge, CH2, and CH3 domains but lacks CH1 and CL domains. 
     In some embodiments, an anti-CD20 antibody described herein is conjugated or otherwise bound to a therapeutic agent, e.g., a chemotherapeutic agent (e.g., cytoxan, fludarabine, histone deacetylase inhibitor, demethylating agent, peptide vaccine, anti-tumor antibiotic, tyrosine kinase inhibitor, alkylating agent, anti-microtubule or anti-mitotic agent), anti-allergic agent, anti-nausea agent (or anti-emetic), pain reliever, or cytoprotective agent described herein. 
     In embodiments, a CAR-expressing cell described herein is administered to a subject in combination with a B-cell lymphoma 2 (BCL-2) inhibitor (e.g., venetoclax, also called ABT-199 or GDC-0199;) and/or rituximab. In embodiments, a CAR-expressing cell described herein is administered to a subject in combination with venetoclax and rituximab. Venetoclax is a small molecule that inhibits the anti-apoptotic protein, BCL-2. The structure of venetoclax (4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide) is shown below. 
     
       
         
         
             
             
         
       
     
     In embodiments, the subject has CLL. In embodiments, the subject has relapsed CLL, e.g., the subject has previously been administered a cancer therapy. In embodiments, venetoclax is administered at a dosage of about 15-600 mg (e.g., 15-20, 20-50, 50-75, 75-100, 100-200, 200-300, 300-400, 400-500, or 500-600 mg), e.g., daily. In embodiments, rituximab is administered at a dosage of about 350-550 mg/m2 (e.g., 350-375, 375-400, 400-425, 425-450, 450-475, or 475-500 mg/m2), e.g., intravenously, e.g., monthly 
     In an embodiment, cells expressing a CAR described herein are administered to a subject in combination with a molecule that decreases the Treg cell population. Methods that decrease the number of (e.g., deplete) Treg cells are known in the art and include, e.g., CD25 depletion, cyclophosphamide administration, modulating GITR function. Without wishing to be bound by theory, it is believed that reducing the number of Treg cells in a subject prior to apheresis or prior to administration of a CAR-expressing cell described herein reduces the number of unwanted immune cells (e.g., Tregs) in the tumor microenvironment and reduces the subject&#39;s risk of relapse. In one embodiment, cells expressing a CAR described herein are administered to a subject in combination with a molecule targeting GITR and/or modulating GITR functions, such as a GITR agonist and/or a GITR antibody that depletes regulatory T cells (Tregs). In embodiments, cells expressing a CAR described herein are administered to a subject in combination with cyclophosphamide. In one embodiment, the GITR binding molecules and/or molecules modulating GITR functions (e.g., GITR agonist and/or Treg depleting GITR antibodies) are administered prior to administration of the CAR-expressing cell. For example, in one embodiment, the GITR agonist can be administered prior to apheresis of the cells. In embodiments, cyclophosphamide is administered to the subject prior to administration (e.g., infusion or re-infusion) of the CAR-expressing cell or prior to aphersis of the cells. In embodiments, cyclophosphamide and an anti-GITR antibody are administered to the subject prior to administration (e.g., infusion or re-infusion) of the CAR-expressing cell or prior to apheresis of the cells. In one embodiment, the subject has cancer (e.g., a solid cancer or a hematological cancer such as ALL or CLL). In an embodiment, the subject has CLL. In embodiments, the subject has ALL. In embodiments, the subject has a solid cancer, e.g., a solid cancer described herein. Exemplary GITR agonists include, e.g., GITR fusion proteins and anti-GITR antibodies (e.g., bivalent anti-GITR antibodies) such as, e.g., a GITR fusion protein described in U.S. Pat. No. 6,111,090, European Patent No.: 090505B1, U.S. Pat. No. 8,586,023, PCT Publication Nos.: WO 2010/003118 and 2011/090754, or an anti-GITR antibody described, e.g., in U.S. Pat. No. 7,025,962, European Patent No.: 1947183B1, U.S. Pat. Nos. 7,812,135, 8,388,967, 8,591,886, European Patent No.: EP 1866339, PCT Publication No.: WO 2011/028683, PCT Publication No.:WO 2013/039954, PCT Publication No.: WO2005/007190, PCT Publication No.: WO 2007/133822, PCT Publication No.: WO2005/055808, PCT Publication No.: WO 99/40196, PCT Publication No.: WO 2001/03720, PCT Publication No.: WO99/20758, PCT Publication No.: WO2006/083289, PCT Publication No.: WO 2005/115451, U.S. Pat. No. 7,618,632, and PCT Publication No.: WO 2011/051726. 
     In one embodiment, a CAR expressing cell described herein is administered to a subject in combination with an mTOR inhibitor, e.g., an mTOR inhibitor described herein, e.g., a rapalog such as everolimus. In one embodiment, the mTOR inhibitor is administered prior to the CAR-expressing cell. For example, in one embodiment, the mTOR inhibitor can be administered prior to apheresis of the cells. In one embodiment, the subject has CLL. 
     In one embodiment, a CAR expressing cell described herein is administered to a subject in combination with a GITR agonist, e.g., a GITR agonist described herein. In one embodiment, the GITR agonist is administered prior to the CAR-expressing cell. For example, in one embodiment, the GITR agonist can be administered prior to apheresis of the cells. In one embodiment, the subject has CLL. 
     In one embodiment, a CAR-expressing cell described herein can be used in combination with a kinase inhibitor. In one embodiment, the kinase inhibitor is a CDK4 inhibitor, e.g., a CDK4 inhibitor described herein, e.g., a CD4/6 inhibitor, such as, e.g., 6-Acetyl-8-cyclopentyl-5-methyl-2-(5-piperazin-1-yl-pyridin-2-ylamino)-8H-pyrido[2,3-d]pyrimidin-7-one, hydrochloride (also referred to as palbociclib or PD0332991). In one embodiment, the kinase inhibitor is a BTK inhibitor, e.g., a BTK inhibitor described herein, such as, e.g., ibrutinib. In one embodiment, the kinase inhibitor is an mTOR inhibitor, e.g., an mTOR inhibitor described herein, such as, e.g., rapamycin, a rapamycin analog, OSI-027. The mTOR inhibitor can be, e.g., an mTORC1 inhibitor and/or an mTORC2 inhibitor, e.g., an mTORC1 inhibitor and/or mTORC2 inhibitor described herein. In one embodiment, the kinase inhibitor is a MNK inhibitor, e.g., a MNK inhibitor described herein, such as, e.g., 4-amino-5-(4-fluoroanilino)-pyrazolo [3,4-d]pyrimidine. The MNK inhibitor can be, e.g., a MNK1a, MNK1b, MNK2a and/or MNK2b inhibitor. In one embodiment, the kinase inhibitor is a dual PI3K/mTOR inhibitor described herein, such as, e.g., PF-04695102. 
     In one embodiment, the kinase inhibitor is a CDK4 inhibitor selected from aloisine A; flavopiridol or HMR-1275, 2-(2-chlorophenyl)-5,7-dihydroxy-8-[(3S,4R)-3-hydroxy-1-methyl-4-piperidinyl]-4-chromenone; crizotinib (PF-02341066; 2-(2-Chlorophenyl)-5,7-dihydroxy-8-[(2R,3S)-2-(hydroxymethyl)-1-methyl-3-pyrrolidinyl]-4H-1-benzopyran-4-one, hydrochloride (P276-00); 1-methyl-5-[[2-[5-(trifluoromethyl)-1H-imidazol-2-yl]-4-pyridinyl]oxy]-N-[4-(trifluoromethyl)phenyl]-1H-benzimidazol-2-amine (RAF265); indisulam (E7070); roscovitine (CYC202); palbociclib (PD0332991); dinaciclib (SCH727965); N-[5-[[(5-tert-butyloxazol-2-yl)methyl]thio]thiazol-2-yl]piperidine-4-carboxamide (BMS 387032); 4-[[9-chloro-7-(2,6-difluorophenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino]-benzoic acid (MLN8054); 5-[3-(4,6-difluoro-1H-benzimidazol-2-yl)-1H-indazol-5-yl]-N-ethyl-4-methyl-3-pyridinemethanamine (AG-024322); 4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-carboxylic acid N-(piperidin-4-yl)amide (AT7519); 4-[2-methyl-1-(1-methylethyl)-1H-imidazol-5-yl]-N-[4-(methylsulfonyl)phenyl]-2-pyrimidinamine (AZD5438); and XL281 (BMS908662). 
     In one embodiment, the kinase inhibitor is a CDK4 inhibitor, e.g., palbociclib (PD0332991), and the palbociclib is administered at a dose of about 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg, 130 mg, 135 mg (e.g., 75 mg, 100 mg or 125 mg) daily for a period of time, e.g., daily for 14-21 days of a 28 day cycle, or daily for 7-12 days of a 21 day cycle. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of palbociclib are administered. 
     In embodiments, a CAR-expressing cell described herein is administered to a subject in combination with a cyclin-dependent kinase (CDK) 4 or 6 inhibitor, e.g., a CDK4 inhibitor or a CDK6 inhibitor described herein. In embodiments, a CAR-expressing cell described herein is administered to a subject in combination with a CDK4/6 inhibitor (e.g., an inhibitor that targets both CDK4 and CDK6), e.g., a CDK4/6 inhibitor described herein. In an embodiment, the subject has MCL. MCL is an aggressive cancer that is poorly responsive to currently available therapies, i.e., essentially incurable. In many cases of MCL, cyclin D1 (a regulator of CDK4/6) is expressed (e.g., due to chromosomal translocation involving immunoglobulin and Cyclin D1 genes) in MCL cells. Thus, without being bound by theory, it is thought that MCL cells are highly sensitive to CDK4/6 inhibition with high specificity (i.e., minimal effect on normal immune cells). CDK4/6 inhibitors alone have had some efficacy in treating MCL, but have only achieved partial remission with a high relapse rate. An exemplary CDK4/6 inhibitor is LEE011 (also called ribociclib), the structure of which is shown below. 
     
       
         
         
             
             
         
       
     
     Without being bound by theory, it is believed that administration of a CAR-expressing cell described herein with a CDK4/6 inhibitor (e.g., LEE011 or other CDK4/6 inhibitor described herein) can achieve higher responsiveness, e.g., with higher remission rates and/or lower relapse rates, e.g., compared to a CDK4/6 inhibitor alone. 
     In one embodiment, the kinase inhibitor is a BTK inhibitor selected from ibrutinib (PCI-32765); GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059; CNX-774; and LFM-A13. In a preferred embodiment, the BTK inhibitor does not reduce or inhibit the kinase activity of interleukin-2-inducible kinase (ITK), and is selected from GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059; CNX-774; and LFM-A13. 
     In one embodiment, the kinase inhibitor is a BTK inhibitor, e.g., ibrutinib (PCI-32765). In embodiments, a CAR-expressing cell described herein is administered to a subject in combination with a BTK inhibitor (e.g., ibrutinib). In embodiments, a CAR-expressing cell described herein is administered to a subject in combination with ibrutinib (also called PCI-32765). The structure of ibrutinib (1-[(3R)-3-[4-Amino-3-(4-phenoxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl]prop-2-en-1-one) is shown below. 
     
       
         
         
             
             
         
       
     
     In embodiments, the subject has CLL, mantle cell lymphoma (MCL), or small lymphocytic lymphoma (SLL). For example, the subject has a deletion in the short arm of chromosome 17 (del(17p), e.g., in a leukemic cell). In other examples, the subject does not have a del(17p). In embodiments, the subject has relapsed CLL or SLL, e.g., the subject has previously been administered a cancer therapy (e.g., previously been administered one, two, three, or four prior cancer therapies). In embodiments, the subject has refractory CLL or SLL. In other embodiments, the subject has follicular lymphoma, e.g., relapse or refractory follicular lymphoma. In some embodiments, ibrutinib is administered at a dosage of about 300-600 mg/day (e.g., about 300-350, 350-400, 400-450, 450-500, 500-550, or 550-600 mg/day, e.g., about 420 mg/day or about 560 mg/day), e.g., orally. In embodiments, the ibrutinib is administered at a dose of about 250 mg, 300 mg, 350 mg, 400 mg, 420 mg, 440 mg, 460 mg, 480 mg, 500 mg, 520 mg, 540 mg, 560 mg, 580 mg, 600 mg (e.g., 250 mg, 420 mg or 560 mg) daily for a period of time, e.g., daily for 21 day cycle, or daily for 28 day cycle. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of ibrutinib are administered. Without being bound by theory, it is thought that the addition of ibrutinib enhances the T cell proliferative response and may shift T cells from a T-helper-2 (Th2) to T-helper-1 (Th1) phenotype. Th1 and Th2 are phenotypes of helper T cells, with Th1 versus Th2 directing different immune response pathways. A Th1 phenotype is associated with proinflammatory responses, e.g., for killing cells, such as intracellular pathogens/viruses or cancerous cells, or perpetuating autoimmune responses. A Th2 phenotype is associated with eosinophil accumulation and anti-inflammatory responses. 
     In one embodiment, the kinase inhibitor is an mTOR inhibitor selected from temsirolimus; ridaforolimus (1R,2R,4S)-4-[(2R)-2 [(1R,9S,12S,15R,16E,18R,19R,21R, 23S,24E,26E,28Z,30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23, 29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.0 4,9 ] hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyl dimethylphosphinate, also known as AP23573 and MK8669; everolimus (RAD001); rapamycin (AY22989); simapimod; (5-{2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl}-2-methoxyphenyl)methanol (AZD8055); 2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one (PF04691502); and N 2 -[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4-yl]methoxy]butyl]-L-arginylglycyl-L-α-aspartylL-serine-, inner salt (SF1126) (SEQ ID NO: 1262); and XL765. 
     In one embodiment, the kinase inhibitor is an mTOR inhibitor, e.g., rapamycin, and the rapamycin is administered at a dose of about 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg (e.g., 6 mg) daily for a period of time, e.g., daily for 21 day cycle, or daily for 28 day cycle. 
     In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of rapamycin are administered. In one embodiment, the kinase inhibitor is an mTOR inhibitor, e.g., everolimus and the everolimus is administered at a dose of about 2 mg, 2.5 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg (e.g., 10 mg) daily for a period of time, e.g., daily for 28 day cycle. In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of everolimus are administered. 
     In one embodiment, the kinase inhibitor is an MNK inhibitor selected from CGP052088; 4-amino-3-(p-fluorophenylamino)-pyrazolo [3,4-d] pyrimidine (CGP57380); cercosporamide; ETC-1780445-2; and 4-amino-5-(4-fluoroanilino)-pyrazolo [3,4-d] pyrimidine. 
     In embodiments, a CAR-expressing cell described herein is administered to a subject in combination with a phosphoinositide 3-kinase (PI3K) inhibitor (e.g., a PI3K inhibitor described herein, e.g., idelalisib or duvelisib) and/or rituximab. In embodiments, a CAR-expressing cell described herein is administered to a subject in combination with idelalisib and rituximab. In embodiments, a CAR-expressing cell described herein is administered to a subject in combination with duvelisib and rituximab. Idelalisib (also called GS-1101 or CAL-101; Gilead) is a small molecule that blocks the delta isoform of PI3K. The structure of idelalisib (5-Fluoro-3-phenyl-2-[(1S)-1-(7H-purin-6-ylamino)propyl]-4(3H)-quinazolinone) is shown below. 
     
       
         
         
             
             
         
       
     
     Duvelisib (also called IPI-145; Infinity Pharmaceuticals and Abbvie) is a small molecule that blocks PI3K-δ,γ. The structure of duvelisib (8-Chloro-2-phenyl-3-[(1S)-1-(9H-purin-6-ylamino)ethyl]-1(2H)-isoquinolinone) is shown below. 
     
       
         
         
             
             
         
       
     
     In embodiments, the subject has CLL. In embodiments, the subject has relapsed CLL, e.g., the subject has previously been administered a cancer therapy (e.g., previously been administered an anti-CD20 antibody or previously been administered ibrutinib). For example, the subject has a deletion in the short arm of chromosome 17 (del(17p), e.g., in a leukemic cell). In other examples, the subject does not have a del(17p). In embodiments, the subject comprises a leukemic cell comprising a mutation in the immunoglobulin heavy-chain variable-region (IgV H ) gene. In other embodiments, the subject does not comprise a leukemic cell comprising a mutation in the immunoglobulin heavy-chain variable-region (IgV H ) gene. In embodiments, the subject has a deletion in the long arm of chromosome 11 (del(11q)). In other embodiments, the subject does not have a del(11q). In embodiments, idelalisib is administered at a dosage of about 100-400 mg (e.g., 100-125, 125-150, 150-175, 175-200, 200-225, 225-250, 250-275, 275-300, 325-350, 350-375, or 375-400 mg), e.g., BID. In embodiments, duvelisib is administered at a dosage of about 15-100 mg (e.g., about 15-25, 25-50, 50-75, or 75-100 mg), e.g., twice a day. In embodiments, rituximab is administered at a dosage of about 350-550 mg/m 2  (e.g., 350-375, 375-400, 400-425, 425-450, 450-475, or 475-500 mg/m 2 ), e.g., intravenously. 
     In one embodiment, the kinase inhibitor is a dual phosphatidylinositol 3-kinase (PI3K) and mTOR inhibitor selected from 2-Amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one (PF-04691502); N-[4-[[4-(Dimethylamino)-1-piperidinyl]carbonyl]phenyl]-N-[4-(4,6-di-4-morpholinyl-1,3,5-triazin-2-yl)phenyl]urea (PF-05212384, PKI-587); 2-Methyl-2-{4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydro-1H-imidazo[4,5-c]quinolin-1-yl]phenyl}propanenitrile (BEZ-235); apitolisib (GDC-0980, RG7422); 2,4-Difluoro-N-{2-(methyloxy)-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-pyridinyl}benzenesulfonamide (GSK2126458); 8-(6-methoxypyridin-3-yl)-3-methyl-1-(4-(piperazin-1-yl)-3-(trifluoromethyl)phenyl)-1H-imidazo[4,5-c]quinolin-2(3H)-one Maleic acid (NVP-BGT226); 3-[4-(4-Morpholinylpyrido[3′,2′:4,5]furo[3,2-d]pyrimidin-2-yl]phenol (PI-103); 5-(9-isopropyl-8-methyl-2-morpholino-9H-purin-6-yl)pyrimidin-2-amine (VS-5584, SB2343); and N-[2-[(3,5-Dimethoxyphenyl)amino]quinoxalin-3-yl]-4-[(4-methyl-3-methoxyphenyl)carbonyl]aminophenylsulfonamide (XL765). 
     In embodiments, a CAR-expressing cell described herein is administered to a subject in combination with an anaplastic lymphoma kinase (ALK) inhibitor. Exemplary ALK kinases include but are not limited to crizotinib (Pfizer), ceritinib (Novartis), alectinib (Chugai), brigatinib (also called AP26113; Ariad), entrectinib (Ignyta), PF-06463922 (Pfizer), TSR-011 (Tesaro) (see, e.g., Clinical Trial Identifier No. NCT02048488), CEP-37440 (Teva), and X-396 (Xcovery). In some embodiments, the subject has a solid cancer, e.g., a solid cancer described herein, e.g., lung cancer. 
     The chemical name of crizotinib is 3-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-(1-piperidin-4-ylpyrazol-4-yl)pyridin-2-amine. The chemical name of ceritinib is 5-Chloro-N 2 -[2-isopropoxy-5-methyl-4-(4-piperidinyl)phenyl]-N4-[2-(isopropylsulfonyl)phenyl]-2,4-pyrimidinediamine. The chemical name of alectinib is 9-ethyl-6,6-dimethyl-8-(4-morpholinopiperidin-1-yl)-11-oxo-6,11-dihydro-5H-benzo[b]carbazole-3-carbonitrile. The chemical name of brigatinib is 5-Chloro-N 2 -{4-[4-(dimethylamino)-1-piperidinyl]-2-methoxyphenyl}-N 4 -[2-(dimethylphosphoryl)phenyl]-2,4-pyrimidinediamine. The chemical name of entrectinib is N-(5-(3,5-difluorobenzyl)-1H-indazol-3-yl)-4-(4-methylpiperazin-1-yl)-2-((tetrahydro-2H-pyran-4-yl)amino)benzamide. The chemical name of PF-06463922 is (10R)-7-Amino-12-fluoro-2,10,16-trimethyl-15-oxo-10,15,16,17-tetrahydro-2H-8,4-(metheno)pyrazolo[4,3-h][2,5,11]-benzoxadiazacyclotetradecine-3-carbonitrile. The chemical structure of CEP-37440 is (S)-2-((5-chloro-2-((6-(4-(2-hydroxyethyl)piperazin-1-yl)-1-methoxy-6,7,8,9-tetrahydro-5H-benzo[7]annulen-2-yl)amino)pyrimidin-4-yl)amino)-N-methylbenzamide. The chemical name of X-396 is (R)-6-amino-5-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)-N-(4-(4-methylpiperazine-1-carbonyl)phenyl)pyridazine-3-carboxamide. 
     Drugs that inhibit either the calcium dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase that is important for growth factor induced signaling (rapamycin). (Liu et al., Cell 66:807-815, 1991; Henderson et al., Immun. 73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773, 1993) can also be used. In a further aspect, the cell compositions of the present invention may be administered to a patient in conjunction with (e.g., before, simultaneously or following) bone marrow transplantation, T cell ablative therapy using chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, and/or antibodies such as OKT3 or CAMPATH. In one aspect, the cell compositions of the present invention are administered following B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan. For example, in one embodiment, subjects may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, following the transplant, subjects receive an infusion of the expanded immune cells of the present invention. In an additional embodiment, expanded cells are administered before or following surgery. 
     In embodiments, a CAR-expressing cell described herein is administered to a subject in combination with an indoleamine 2,3-dioxygenase (IDO) inhibitor. IDO is an enzyme that catalyzes the degradation of the amino acid, L-tryptophan, to kynurenine. Many cancers overexpress IDO, e.g., prostatic, colorectal, pancreatic, cervical, gastric, ovarian, head, and lung cancer. pDCs, macrophages, and dendritic cells (DCs) can express IDO. Without being bound by theory, it is thought that a decrease in L-tryptophan (e.g., catalyzed by IDO) results in an immunosuppressive milieu by inducing T-cell anergy and apoptosis. Thus, without being bound by theory, it is thought that an IDO inhibitor can enhance the efficacy of a CAR-expressing cell described herein, e.g., by decreasing the suppression or death of a CAR-expressing immune cell. In embodiments, the subject has a solid tumor, e.g., a solid tumor described herein, e.g., prostatic, colorectal, pancreatic, cervical, gastric, ovarian, head, or lung cancer. Exemplary inhibitors of IDO include but are not limited to 1-methyl-tryptophan, indoximod (NewLink Genetics) (see, e.g., Clinical Trial Identifier Nos. NCT01191216; NCT01792050), and INCB024360 (Incyte Corp.) (see, e.g., Clinical Trial Identifier Nos. NCT01604889; NCT01685255) 
     In embodiments, a CAR-expressing cell described herein is administered to a subject in combination with a modulator of myeloid-derived suppressor cells (MDSCs). MDSCs accumulate in the periphery and at the tumor site of many solid tumors. These cells suppress T cell responses, thereby hindering the efficacy of CAR-expressing cell therapy. Without being bound by theory, it is thought that administration of a MDSC modulator enhances the efficacy of a CAR-expressing cell described herein. In an embodiment, the subject has a solid tumor, e.g., a solid tumor described herein, e.g., glioblastoma. Exemplary modulators of MDSCs include but are not limited to MCS 110 and BLZ945. MCS 110 is a monoclonal antibody (mAb) against macrophage colony-stimulating factor (M-CSF). See, e.g., Clinical Trial Identifier No. NCT00757757. BLZ945 is a small molecule inhibitor of colony stimulating factor 1 receptor (CSF1R). See, e.g., Pyonteck et al. Nat. Med. 19(2013):1264-72. The structure of BLZ945 is shown below. 
     
       
         
         
             
             
         
       
     
     In embodiments, a CAR-expressing cell described herein is administered to a subject in combination with a CD19 CART cell (e.g., CTL019, e.g., as described in WO2012/079000, incorporated herein by reference). In embodiments, the subject has a CD19+ lymphoma, e.g., a CD19+ Non-Hodgkin&#39;s Lymphoma (NHL), a CD19+FL, or a CD19+ DLBCL. In embodiments, the subject has a relapsed or refractory CD19+ lymphoma. In embodiments, a lymphodepleting chemotherapy is administered to the subject prior to, concurrently with, or after administration (e.g., infusion) of CD19 CART cells. In an example, the lymphodepleting chemotherapy is administered to the subject prior to administration of CD19 CART cells. For example, the lymphodepleting chemotherapy ends 1-4 days (e.g., 1, 2, 3, or 4 days) prior to CD19 CART cell infusion. In embodiments, multiple doses of CD19 CART cells are administered, e.g., as described herein. For example, a single dose comprises about 5×10 8  CD19 CART cells. In embodiments, a lymphodepleting chemotherapy is administered to the subject prior to, concurrently with, or after administration (e.g., infusion) of a CAR-expressing cell described herein, e.g., a non-CD19 CAR-expressing cell. In embodiments, a CD19 CART is administered to the subject prior to, concurrently with, or after administration (e.g., infusion) of a non-CD19 CAR-expressing cell, e.g., a non-CD19 CAR-expressing cell described herein. 
     In some embodiments, a CAR-expressing cell described herein is administered to a subject in combination with a interleukin-15 (IL-15) polypeptide, a interleukin-15 receptor alpha (IL-15Ra) polypeptide, or a combination of both a IL-15 polypeptide and a IL-15Ra polypeptide e.g., hetIL-15 (Admune Therapeutics, LLC). hetIL-15 is a heterodimeric non-covalent complex of IL-15 and IL-15Ra. hetIL-15 is described in, e.g., U.S. Pat. No. 8,124,084, U.S. 2012/0177598, U.S. 2009/0082299, U.S. 2012/0141413, and U.S. 2011/0081311, incorporated herein by reference. In embodiments, het-IL-15 is administered subcutaneously. In embodiments, the subject has a cancer, e.g., solid cancer, e.g., melanoma or colon cancer. In embodiments, the subject has a metastatic cancer. 
     In one embodiment, the subject can be administered an agent which reduces or ameliorates a side effect associated with the administration of a CAR-expressing cell. Side effects associated with the administration of a CAR-expressing cell include, but are not limited to CRS, and hemophagocytic lymphohistiocytosis (HLH), also termed Macrophage Activation Syndrome (MAS). Symptoms of CRS include high fevers, nausea, transient hypotension, hypoxia, and the like. CRS may include clinical constitutional signs and symptoms such as fever, fatigue, anorexia, myalgias, arthalgias, nausea, vomiting, and headache. CRS may include clinical skin signs and symptoms such as rash. CRS may include clinical gastrointestinal signs and symptoms such as nausea, vomiting and diarrhea. CRS may include clinical respiratory signs and symptoms such as tachypnea and hypoxemia. CRS may include clinical cardiovascular signs and symptoms such as tachycardia, widened pulse pressure, hypotension, increased cardiac output (early) and potentially diminished cardiac output (late). CRS may include clinical coagulation signs and symptoms such as elevated d-dimer, hyperfibrinogenemia with or without bleeding. CRS may include clinical renal signs and symptoms such as azotemia. CRS may include clinical hepatic signs and symptoms such as transaminitis and hyperbilirubinemia. CRS may include clinical neurologic signs and symptoms such as headache, mental status changes, confusion, delirium, word finding difficulty or frank aphasia, hallucinations, tremor, dymetria, altered gait, and seizures. 
     Accordingly, the methods described herein can comprise administering a CAR-expressing cell described herein to a subject and further administering one or more agents to manage elevated levels of a soluble factor resulting from treatment with a CAR-expressing cell. In one embodiment, the soluble factor elevated in the subject is one or more of IFN-γ, TNFα, IL-2 and IL-6. In an embodiment, the factor elevated in the subject is one or more of IL-1, GM-CSF, IL-10, IL-8, IL-5 and fraktalkine. Therefore, an agent administered to treat this side effect can be an agent that neutralizes one or more of these soluble factors. In one embodiment, the agent that neutralizes one or more of these soluble forms is an antibody or antigen binding fragment thereof. Examples of such agents include, but are not limited to a steroid (e.g., corticosteroid), an inhibitor of TNFα, and an inhibitor of IL-6. An example of a TNFα inhibitor is an anti-TNFa antibody molecule such as, infliximab, adalimumab, certolizumab pegol, and golimumab. Another example of a TNFα inhibitor is a fusion protein such as entanercept. Small molecule inhibitors of TNFα include, but are not limited to, xanthine derivatives (e.g. pentoxifylline) and bupropion. An example of an IL-6 inhibitor is an anti-IL-6 antibody molecule or an anti-IL-6 receptor antibody molecule such as tocilizumab (toc), sarilumab, elsilimomab, CNTO 328, ALD518/BMS-945429, CNTO 136, CPSI-2364, CDP6038, VX30, ARGX-109, FE301, and FM101. In one embodiment, the anti-IL-6 receptor antibody molecule is tocilizumab. An example of an IL-1R based inhibitor is anakinra. 
     In one embodiment, the subject can be administered an agent which enhances the activity of a CAR-expressing cell. For example, in one embodiment, the agent can be an agent which inhibits an inhibitory molecule. Inhibitory molecules, e.g., Programmed Death 1 (PD-1), can, in some embodiments, decrease the ability of a CAR-expressing cell to mount an immune effector response. Examples of inhibitory molecules include PD-1, PD-L1, CTLA-4, TIM-3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGF beta. Inhibition of an inhibitory molecule, e.g., by inhibition at the DNA, RNA or protein level, can optimize a CAR-expressing cell performance. In embodiments, an inhibitory nucleic acid, e.g., an inhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA or shRNA, a clustered regularly interspaced short palindromic repeats (CRISPR), a transcription-activator like effector nuclease (TALEN), or a zinc finger endonuclease (ZFN), e.g., as described herein, can be used to inhibit expression of an inhibitory molecule in the CAR-expressing cell. In an embodiment the inhibitor is an shRNA. In an embodiment, the inhibitory molecule is inhibited within a CAR-expressing cell. In these embodiments, a dsRNA molecule that inhibits expression of the inhibitory molecule is linked to the nucleic acid that encodes a component, e.g., all of the components, of the CAR. In one embodiment, the inhibitor of an inhibitory signal can be, e.g., an antibody or antibody fragment that binds to an inhibitory molecule. For example, the agent can be an antibody or antibody fragment that binds to PD-1, PD-L1, PD-L2 or CTLA4 (e.g., ipilimumab (also referred to as MDX-010 and MDX-101, and marketed as Yervoy®; Bristol-Myers Squibb; Tremelimumab (IgG2 monoclonal antibody available from Pfizer, formerly known as ticilimumab, CP-675,206).). In an embodiment, the agent is an antibody or antibody fragment that binds to TIM3. In an embodiment, the agent is an antibody or antibody fragment that binds to CEACAM (CEACAM-1, CEACAM-3, and/or CEACAM-5). In an embodiment, the agent is an antibody or antibody fragment that binds to LAG3. 
     PD-1 is an inhibitory member of the CD28 family of receptors that also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed on activated B cells, T cells and myeloid cells (Agata et al. 1996 Int. Immunol 8:765-75). Two ligands for PD-1, PD-L1 and PD-L2 have been shown to downregulate T cell activation upon binding to PD-1 (Freeman et a. 2000 J Exp Med 192:1027-34; Latchman et al. 2001 Nat Immunol 2:261-8; Carter et al. 2002 Eur J Immunol 32:634-43). PD-L1 is abundant in human cancers (Dong et al. 2003 J Mol Med 81:281-7; Blank et al. 2005 Cancer Immunol. Immunother 54:307-314; Konishi et al. 2004 Clin Cancer Res 10:5094). Immune suppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1. Antibodies, antibody fragments, and other inhibitors of PD-1, PD-L1 and PD-L2 are available in the art and may be used combination with a cars of the present invention described herein. For example, nivolumab (also referred to as BMS-936558 or MDX1106; Bristol-Myers Squibb) is a fully human IgG4 monoclonal antibody which specifically blocks PD-1. Nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PD-1 are disclosed in U.S. Pat. No. 8,008,449 and WO2006/121168. Pidilizumab (CT-011; Cure Tech) is a humanized IgG1k monoclonal antibody that binds to PD-1. Pidilizumab and other humanized anti-PD-1 monoclonal antibodies are disclosed in WO2009/101611. Pembrolizumab (formerly known as lambrolizumab, and also referred to as MK03475; Merck) is a humanized IgG4 monoclonal antibody that binds to PD-1. Pembrolizumab and other humanized anti-PD-1 antibodies are disclosed in U.S. Pat. No. 8,354,509 and WO2009/114335. MEDI4736 (Medimmune) is a human monoclonal antibody that binds to PDL1, and inhibits interaction of the ligand with PD1. MDPL3280A (Genentech/Roche) is a human Fc optimized IgG1 monoclonal antibody that binds to PD-L1. MDPL3280A and other human monoclonal antibodies to PD-L1 are disclosed in U.S. Pat. No. 7,943,743 and U.S Publication No.: 20120039906. Other anti-PD-L1 binding agents include YW243.55.S70 (heavy and light chain variable regions are shown in SEQ ID NOs 20 and 21 in WO2010/077634) and MDX-1 105 (also referred to as BMS-936559, and, e.g., anti-PD-L1 binding agents disclosed in WO2007/005874). AMP-224 (B7-DCIg; Amplimmune; e.g., disclosed in WO2010/027827 and WO2011/066342), is a PD-L2 Fc fusion soluble receptor that blocks the interaction between PD-1 and B7-H1. Other anti-PD-1 antibodies include AMP 514 (Amplimmune), among others, e.g., anti-PD-1 antibodies disclosed in U.S. Pat. No. 8,609,089, US 2010028330, and/or US 20120114649. 
     TIM-3 (T cell immunoglobulin-3) also negatively regulates T cell function, particularly in IFN-g-secreting CD4+ T helper 1 and CD8+ T cytotoxic 1 cells, and plays a critical role in T cell exhaustion. Inhibition of the interaction between TIM3 and its ligands, e.g., galectin-9 (Gal9), phosphotidylserine (PS), and HMGB1, can increase immune response. Antibodies, antibody fragments, and other inhibitors of TIM3 and its ligands are available in the art and may be used combination with a CD19 CAR described herein. For example, antibodies, antibody fragments, small molecules, or peptide inhibitors that target TIM3 binds to the IgV domain of TIM3 to inhibit interaction with its ligands. Antibodies and peptides that inhibit TIM3 are disclosed in WO2013/006490 and US20100247521. Other anti-TIM3 antibodies include humanized versions of RMT3-23 (disclosed in Ngiow et al., 2011, Cancer Res, 71:3540-3551), and clone 8B.2C12 (disclosed in Monney et al., 2002, Nature, 415:536-541). Bi-specific antibodies that inhibit TIM3 and PD-1 are disclosed in US20130156774. 
     In other embodiments, the agent that enhances the activity of a CAR-expressing cell is a CEACAM inhibitor (e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5 inhibitor). In one embodiment, the inhibitor of CEACAM is an anti-CEACAM antibody molecule. Exemplary anti-CEACAM-1 antibodies are described in WO 2010/125571, WO 2013/082366 WO 2014/059251 and WO 2014/022332, e.g., a monoclonal antibody 34B1, 26H7, and 5F4; or a recombinant form thereof, as described in, e.g., US 2004/0047858, U.S. Pat. No. 7,132,255 and WO 99/052552. In other embodiments, the anti-CEACAM antibody binds to CEACAM-5 as described in, e.g., Zheng et al.  PLoS One.  2010 Sep. 2; 5(9). pii: e12529 (DOI:10:1371/journal.pone.0021146), or crossreacts with CEACAM-1 and CEACAM-5 as described in, e.g., WO 2013/054331 and US 2014/0271618. 
     Without wishing to be bound by theory, carcinoembryonic antigen cell adhesion molecules (CEACAM), such as CEACAM-1 and CEACAM-5, are believed to mediate, at least in part, inhibition of an anti-tumor immune response (see e.g., Markel et al. J Immunol. 2002 Mar. 15; 168(6):2803-10; Markel et al. J Immunol. 2006 Nov. 1; 177(9):6062-71; Markel et al. Immunology. 2009 February; 126(2):186-200; Markel et al. Cancer Immunol Immunother. 2010 Feb.; 59(2):215-30; Ortenberg et al. Mol Cancer Ther. 2012 Jun.; 11(6):1300-10; Stern et al. J Immunol. 2005 Jun. 1; 174(11):6692-701; Zheng et al. PLoS One. 2010 Sep. 2; 5(9). pii: e12529). For example, CEACAM-1 has been described as a heterophilic ligand for TIM-3 and as playing a role in TIM-3-mediated T cell tolerance and exhaustion (see e.g., WO 2014/022332; Huang, et al. (2014) Nature doi:10.1038/nature13848). In embodiments, co-blockade of CEACAM-1 and TIM-3 has been shown to enhance an anti-tumor immune response in xenograft colorectal cancer models (see e.g., WO 2014/022332; Huang, et al. (2014), supra). In other embodiments, co-blockade of CEACAM-1 and PD-1 reduce T cell tolerance as described, e.g., in WO 2014/059251. Thus, CEACAM inhibitors can be used with the other immunomodulators described herein (e.g., anti-PD-1 and/or anti-TIM-3 inhibitors) to enhance an immune response against a cancer, e.g., a melanoma, a lung cancer (e.g., NSCLC), a bladder cancer, a colon cancer an ovarian cancer, and other cancers as described herein. 
     LAG-3 (lymphocyte activation gene-3 or CD223) is a cell surface molecule expressed on activated T cells and B cells that has been shown to play a role in CD8+ T cell exhaustion. Antibodies, antibody fragments, and other inhibitors of LAG-3 and its ligands are available in the art and may be used combination with a CD19 CAR described herein. For example, BMS-986016 (Bristol-Myers Squib) is a monoclonal antibody that targets LAG3. IMP701 (Immutep) is an antagonist LAG-3 antibody and IMP731 (Immutep and GlaxoSmithKline) is a depleting LAG-3 antibody. Other LAG-3 inhibitors include IMP321 (Immutep), which is a recombinant fusion protein of a soluble portion of LAG3 and Ig that binds to MHC class II molecules and activates antigen presenting cells (APC). Other antibodies are disclosed, e.g., in WO2010/019570. 
     In some embodiments, the agent which enhances the activity of a CAR-expressing cell can be, e.g., a fusion protein comprising a first domain and a second domain, wherein the first domain is an inhibitory molecule, or fragment thereof, and the second domain is a polypeptide that is associated with a positive signal, e.g., a polypeptide comprising an antracellular signaling domain as described herein. In some embodiments, the polypeptide that is associated with a positive signal can include a costimulatory domain of CD28, CD27, ICOS, e.g., an intracellular signaling domain of CD28, CD27 and/or ICOS, and/or a primary signaling domain, e.g., of CD3 zeta, e.g., described herein. In one embodiment, the fusion protein is expressed by the same cell that expressed the CAR. In another embodiment, the fusion protein is expressed by a cell, e.g., a T cell that does not express a CAR of the present invention. 
     In one embodiment, the agent which enhances activity of a CAR-expressing cell described herein is miR-17-92. 
     In one embodiment, the agent which enhances activity of a CAR-described herein is a cytokine. Cytokines have important functions related to T cell expansion, differentiation, survival, and homeostatis. Cytokines that can be administered to the subject receiving a CAR-expressing cell described herein include: IL-2, IL-4, IL-7, IL-9, IL-15, IL-18, and IL-21, or a combination thereof. In preferred embodiments, the cytokine administered is IL-7, IL-15, or IL-21, or a combination thereof. The cytokine can be administered once a day or more than once a day, e.g., twice a day, three times a day, or four times a day. The cytokine can be administered for more than one day, e.g. the cytokine is administered for 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks. For example, the cytokine is administered once a day for 7 days. 
     In embodiments, the cytokine is administered in combination with CAR-expressing T cells. The cytokine can be administered simultaneously or concurrently with the CAR-expressing T cells, e.g., administered on the same day. The cytokine may be prepared in the same pharmaceutical composition as the CAR-expressing T cells, or may be prepared in a separate pharmaceutical composition. Alternatively, the cytokine can be administered shortly after administration of the CAR-expressing T cells, e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after administration of the CAR-expressing T cells. In embodiments where the cytokine is administered in a dosing regimen that occurs over more than one day, the first day of the cytokine dosing regimen can be on the same day as administration with the CAR-expressing T cells, or the first day of the cytokine dosing regimen can be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after administration of the CAR-expressing T cells. In one embodiment, on the first day, the CAR-expressing T cells are administered to the subject, and on the second day, a cytokine is administered once a day for the next 7 days. In a preferred embodiment, the cytokine to be administered in combination with CAR-expressing T cells is IL-7, IL-15, or IL-21. 
     In other embodiments, the cytokine is administered a period of time after administration of CAR-expressing cells, e.g., at least 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year or more after administration of CAR-expressing cells. In one embodiment, the cytokine is administered after assessment of the subject&#39;s response to the CAR-expressing cells. For example, the subject is administered CAR-expressing cells according to the dosage and regimens described herein. The response of the subject to CAR-expressing cell therapy is assessed at 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year or more after administration of CAR-expressing cells, using any of the methods described herein, including inhibition of tumor growth, reduction of circulating tumor cells, or tumor regression. Subjects that do not exhibit a sufficient response to CAR-expressing cell therapy can be administered a cytokine. Administration of the cytokine to the subject that has sub-optimal response to the CAR-expressing cell therapy improves CAR-expressing cell efficacy or anti-cancer activity. In a preferred embodiment, the cytokine administered after administration of CAR-expressing cells is IL-7. 
     Combination with a Low Dose of an mTOR Inhibitor 
     In one embodiment, the cells expressing a CAR molecule, e.g., a CAR molecule described herein, are administered in combination with a low, immune enhancing dose of an mTOR inhibitor. 
     In an embodiment, a dose of an mTOR inhibitor is associated with, or provides, mTOR inhibition of at least 5 but no more than 90%, at least 10 but no more than 90%, at least 15, but no more than 90%, at least 20 but no more than 90%, at least 30 but no more than 90%, at least 40 but no more than 90%, at least 50 but no more than 90%, at least 60 but no more than 90%, or at least 70 but no more than 90%. 
     In an embodiment, a dose of an mTOR inhibitor is associated with, or provides, mTOR inhibition of at least 5 but no more than 80%, at least 10 but no more than 80%, at least 15, but no more than 80%, at least 20 but no more than 80%, at least 30 but no more than 80%, at least 40 but no more than 80%, at least 50 but no more than 80%, or at least 60 but no more than 80%. 
     In an embodiment, a dose of an mTOR inhibitor is associated with, or provides, mTOR inhibition of at least 5 but no more than 70%, at least 10 but no more than 70%, at least 15, but no more than 70%, at least 20 but no more than 70%, at least 30 but no more than 70%, at least 40 but no more than 70%, or at least 50 but no more than 70%. 
     In an embodiment, a dose of an mTOR inhibitor is associated with, or provides, mTOR inhibition of at least 5 but no more than 60%, at least 10 but no more than 60%, at least 15, but no more than 60%, at least 20 but no more than 60%, at least 30 but no more than 60%, or at least 40 but no more than 60%. 
     In an embodiment, a dose of an mTOR inhibitor is associated with, or provides, mTOR inhibition of at least 5 but no more than 50%, at least 10 but no more than 50%, at least 15, but no more than 50%, at least 20 but no more than 50%, at least 30 but no more than 50%, or at least 40 but no more than 50%. 
     In an embodiment, a dose of an mTOR inhibitor is associated with, or provides, mTOR inhibition of at least 5 but no more than 40%, at least 10 but no more than 40%, at least 15, but no more than 40%, at least 20 but no more than 40%, at least 30 but no more than 40%, or at least 35 but no more than 40%. 
     In an embodiment, a dose of an mTOR inhibitor is associated with, or provides, mTOR inhibition of at least 5 but no more than 30%, at least 10 but no more than 30%, at least 15, but no more than 30%, at least 20 but no more than 30%, or at least 25 but no more than 30%. 
     In an embodiment, a dose of an mTOR inhibitor is associated with, or provides, mTOR inhibition of at least 1, 2, 3, 4 or 5 but no more than 20%, at least 1, 2, 3, 4 or 5 but no more than 30%, at least 1, 2, 3, 4 or 5, but no more than 35, at least 1, 2, 3, 4 or 5 but no more than 40%, or at least 1, 2, 3, 4 or 5 but no more than 45%. 
     In an embodiment, a dose of an mTOR inhibitor is associated with, or provides, mTOR inhibition of at least 1, 2, 3, 4 or 5 but no more than 90%. 
     As is discussed herein, the extent of mTOR inhibition can be expressed as the extent of P70 S6 kinase inhibition, e.g., the extent of mTOR inhibition can be determined by the level of decrease in P70 S6 kinase activity, e.g., by the decrease in phosphorylation of a P70 S6 kinase substrate. The level of mTOR inhibition can be evaluated by a method described herein, e.g. by the Boulay assay, or measurement of phosphorylated S6 levels by western blot. 
     Exemplary mTOR Inhibitors 
     As used herein, the term “mTOR inhibitor” refers to a compound or ligand, or a pharmaceutically acceptable salt thereof, which inhibits the mTOR kinase in a cell. In an embodiment an mTOR inhibitor is an allosteric inhibitor. In an embodiment an mTOR inhibitor is a catalytic inhibitor. 
     Allosteric mTOR inhibitors include the neutral tricyclic compound rapamycin (sirolimus), rapamycin-related compounds, that is compounds having structural and functional similarity to rapamycin including, e.g., rapamycin derivatives, rapamycin analogs (also referred to as rapalogs) and other macrolide compounds that inhibit mTOR activity. 
     Rapamycin is a known macrolide antibiotic produced by  Streptomyces hygroscopicus  having the structure shown in Formula A. 
     
       
         
         
             
             
         
       
     
     See, e.g., McAlpine, J. B., et al., J. Antibiotics (1991) 44: 688; Schreiber, S. L., et al., J. Am. Chem. Soc. (1991) 113: 7433; U.S. Pat. No. 3,929,992. There are various numbering schemes proposed for rapamycin. To avoid confusion, when specific rapamycin analogs are named herein, the names are given with reference to rapamycin using the numbering scheme of formula A. 
     Rapamycin analogs useful in the invention are, for example, 0-substituted analogs in which the hydroxyl group on the cyclohexyl ring of rapamycin is replaced by OR 1  in which R 1  is hydroxyalkyl, hydroxyalkoxyalkyl, acylaminoalkyl, or aminoalkyl; e.g. RAD001, also known as, everolimus as described in U.S. Pat. No. 5,665,772 and WO94/09010 the contents of which are incorporated by reference. Other suitable rapamycin analogs include those substituted at the 26- or 28-position. The rapamycin analog may be an epimer of an analog mentioned above, particularly an epimer of an analog substituted in position 40, 28 or 26, and may optionally be further hydrogenated, e.g. as described in U.S. Pat. No. 6,015,815, WO95/14023 and WO99/15530 the contents of which are incorporated by reference, e.g. ABT578 also known as zotarolimus or a rapamycin analog described in U.S. Pat. No. 7,091,213, WO98/02441 and WO01/14387 the contents of which are incorporated by reference, e.g. AP23573 also known as ridaforolimus. 
     Examples of rapamycin analogs suitable for use in the present invention from U.S. Pat. No. 5,665,772 include, but are not limited to, 40-O-benzyl-rapamycin, 40-O-(4′-hydroxymethyl)benzyl-rapamycin, 40-O-[4′-(1,2-dihydroxyethyl)]benzyl-rapamycin, 40-O-allyl-rapamycin, 40-O-[3′-(2,2-dimethyl-1,3-dioxolan-4(S)-yl)-prop-2′-en-1′-yl]-rapamycin, (2′E,4&#39;S)-40-O-(4′,5′-dihydroxypent-2′-en-1′-yl)-rapamycin, 40-O-(2-hydroxy)ethoxycarbonylmethyl-rapamycin, 40-O-(2-hydroxy)ethyl-rapamycin, 40-O-(3-hydroxy)propyl-rapamycin, 40-O-(6-hydroxy)hexyl-rapamycin, 40-O-[2-(2-hydroxy)ethoxy]ethyl-rapamycin, 40-O-[(3S)-2,2-dimethyldioxolan-3-yl]methyl-rapamycin, 40-O-[(2S)-2,3-dihydroxyprop-1-yl]-rapamycin, 40-O-(2-acetoxy)ethyl-rapamycin, 40-O-(2-nicotinoyloxy)ethyl-rapamycin, 40-O-[2-(N-morpholino)acetoxy]ethyl-rapamycin, 40-O-(2-N-imidazolylacetoxy)ethyl-rapamycin, 40-O-[2-(N-methyl-N′-piperazinyl)acetoxy]ethyl-rapamycin, 39-O-desmethyl-39,40-O,O-ethylene-rapamycin, (26R)-26-dihydro-40-O-(2-hydroxy)ethyl-rapamycin, 40-O-(2-aminoethyl)-rapamycin, 40-O-(2-acetaminoethyl)-rapamycin, 40-O-(2-nicotinamidoethyl)-rapamycin, 40-O-(2-(N-methyl-imidazo-2′-ylcarbethoxamido)ethyl)-rapamycin, 40-O-(2-ethoxycarbonylaminoethyl)-rapamycin, 40-O-(2-tolylsulfonamidoethyl)-rapamycin and 40-O-[2-(4′,5′-dicarboethoxy-1′,2′,3′-triazol-1′-yl)-ethyl]-rapamycin. 
     Other rapamycin analogs useful in the present invention are analogs where the hydroxyl group on the cyclohexyl ring of rapamycin and/or the hydroxy group at the 28 position is replaced with an hydroxyester group are known, for example, rapamycin analogs found in U.S. RE44,768, e.g. temsirolimus. 
     Other rapamycin analogs useful in the preset invention include those wherein the methoxy group at the 16 position is replaced with another substituent, preferably (optionally hydroxy-substituted) alkynyloxy, benzyl, orthomethoxybenzyl or chlorobenzyl and/or wherein the mexthoxy group at the 39 position is deleted together with the 39 carbon so that the cyclohexyl ring of rapamycin becomes a cyclopentyl ring lacking the 39 position methyoxy group; e.g. as described in WO95/16691 and WO96/41807 the contents of which are incorporated by reference. The analogs can be further modified such that the hydroxy at the 40-position of rapamycin is alkylated and/or the 32-carbonyl is reduced. 
     Rapamycin analogs from WO95/16691 include, but are not limited to, 16-demthoxy-16-(pent-2-ynyl)oxy-rapamycin, 16-demthoxy-16-(but-2-ynyl)oxy-rapamycin, 16-demthoxy-16-(propargyl)oxy-rapamycin, 16-demethoxy-16-(4-hydroxy-but-2-ynyl)oxy-rapamycin, 16-demthoxy-16-benzyloxy-40-O-(2-hydroxyethyl)-rapamycin, 16-demthoxy-16-benzyloxy-rapamycin, 16-demethoxy-16-ortho-methoxybenzyl-rapamycin, 16-demethoxy-40-O-(2-methoxyethyl)-16-pent-2-ynyl)oxy-rapamycin, 39-demethoxy-40-desoxy-39-formyl-42-nor-rapamycin, 39-demethoxy-40-desoxy-39-hydroxymethyl-42-nor-rapamycin, 39-demethoxy-40-desoxy-39-carboxy-42-nor-rapamycin, 39-demethoxy-40-desoxy-39-(4-methyl-piperazin-1-yl)carbonyl-42-nor-rapamycin, 39-demethoxy-40-desoxy-39-(morpholin-4-yl)carbonyl-42-nor-rapamycin, 39-demethoxy-40-desoxy-39-[N-methyl, N-(2-pyridin-2-yl-ethyl)]carbamoyl-42-nor-rapamycin and 39-demethoxy-40-desoxy-39-(p-toluenesulfonylhydrazonomethyl)-42-nor-rapamycin. 
     Rapamycin analogs from WO96/41807 include, but are not limited to, 32-deoxo-rapamycin, 16-O-pent-2-ynyl-32-deoxo-rapamycin, 16-O-pent-2-ynyl-32-deoxo-40-O-(2-hydroxy-ethyl)-rapamycin, 16-O-pent-2-ynyl-32-(S)-dihydro-40-O-(2-hydroxyethyl)-rapamycin, 32(S)-dihydro-40-O-(2-methoxy)ethyl-rapamycin and 32(S)-dihydro-40-O-(2-hydroxyethyl)-rapamycin. 
     Another suitable rapamycin analog is umirolimus as described in US2005/0101624 the contents of which are incorporated by reference. 
     RAD001, otherwise known as everolimus (Afinitor®), has the chemical name (1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-1,18-dihydroxy-12-{(1R)-2-[(1S,3R,4R)-4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl}-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-11,36-dioxa-4-aza-tricyclo[30.3.1.0 4,9 ]hexatriaconta-16,24,26,28-tetraene-2,3,10,14,20-pentaone 
     Further examples of allosteric mTOR inhibitors include sirolimus (rapamycin, AY-22989), 40-[3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate]-rapamycin (also called temsirolimus or CCI-779) and ridaforolimus (AP-23573/MK-8669). Other examples of allosteric mTor inhibitors include zotarolimus (ABT578) and umirolimus. 
     Alternatively or additionally, catalytic, ATP-competitive mTOR inhibitors have been found to target the mTOR kinase domain directly and target both mTORC1 and mTORC2. These are also more effective inhibitors of mTORC1 than such allosteric mTOR inhibitors as rapamycin, because they modulate rapamycin-resistant mTORC1 outputs such as 4EBP1-T37/46 phosphorylation and cap-dependent translation. 
     Catalytic inhibitors include: BEZ235 or 2-methyl-2-[4-(3-methyl-2-oxo-8-quinolin-3-yl-2,3-dihydro-imidazo[4,5-c]quinolin-1-yl)-phenyl]-propionitrile, or the monotosylate salt form. the synthesis of BEZ235 is described in WO2006/122806; CCG168 (otherwise known as AZD-8055, Chresta, C. M., et al., Cancer Res, 2010, 70(1), 288-298) which has the chemical name {5-[2,4-bis-((S)-3-methyl-morpholin-4-yl)-pyrido[2,3d]pyrimidin-7-yl]-2-methoxy-phenyl}-methanol; 3-[2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl]-N-methylbenzamide (WO09104019); 3-(2-aminobenzo[d]oxazol-5-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine (WO10051043 and WO2013023184); A N-(3-(N-(3-((3,5-dimethoxyphenyl)amino)quinoxaline-2-yl)sulfamoyl)phenyl)-3-methoxy-4-methylbenzamide (WO07044729 and WO12006552); PKI-587 (Venkatesan, A. M., J. Med.Chem., 2010, 53, 2636-2645) which has the chemical name 1-[4-[4-(dimethylamino)piperidine-1-carbonyl]phenyl]-3-[4-(4,6-dimorpholino-1,3,5-triazin-2-yl)phenyl]urea; GSK-2126458 (ACS Med. Chem. Lett., 2010, 1, 39-43) which has the chemical name 2,4-difluoro-N-{2-methoxy-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-pyridinyl}benzenesulfonamide; 5-(9-isopropyl-8-methyl-2-morpholino-9H-purin-6-yl)pyrimidin-2-amine (WO10114484); (E)-N-(8-(6-amino-5-(trifluoromethyl)pyridin-3-yl)-1-(6-(2-cyanopropan-2-yl)pyridin-3-yl)-3-methyl-1H-imidazo[4,5-c]quinolin-2(3H)-ylidene)cyanamide (WO12007926). 
     Further examples of catalytic mTOR inhibitors include 8-(6-methoxy-pyridin-3-yl)-3-methyl-1-(4-piperazin-1-yl-3-trifluoromethyl-phenyl)-1,3-dihydro-imidazo[4,5-c]quinolin-2-one (WO2006/122806) and Ku-0063794 (Garcia-Martinez J M, et al., Biochem J., 2009, 421(1), 29-42. Ku-0063794 is a specific inhibitor of the mammalian target of rapamycin (mTOR).) WYE-354 is another example of a catalytic mTor inhibitor (Yu K, et al. (2009). Biochemical, Cellular, and In vivo Activity of Novel ATP-Competitive and Selective Inhibitors of the Mammalian Target of Rapamycin. Cancer Res. 69(15): 6232-6240). 
     mTOR inhibitors useful according to the present invention also include prodrugs, derivatives, pharmaceutically acceptable salts, or analogs thereof of any of the foregoing. 
     mTOR inhibitors, such as RAD001, may be formulated for delivery based on well-established methods in the art based on the particular dosages described herein. In particular, U.S. Pat. No. 6,004,973 (incorporated herein by reference) provides examples of formulations useable with the mTOR inhibitors described herein. 
     Evaluation of mTOR Inhibition 
     mTOR phosphorylates the kinase P70 S6, thereby activating P70 S6 kinase and allowing it to phosphorylate its substrate. The extent of mTOR inhibition can be expressed as the extent of P70 S6 kinase inhibition, e.g., the extent of mTOR inhibition can be determined by the level of decrease in P70 S6 kinase activity, e.g., by the decrease in phosphorylation of a P70 S6 kinase substrate. One can determine the level of mTOR inhibition, by measuring P70 S6 kinase activity (the ability of P70 S6 kinase to phsophorylate a substrate), in the absence of inhibitor, e.g., prior to administration of inhibitor, and in the presences of inhibitor, or after the administration of inhibitor. The level of inhibition of P70 S6 kinase gives the level of mTOR inhibition. Thus, if P70 S6 kinase is inhibited by 40%, mTOR activity, as measured by P70 S6 kinase activity, is inhibited by 40%. The extent or level of inhibition referred to herein is the average level of inhibition over the dosage interval. By way of example, if the inhibitor is given once per week, the level of inhibition is given by the average level of inhibition over that interval, namely a week. 
     Boulay et al.,  Cancer Res,  2004, 64:252-61, hereby incorporated by reference, teaches an assay that can be used to assess the level of mTOR inhibition (referred to herein as the Boulay assay). In an embodiment, the assay relies on the measurement of P70 S6 kinase activity from biological samples before and after administration of an mTOR inhibitor, e.g., RAD001. Samples can be taken at preselected times after treatment with an mTOR inhibitor, e.g., 24, 48, and 72 hours after treatment. Biological samples, e.g., from skin or peripheral blood mononuclear cells (PBMCs) can be used. Total protein extracts are prepared from the samples. P70 S6 kinase is isolated from the protein extracts by immunoprecipitation using an antibody that specifically recognizes the P70 S6 kinase. Activity of the isolated P70 S6 kinase can be measured in an in vitro kinase assay. The isolated kinase can be incubated with 40S ribosomal subunit substrates (which is an endogenous substrate of P70 S6 kinase) and gamma- 32 P under conditions that allow phosphorylation of the substrate. Then the reaction mixture can be resolved on an SDS-PAGE gel, and  32 P signal analyzed using a Phosphorlmager. A  32 P signal corresponding to the size of the 40S ribosomal subunit indicates phosphorylated substrate and the activity of P70 S6 kinase. Increases and decreases in kinase activity can be calculated by quantifying the area and intensity of the  32 P signal of the phosphorylated substrate (e.g., using ImageQuant, Molecular Dynamics), assigning arbitrary unit values to the quantified signal, and comparing the values from after administration with values from before administration or with a reference value. For example, percent inhibition of kinase activity can be calculated with the following formula: 1-(value obtained after administration/value obtained before administration)×100. As described above, the extent or level of inhibition referred to herein is the average level of inhibition over the dosage interval. 
     Methods for the evaluation of kinase activity, e.g., P70 S6 kinase activity, are also provided in U.S. Pat. No. 7,727,950, hereby incorporated by reference. 
     The level of mTOR inhibition can also be evaluated by a change in the ration of PD1 negative to PD1 positive T cells. T cells from peripheral blood can be identified as PD1 negative or positive by art-known methods. 
     Low-Dose mTOR Inhibitors 
     Methods described herein use low, immune enhancing, dose mTOR inhibitors, doses of mTOR inhibitors, e.g., allosteric mTOR inhibitors, including rapalogs such as RAD001. In contrast, levels of inhibitor that fully or near fully inhibit the mTOR pathway are immunosuppressive and are used, e.g., to prevent organ transplant rejection. In addition, high doses of rapalogs that fully inhibit mTOR also inhibit tumor cell growth and are used to treat a variety of cancers (See, e.g., Antineoplastic effects of mammalian target of rapamycine inhibitors. Salvadori M. World J Transplant. 2012 Oct. 24; 2(5):74-83; Current and Future Treatment Strategies for Patients with Advanced Hepatocellular Carcinoma: Role of mTOR Inhibition. Finn RS. Liver Cancer. 2012 Nov.; 1(3-4):247-256; Emerging Signaling Pathways in Hepatocellular Carcinoma. Moeini A, Cornella H, Villanueva A. Liver Cancer. 2012 Sep.; 1(2):83-93; Targeted cancer therapy—Are the days of systemic chemotherapy numbered?Joo W D, Visintin I, Mor G. Maturitas. 2013 Sep. 20 .; Role of natural and adaptive immunity in renal cell carcinoma response to VEGFR - TKIs and mTOR inhibitor . Santoni M, Berardi R, Amantini C, Burattini L, Santini D, Santoni G, Cascinu S. Int J Cancer. 2013 Oct. 2). 
     The present invention is based, at least in part, on the surprising finding that doses of mTOR inhibitors well below those used in current clinical settings had a superior effect in increasing an immune response in a subject and increasing the ratio of PD-1 negative T cells/PD-1 positive T cells. It was surprising that low doses of mTOR inhibitors, producing only partial inhibition of mTOR activity, were able to effectively improve immune responses in human human subjects and increase the ratio of PD-1 negative T cells/PD-1 positive T cells. 
     Alternatively, or in addition, without wishing to be bound by any theory, it is believed that low, a low, immune enhancing, dose of an mTOR inhibitor can increase naive T cell numbers, e.g., at least transiently, e.g., as compared to a non-treated subject. Alternatively or additionally, again while not wishing to be bound by theory, it is believed that treatment with an mTOR inhibitor after a sufficient amount of time or sufficient dosing results in one or more of the following:
         an increase in the expression of one or more of the following markers: CD62 high  CD127 high , CD27+, and BCL2, e.g., on memory T cells, e.g., memory T cell precursors;   a decrease in the expression of KLRG1, e.g., on memory T cells, e.g., memory T cell precursors; and   an increase in the number of memory T cell precursors, e.g., cells with any one or combination of the following characteristics: increased CD62 high  increased CD127 high  increased CD27+, decreased KLRG1, and increased BCL2;   and wherein any of the changes described above occurs, e.g., at least transiently, e.g., as compared to a non-treated subject (Araki, K et al. (2009)  Nature  460:108-112). Memory T cell precursors are memory T cells that are early in the differentiation program. For example, memory T cells have one or more of the following characteristics: increased CD62L high , increased CD127 high , increased CD27 + , decreased KLRG1, and/or increased BCL2.       

     In an embodiment, the invention relates to a composition, or dosage form, of an mTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g., a rapalog, rapamycin, or RAD001, or a catalytic mTOR inhibitor, which, when administered on a selected dosing regimen, e.g., once daily or once weekly, is associated with: a level of mTOR inhibition that is not associated with complete, or significant immune suppression, but is associated with enhancement of the immune response. 
     An mTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g., a rapalog, rapamycin, or RAD001, or a catalytic mTOR inhibitor, can be provided in a sustained release formulation. Any of the compositions or unit dosage forms described herein can be provided in a sustained release formulation. In some embodiments, a sustained release formulation will have lower bioavailability than an immediate release formulation. E.g., in embodiments, to attain a similar therapeutic effect of an immediate release formulation a sustained release formulation will have from about 2 to about 5, about 2.5 to about 3.5, or about 3 times the amount of inhibitor provided in the immediate release formulation. 
     In an embodiment, immediate release forms, e.g., of RAD001, typically used for one administration per week, having 0.1 to 20, 0.5 to 10, 2.5 to 7.5, 3 to 6, or about 5, mgs per unit dosage form, are provided. For once per week administrations, these immediate release formulations correspond to sustained release forms, having, respectively, 0.3 to 60, 1.5 to 30, 7.5 to 22.5, 9 to 18, or about 15 mgs of an mTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g., rapamycin or RAD001. In embodiments both forms are administered on a once/week basis. 
     In an embodiment, immediate release forms, e.g., of RAD001, typically used for one administration per day, having 0.005 to 1.5, 0.01 to 1.5, 0.1 to 1.5, 0.2 to 1.5, 0.3 to 1.5, 0.4 to 1.5, 0.5 to 1.5, 0.6 to 1.5, 0.7 to 1.5, 0.8 to 1.5, 1.0 to 1.5, 0.3 to 0.6, or about 0.5 mgs per unit dosage form, are provided. For once per day administrations, these immediate release forms correspond to sustained release forms, having, respectively, 0.015 to 4.5, 0.03 to 4.5, 0.3 to 4.5, 0.6 to 4.5, 0.9 to 4.5, 1.2 to 4.5, 1.5 to 4.5, 1.8 to 4.5, 2.1 to 4.5, 2.4 to 4.5, 3.0 to 4.5, 0.9 to 1.8, or about 1.5 mgs of an mTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g., rapamycin or RAD001. For once per week administrations, these immediate release forms correspond to sustained release forms, having, respectively, 0.1 to 30, 0.2 to 30, 2 to 30, 4 to 30, 6 to 30, 8 to 30, 10 to 30, 1.2 to 30, 14 to 30, 16 to 30, 20 to 30, 6 to 12, or about 10 mgs of an mTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g., rapamycin or RAD001. 
     In an embodiment, immediate release forms, e.g., of RAD001, typically used for one administration per day, having 0.01 to 1.0 mgs per unit dosage form, are provided. For once per day administrations, these immediate release forms correspond to sustained release forms, having, respectively, 0.03 to 3 mgs of an mTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g., rapamycin or RAD001. For once per week administrations, these immediate release forms correspond to sustained release forms, having, respectively, 0.2 to 20 mgs of an mTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g., rapamycin or RAD001. 
     In an embodiment, immediate release forms, e.g., of RAD001, typically used for one administration per week, having 0.5 to 5.0 mgs per unit dosage form, are provided. For once per week administrations, these immediate release forms correspond to sustained release forms, having, respectively, 1.5 to 15 mgs of an mTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g., rapamycin or RAD001. 
     As described above, one target of the mTOR pathway is the P70 S6 kinase. Thus, doses of mTOR inhibitors which are useful in the methods and compositions described herein are those which are sufficient to achieve no greater than 80% inhibition of P70 S6 kinase activity relative to the activity of the P70 S6 kinase in the absence of an mTOR inhibitor, e.g., as measured by an assay described herein, e.g., the Boulay assay. In a further aspect, the invention provides an amount of an mTOR inhibitor sufficient to achieve no greater than 38% inhibition of P70 S6 kinase activity relative to P70 S6 kinase activity in the absence of an mTOR inhibitor. 
     In one aspect the dose of mTOR inhibitor useful in the methods and compositions of the invention is sufficient to achieve, e.g., when administered to a human subject, 90+/−5% (i.e., 85-95%), 89+/−5%, 88+/−5%, 87+/−5%, 86+/−5%, 85+/−5%, 84+/−5%, 83+/−5%, 82+/−5%, 81+/−5%, 80+/−5%, 79+/−5%, 78+/−5%, 77+/−5%, 76+/−5%, 75+/−5%, 74+/−5%, 73+/−5%, 72+/−5%, 71+/−5%, 70+/−5%, 69+/−5%, 68+/−5%, 67+/−5%, 66+/−5%, 65+/−5%, 64+/−5%, 63+/−5%, 62+/−5%, 61+/−5%, 60+/−5%, 59+/−5%, 58+/−5%, 57+/−5%, 56+/−5%, 55+/−5%, 54+/−5%, 54+/−5%, 53+/−5%, 52+/−5%, 51+/−5%, 50+/−5%, 49+/−5%, 48+/−5%, 47+/−5%, 46+/−5%, 45+/−5%, 44+/−5%, 43+/−5%, 42+/−5%, 41+/−5%, 40+/−5%, 39+/−5%, 38+/−5%, 37+/−5%, 36+/−5%, 35+/−5%, 34+/−5%, 33+/−5%, 32+/−5%, 31+/−5%, 30+/−5%, 29+/−5%, 28+/−5%, 27+/−5%, 26+/−5%, 25+/−5%, 24+/−5%, 23+/−5%, 22+/−5%, 21+/−5%, 20+/−5%, 19+/−5%, 18+/−5%, 17+/−5%, 16+/−5%, 15+/−5%, 14+/−5%, 13+/−5%, 12+/−5%, 11+/−5%, or 10 +/−5%, inhibition of P70 S6 kinase activity, e.g., as measured by an assay described herein, e.g., the Boulay assay. 
     P70 S6 kinase activity in a subject may be measured using methods known in the art, such as, for example, according to the methods described in U.S. Pat. No. 7,727,950, by immunoblot analysis of phosphoP70 S6K levels and/or phosphoP70 S6 levels or by in vitro kinase activity assays. 
     As used herein, the term “about” in reference to a dose of mTOR inhibitor refers to up to a +/−10% variability in the amount of mTOR inhibitor, but can include no variability around the stated dose. 
     In some embodiments, the invention provides methods comprising administering to a subject an mTOR inhibitor, e.g., an allosteric inhibitor, e.g., RAD001, at a dosage within a target trough level. In some embodiments, the trough level is significantly lower than trough levels associated with dosing regimens used in organ transplant and cancer patients. In an embodiment mTOR inhibitor, e.g., RAD001, or rapamycin, is administered to result in a trough level that is less than ½, ¼, 1/10, or 1/20 of the trough level that results in immunosuppression or an anticancer effect. In an embodiment mTOR inhibitor, e.g., RAD001, or rapamycin, is administered to result in a trough level that is less than ½, ¼, 1/10, or 1/20 of the trough level provided on the FDA approved packaging insert for use in immunosuppression or an anticancer indications. 
     In an embodiment a method disclosed herein comprises administering to a subject an mTOR inhibitor, e.g., an allosteric inhibitor, e.g., RAD001, at a dosage that provides a target trough level of 0.1 to 10 ng/ml, 0.1 to 5 ng/ml, 0.1 to 3 ng/ml, 0.1 to 2 ng/ml, or 0.1 to 1 ng/ml. 
     In an embodiment a method disclosed herein comprises administering to a subject an mTOR inhibitor, e.g., an allosteric inhibitor, e.g., RAD001, at a dosage that provides a target trough level of 0.2 to 10 ng/ml, 0.2 to 5 ng/ml, 0.2 to 3 ng/ml, 0.2 to 2 ng/ml, or 0.2 to 1 ng/ml. 
     In an embodiment a method disclosed herein comprises administering to a subject an mTOR inhibitor, e.g. an, allosteric inhibitor, e.g., RAD001, at a dosage that provides a target trough level of 0.3 to 10 ng/ml, 0.3 to 5 ng/ml, 0.3 to 3 ng/ml, 0.3 to 2 ng/ml, or 0.3 to 1 ng/ml. 
     In an embodiment a method disclosed herein comprises administering to a subject an mTOR inhibitor, e.g., an allosteric inhibitor, e.g., RAD001, at a dosage that provides a target trough level of 0.4 to 10 ng/ml, 0.4 to 5 ng/ml, 0.4 to 3 ng/ml, 0.4 to 2 ng/ml, or 0.4 to 1 ng/ml. 
     In an embodiment a method disclosed herein comprises administering to a subject an mTOR inhibitor, e.g., an allosteric inhibitor, e.g., RAD001, at a dosage that provides a target trough level of 0.5 to 10 ng/ml, 0.5 to 5 ng/ml, 0.5 to 3 ng/ml, 0.5 to 2 ng/ml, or 0.5 to 1 ng/ml. 
     In an embodiment a method disclosed herein comprises administering to a subject an mTOR inhibitor, e.g., an allosteric inhibitor, e.g., RAD001, at a dosage that provides a target trough level of 1 to 10 ng/ml, 1 to 5 ng/ml, 1 to 3 ng/ml, or 1 to 2 ng/ml. 
     As used herein, the term “trough level” refers to the concentration of a drug in plasma just before the next dose, or the minimum drug concentration between two doses. 
     In some embodiments, a target trough level of RAD001 is in a range of between about 0.1 and 4.9 ng/ml. In an embodiment, the target trough level is below 3 ng/ml, e.g., is between 0.3 or less and 3 ng/ml. In an embodiment, the target trough level is below 3 ng/ml, e.g., is between 0.3 or less and 1 ng/ml. 
     In a further aspect, the invention can utilize an mTOR inhibitor other than RAD001 in an amount that is associated with a target trough level that is bioequivalent to the specified target trough level for RAD001. In an embodiment, the target trough level for an mTOR inhibitor other than RAD001, is a level that gives the same level of mTOR inhibition (e.g., as measured by a method described herein, e.g., the inhibition of P70 S6) as does a trough level of RAD001 described herein. 
     Pharmaceutical Compositions: mTOR Inhibitors 
     In one aspect, the present invention relates to pharmaceutical compositions comprising an mTOR inhibitor, e.g., an mTOR inhibitor as described herein, formulated for use in combination with CAR cells described herein. 
     In some embodiments, the mTOR inhibitor is formulated for administration in combination with an additional, e.g., as described herein. 
     In general, compounds of the invention will be administered in therapeutically effective amounts as described above via any of the usual and acceptable modes known in the art, either singly or in combination with one or more therapeutic agents. 
     The pharmaceutical formulations may be prepared using conventional dissolution and mixing procedures. For example, the bulk drug substance (e.g., an mTOR inhibitor or stabilized form of the compound (e.g., complex with a cyclodextrin derivative or other known complexation agent) is dissolved in a suitable solvent in the presence of one or more of the excipients described herein. The mTOR inhibitor is typically formulated into pharmaceutical dosage forms to provide an easily controllable dosage of the drug and to give the patient an elegant and easily handleable product. 
     Compounds of the invention can be administered as pharmaceutical compositions by any conventional route, in particular enterally, e.g., orally, e.g., in the form of tablets or capsules, or parenterally, e.g., in the form of injectable solutions or suspensions, topically, e.g., in the form of lotions, gels, ointments or creams, or in a nasal or suppository form. Where an mTOR inhibitor is administered in combination with (either simultaneously with or separately from) another agent as described herein, in one aspect, both components can be administered by the same route (e.g., parenterally). Alternatively, another agent may be administered by a different route relative to the mTOR inhibitor. For example, an mTOR inhibitor may be administered orally and the other agent may be administered parenterally. 
     Sustained Release 
     mTOR inhibitors, e.g., allosteric mTOR inhibitors or catalytic mTOR inhibitors, disclosed herein can be provided as pharmaceutical formulations in form of oral solid dosage forms comprising an mTOR inhibitor disclosed herein, e.g., rapamycin or RAD001, which satisfy product stability requirements and/or have favorable pharmacokinetic properties over the immediate release (IR) tablets, such as reduced average plasma peak concentrations, reduced inter- and intra-patient variability in the extent of drug absorption and in the plasma peak concentration, reduced C max /C min  ratio and/or reduced food effects. Provided pharmaceutical formulations may allow for more precise dose adjustment and/or reduce frequency of adverse events thus providing safer treatments for patients with an mTOR inhibitor disclosed herein, e.g., rapamycin or RAD001. 
     In some embodiments, the present disclosure provides stable extended release formulations of an mTOR inhibitor disclosed herein, e.g., rapamycin or RAD001, which are multi-particulate systems and may have functional layers and coatings. 
     The term “extended release, multi-particulate formulation as used herein refers to a formulation which enables release of an mTOR inhibitor disclosed herein, e.g., rapamycin or RAD001, over an extended period of time e.g. over at least 1, 2, 3, 4, 5 or 6 hours. The extended release formulation may contain matrices and coatings made of special excipients, e.g., as described herein, which are formulated in a manner as to make the active ingredient available over an extended period of time following ingestion. 
     The term “extended release” can be interchangeably used with the terms “sustained release” (SR) or “prolonged release”. The term “extended release” relates to a pharmaceutical formulation that does not release active drug substance immediately after oral dosing but over an extended in accordance with the definition in the pharmacopoeias Ph. Eur. (7 th  edition) mongraph for tablets and capsules and USP general chapter &lt;1151&gt; for pharmaceutical dosage forms. The term “Immediate Release” (IR) as used herein refers to a pharmaceutical formulation which releases 85% of the active drug substance within less than 60 minutes in accordance with the definition of “Guidance for Industry: “Dissolution Testing of Immediate Release Solid Oral Dosage Forms” (FDA CDER, 1997). In some embodiments, the term “immediate release” means release of everolismus from tablets within the time of 30 minutes, e.g., as measured in the dissolution assay described herein. 
     Stable extended release formulations of an mTOR inhibitor disclosed herein, e.g., rapamycin or RAD001, can be characterized by an in-vitro release profile using assays known in the art, such as a dissolution assay as described herein: a dissolution vessel filled with 900 mL phosphate buffer pH 6.8 containing sodium dodecyl sulfate 0.2% at 37° C. and the dissolution is performed using a paddle method at 75 rpm according to USP by according to USP testing monograph 711, and Ph.Eur. testing monograph 2.9.3. respectively. 
     In some embodiments, stable extended release formulations of an mTOR inhibitor disclosed herein, e.g., rapamycin or RAD001, release the mTOR inhibitor in the in-vitro release assay according to following release specifications: 
     0.5 h: &lt;45%, or &lt;40, e.g., &lt;30% 
     1 h: 20-80%, e.g., 30-60%
         2 h: &gt;50%, or &gt;70%, e.g., &gt;75%   3 h: &gt;60%, or &gt;65%, e.g., &gt;85%, e.g., &gt;90%.       

     In some embodiments, stable extended release formulations of an mTOR inhibitor disclosed herein, e.g., rapamycin or RAD001, release 50% of the mTOR inhibitor not earlier than 45, 60, 75, 90, 105 min or 120 min in the in-vitro dissolution assay. 
     Biopolymer Delivery Methods 
     In some embodiments, one or more CAR-expressing cells as disclosed herein can be administered or delivered to the subject via a biopolymer scaffold, e.g., a biopolymer implant. Biopolymer scaffolds can support or enhance the delivery, expansion, and/or dispersion of the CAR-expressing cells described herein. A biopolymer scaffold comprises a biocompatible (e.g., does not substantially induce an inflammatory or immune response) and/or a biodegradable polymer that can be naturally occurring or synthetic. 
     Examples of suitable biopolymers include, but are not limited to, agar, agarose, alginate, alginate/calcium phosphate cement (CPC), beta-galactosidase (β-GAL), (1,2,3,4,6-pentaacetyl a-D-galactose), cellulose, chitin, chitosan, collagen, elastin, gelatin, hyaluronic acid collagen, hydroxyapatite, poly(3-hydroxybutyrate-co-3-hydroxy-hexanoate) (PHBHHx), poly(lactide), poly(caprolactone) (PCL), poly(lactide-co-glycolide) (PLG), polyethylene oxide (PEO), poly(lactic-co-glycolic acid) (PLGA), polypropylene oxide (PPO), polyvinyl alcohol) (PVA), silk, soy protein, and soy protein isolate, alone or in combination with any other polymer composition, in any concentration and in any ratio. The biopolymer can be augmented or modified with adhesion- or migration-promoting molecules, e.g., collagen-mimetic peptides that bind to the collagen receptor of lymphocytes, and/or stimulatory molecules to enhance the delivery, expansion, or function, e.g., anti-cancer activity, of the cells to be delivered. The biopolymer scaffold can be an injectable, e.g., a gel or a semi-solid, or a solid composition. 
     In some embodiments, CAR-expressing cells described herein are seeded onto the biopolymer scaffold prior to delivery to the subject. In embodiments, the biopolymer scaffold further comprises one or more additional therapeutic agents described herein (e.g., another CAR-expressing cell, an antibody, or a small molecule) or agents that enhance the activity of a CAR-expressing cell, e.g., incorporated or conjugated to the biopolymers of the scaffold. In embodiments, the biopolymer scaffold is injected, e.g., intratumorally, or surgically implanted at the tumor or within a proximity of the tumor sufficient to mediate an anti-tumor effect. Additional examples of biopolymer compositions and methods for their delivery are described in Stephan et al., Nature Biotechnology, 2015, 33:97-101; and WO2014/110591. 
     Pharmaceutical Compositions and Treatments 
     Pharmaceutical compositions of the present invention may comprise a CAR-expressing cell, e.g., a plurality of CAR-expressing cells, as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions of the present invention are in one aspect formulated for intravenous administration. 
     Pharmaceutical compositions of the present invention may be administered in a manner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient&#39;s disease, although appropriate dosages may be determined by clinical trials. 
     In one embodiment, the pharmaceutical composition is substantially free of, e.g., there are no detectable levels of a contaminant, e.g., selected from the group consisting of endotoxin,  mycoplasma , replication competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual anti-CD3/anti-CD28 coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture media components, vector packaging cell or plasmid components, a bacterium and a fungus. In one embodiment, the bacterium is at least one selected from the group consisting of  Alcaligenes faecalis, Candida albicans, Escherichia coli, Haemophilus influenza, Neisseria meningitides, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumonia , and  Streptococcus pyogenes  group A. 
     When “an immunologically effective amount,” “an anti-tumor effective amount,” “a tumor-inhibiting effective amount,” or “therapeutic amount” is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the immune effector cells (e.g., T cells, NK cells) described herein may be administered at a dosage of 10 4  to 10 9  cells/kg body weight, in some instances 10 5  to 10 6  cells/kg body weight, including all integer values within those ranges. T cell compositions may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). 
     In certain aspects, it may be desired to administer activated immune effector cells (e.g., T cells, NK cells) to a subject and then subsequently redraw blood (or have an apheresis performed), activate immune effector cells (e.g., T cells, NK cells) therefrom according to the present invention, and reinfuse the patient with these activated and expanded immune effector cells (e.g., T cells, NK cells). This process can be carried out multiple times every few weeks. In certain aspects, immune effector cells (e.g., T cells, NK cells) can be activated from blood draws of from 10 cc to 400 cc. In certain aspects, immune effector cells (e.g., T cells, NK cells) are activated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc. 
     The administration of the subject compositions may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient trans arterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In one aspect, the T cell compositions of the present invention are administered to a patient by intradermal or subcutaneous injection. In one aspect, the T cell compositions of the present invention are administered by i.v. injection. The compositions of immune effector cells (e.g., T cells, NK cells) may be injected directly into a tumor, lymph node, or site of infection. 
     In a particular exemplary aspect, subjects may undergo leukapheresis, wherein leukocytes are collected, enriched, or depleted ex vivo to select and/or isolate the cells of interest, e.g., T cells. These T cell isolates may be expanded by methods known in the art and treated such that one or more CAR constructs of the invention may be introduced, thereby creating a CAR T cell of the invention. Subjects in need thereof may subsequently undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain aspects, following or concurrent with the transplant, subjects receive an infusion of the expanded CAR T cells of the present invention. In an additional aspect, expanded cells are administered before or following surgery. 
     The dosage of the above treatments to be administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The scaling of dosages for human administration can be performed according to art-accepted practices. The dose for CAMPATH, for example, will generally be in the range 1 to about 100 mg for an adult patient, usually administered daily for a period between 1 and 30 days. The preferred daily dose is 1 to 10 mg per day although in some instances larger doses of up to 40 mg per day may be used (described in U.S. Pat. No. 6,120,766). 
     In one embodiment, the CAR is introduced into immune effector cells (e.g., T cells, NK cells), e.g., using in vitro transcription, and the subject (e.g., human) receives an initial administration of CAR immune effector cells (e.g., T cells, NK cells) of the invention, and one or more subsequent administrations of the CAR immune effector cells (e.g., T cells, NK cells) of the invention, wherein the one or more subsequent administrations are administered less than 15 days, e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previous administration. In one embodiment, more than one administration of the CAR immune effector cells (e.g., T cells, NK cells) of the invention are administered to the subject (e.g., human) per week, e.g., 2, 3, or 4 administrations of the CAR immune effector cells (e.g., T cells, NK cells) of the invention are administered per week. In one embodiment, the subject (e.g., human subject) receives more than one administration of the CAR immune effector cells (e.g., T cells, NK cells) per week (e.g., 2, 3 or 4 administrations per week) (also referred to herein as a cycle), followed by a week of no CAR immune effector cells (e.g., T cells, NK cells) administrations, and then one or more additional administration of the CAR immune effector cells (e.g., T cells, NK cells) (e.g., more than one administration of the CAR immune effector cells (e.g., T cells, NK cells) per week) is administered to the subject. In another embodiment, the subject (e.g., human subject) receives more than one cycle of CAR immune effector cells (e.g., T cells, NK cells), and the time between each cycle is less than 10, 9, 8, 7, 6, 5, 4, or 3 days. In one embodiment, the CAR immune effector cells (e.g., T cells, NK cells) are administered every other day for 3 administrations per week. In one embodiment, the CAR immune effector cells (e.g., T cells, NK cells) of the invention are administered for at least two, three, four, five, six, seven, eight or more weeks. 
     In one aspect, CAR-expressing cells of the present inventions are generated using lentiviral viral vectors, such as lentivirus. Cells, e.g., CARTs, generated that way will have stable CAR expression. 
     In one aspect, CAR-expressing cells, e.g., CARTs, are generated using a viral vector such as a gammaretroviral vector, e.g., a gammaretroviral vector described herein. CARTs generated using these vectors can have stable CAR expression. 
     In one aspect, CARTs transiently express CAR vectors for 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 days after transduction. Transient expression of CARs can be effected by RNA CAR vector delivery. In one aspect, the CAR RNA is transduced into the T cell by electroporation. 
     A potential issue that can arise in patients being treated using transiently expressing CAR immune effector cells (e.g., T cells, NK cells) (particularly with murine scFv bearing CARTs) is anaphylaxis after multiple treatments. 
     Without being bound by this theory, it is believed that such an anaphylactic response might be caused by a patient developing humoral anti-CAR response, i.e., anti-CAR antibodies having an anti-IgE isotype. It is thought that a patient&#39;s antibody producing cells undergo a class switch from IgG isotype (that does not cause anaphylaxis) to IgE isotype when there is a ten to fourteen day break in exposure to antigen. 
     If a patient is at high risk of generating an anti-CAR antibody response during the course of transient CAR therapy (such as those generated by RNA transductions), CART infusion breaks should not last more than ten to fourteen days. 
     Examples 
     The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein. 
     Treatment of Patient with CLL with CART19 
     Patient UPCC04409-10 was treated with autologous CART19 T cells for CLL. The treatment led to complete remission of the CLL. 
     Analysis of CART Cell Population 
     As shown in  FIG.  1   , CART cells in patient UPCC04409-10 were monitored over time by sampling blood. The amount of BBZ expression in cells was determined (red). The number of copies of sequence from the Vbeta5.1 TCR family was determined (blue). Both measurements were made from samples collected on the indicated days after the second infusion of CART cells. As shown in  FIG.  2   , the T-cell receptor repertoire from patient UPCC04409-10 was determined from a sample collected on day 28 ( FIG.  2 A ) or day 51 ( FIG.  2 B ) after CART infusion. This demonstrates the abundance of the TCRBV05-01 family of T-cell receptors at day 51 indicating clonal expansion over time. As shown in  FIG.  3   , The T-cells isolated from patient UPCC04409-10 were analyzed for the simultaneous expression of CAR19 and 2 different TCR family genes over time (day 50 and day 51) and compared to the input dosed material (product): upper panel is TCR family Vb13.1; the lower panel shows TCR family Vb5.1. The data demonstrate that the CAR19 positive cells contain a single TCR family gene (Vb5.1) that becomes rapidly enriched between days 50 and 51. As shown in  FIG.  4   , the T-cell receptor repertoire of CD8 positive cells from patient UPCC04409-10 was determined from a sample collected on day 51 after CART infusion. This demonstrates the abundance of the TCRBV05-01 family of T-cell receptors at day 51 indicating clonal expansion of CD8 positive cells over time. 
     Analysis of Persisting CART Clone 
     As shown in  FIG.  6   , sonically fragmented DNA was generated from T-cells from Patient #. This material was used to amplify genomic sequences adjacent to the CAR19 insertion. The genes indicated were identified as being enriched relative to the infused product (DO) adjacent to CAR19 in the genome. At the different time points after CART infusion indicated (d=day; m=month), a different relative abundance of adjacent genes was seen, with Tet2 abundance peaking in both peripheral blood (PBMC) and CAR+CD8+ T-cells samples at day 51. 
     As shown in  FIG.  7   , the site of insertion of the CAR19 gene was mapped to the Tet2 gene. More specifically, the insertion occurred between exons 9 and 10 of the Tet2 gene. The catalytic domain for Tet2 resides in exon 11. The insertion at this location may lead to expression of aberrant mRNA transcripts or decrease the expression of functional (wild-type) Tet2. 
     As shown in  FIG.  8   , transcripts of the Tet2 gene from mRNA isolated from patient UPCC04409-10 were evaluated by RTPCR using primers spanning the indicated regions of Tet2 or CAR19 or both as indicated in the right hand side of the figure. Rxn 3 contains primers designed to amplify the region of the Tet2 transcript spanning exons 9 and 10. Rxn, 6, 7, 8, 9, and 10 are primers designed to amplify the indicated portions of the CAR19 lentivirus. Rxn 12-16 are pairs of primers that contain exon 9 sequence of the Tet2 transcript as well as sequence from the CAR19 lentiviral construct. These data show that transcripts are made from the Tet2 locus that contains both Tet2 sequence as well as CAR19 sequence. 
     Analysis of Tet2 Function 
     As shown in  FIG.  10   , the enzymatic activity of Tet2 is schematized ( FIG.  10 A ). Tet family protein convert 5-methylcytosine (5-mc) to 5-hydroxymethylcytosine (5-hmc) and then into 5-formylcytosine (5-fmc) resulting in demethylated cytosine. Methylated DNA is an epigenetic state that is known to affect transcriptional profiles. The methylation state of the T-cells from patient UPCC04409-10 was evaluated ( FIG.  10 B ). The patient&#39;s T-cells were stained for TCRVb5.1 (which contain the CAR19 insertion at Tet2) and the 5-hmc and 5-fmc were evaluated in TCRVb5.1 positive (red) and TCRVb5.1 negative (blue) populations by flow cytometry. This data indicates that the cells containing the insertion of CAR19 in the Tet2 gene are defective in demethylation. 
     Treatment of T Cells with shRNA Tet2 Inhibitors 
     Materials and Methods 
       Lenti -Viral Preparation and Infection to the Jurkat Cells 
       Lenti -viruses were prepared from 15 cm 293T cells. Briefly, 10 million 293T cells were seeded onto collagen coated 15 cm dishes at day −1. At day 0, 15ug shRNA vector (i.e., vector comprising sequence encoding the TET2-targeting or control shRNA), 15ug Gag/pol vector, and 5ug VSV-G vector were transfected using Lipofectamin 2000 (Invitrogen). 24 hours later (day 1), media was changed. After changing media, viral supernants were harvested at day 2 and day 3. Viruses were concentrated with  Lenti -X concentrator (3:1 volume ratio, Clonetech, Cat #: 631231). 100ul of viruses were added into either 0.5 million jurkat cells in the presence of 6 ug/ml of polybrene. The cells were spin-infected at 2000 rpm, 90 min at 32° C. After 1 hour incubation at 37 degree incubator, fresh RPMI 1640 media were added and transferred into 24-well plate. At day 6, cells were transferred into 6-well plate in the presence of final concentration 2 ug/ml of puromycin for 6 days. 
     Antibodies 
     Antibodies used for western blotting were as follows: j-actin (clone #: 8H10D10, Cell Signaling); TET2 (clone #: hT2H21F11, Millipore); mouse IgG(H+L) (HRP conjugated, Cat #: 115-035-166, Jackson ImmunoResearch); rabbit IgG(H+L) (HRP conjugated, Cat #: 111-035-114, Jackson ImmunoResearch). 
     Western Blotting 
     To examine TET2 shRNA knockdown efficiency at protein level in jurkat cells, cell lysates were prepared in protease inhibitor cocktails (Sigma) containing RIPA buffer. Protein concentration was measured by BCA protein assay kits (Pierce, Item #: 3603904). 20ug of protein was subjected to SDS-PAGE followed by transferring protein onto nitrocellulose membrane using iBot transfer system (Invitrogen, 20V, 11 min 30 sec). The membrane was blocked in 5% BSA containing TBST for 30 min at room temperature. Antibodies were overnight incubated with membranes at 1:000 dilution at 4° C. After incubation with HRP conjugated secondary antibodies, signal was detected using chemiluminescence detection machine (Chemidoc; Bio-Rad). 
     Quantitative RT-PCR 
     To examine TET2 shRNA knock-down efficiency at DNA level in jurkat cells, quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed. A RNeasy Micro Kit (Qiagen) was used to extract RNA. mRNA was reverse transcribed to single-strand complementary DNA (cDNA) with SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen). Real-time PCR was performed with C1000 Touch Thermal Cycler (Biorad). A SYBR-based protocol was used to detect gene expression (SsoAdvanced Universal SYBR Green Supermix, Biorad). The PCR reactions were done in 96-well plates and run using the manufacture&#39;s recommended cycling parameters with triplicate (95° C. for 3 minutes, followed by 40 cycles of 95° C. for 15 seconds and 60° C. for 30 seconds). Cycle threshold (Ct) values for the genes of interest were normalized to the Ct for p-actin. Primers used for qRT-PCR were as 
     
       
         
           
               
               
            
               
                   
                 follows: β-actin #1 
               
               
                   
                 (fowrard primer: 
               
               
                   
                 (SEQ ID NO: 1263) 
               
               
                   
                 CAT GTA CGT TGC TAT CCA GGC, 
               
               
                   
               
               
                   
                 reverse primer: 
               
               
                   
                 (SEQ ID NO: 1264) 
               
               
                   
                 CTC CTT AAT GTC ACG CAC GAT; 
               
               
                   
                 product size 250 bp); 
               
               
                   
               
               
                   
                 p-actin #2 (fowrard primer: 
               
               
                   
                 (SEQ ID NO: 1265) 
               
               
                   
                 CTC ACC ATG GAT GAT GAT ATC GC, 
               
               
                   
               
               
                   
                 reverse primer: 
               
               
                   
                 (SEQ ID NO: 1266) 
               
               
                   
                 CCA CAT AGG AAT CCT TCT GAC CC; 
               
               
                   
                 product size 169 bp); 
               
               
                   
               
               
                   
                 TET1 
               
               
                   
                 (forward primer: 
               
               
                   
                 (SEQ ID NO: 1267) 
               
               
                   
                 CAG AAC CTA AAC CAC CCG TG, 
               
               
                   
               
               
                   
                 reverse primer: 
               
               
                   
                 (SEQ ID NO: 1268) 
               
               
                   
                 TGC TTC GTA GCG CCA TTG TAA; 
               
               
                   
                 product size 141 bp); 
               
               
                   
               
               
                   
                 TET2 
               
               
                   
                 (forward primer: 
               
               
                   
                 (SEQ ID NO: 1269) 
               
               
                   
                 ATA CCC TGT ATG AAG GGA AGC C, 
               
               
                   
               
               
                   
                 reverse primer: 
               
               
                   
                 (SEQ ID NO: 1270) 
               
               
                   
                 CTT ACC CCG AAG TTA CGT CTT TC; 
               
               
                   
                 product size 197 bp); 
               
               
                   
               
               
                   
                 TET3 
               
               
                   
                 (forward primer: 
               
               
                   
                 (SEQ ID NO: 1271) 
               
               
                   
                 CAC CCG GCT CTA TGA AAC CTT, 
               
               
                   
               
               
                   
                 reverse primer: 
               
               
                   
                 (SEQ ID NO: 1272) 
               
               
                   
                 CCA GCC ACT CGA GGT AGT CA; 
               
               
                   
                 product size 209 bp); 
               
               
                   
                 RPLP1 (Cat#: PPH17813G-200, Qiagen). 
               
            
           
         
       
     
     Flow Cytometry 
     The cells were acquired on a FACS Fortessa (BD). Data processing for presentation was done using FlowJo (Treestar Inc.) program. 
     Results 
     Validation of Knockdown Efficiency of Tet2 shRNAs 
     As shown in  FIG.  27   , the validation of knockdown efficiency of TET2 shRNAs is schematized. TET2 and scramble control shRNA constructs expressing Red Fluorescence Protein (RPF) and puromycin resistant gene were introduced into jurkat cells to validate knockdown efficiency of TET2 shRNAs by qRT-PCR and western blot experiments. 
     As shown in  FIG.  28   , shRNA infected jurkat cells express RFP. RFP expression was determined by FACS on day 6 after puromycin treatment. Based on RFP expression, greater than 99% shRNA introduced jurkat cells were selected by puromycin treatment. Of note, TET2 shRNA #3 and #4 infected jurkat cells did not grow in the presence of puromycin. Therefore, TET2 shRNA #3 and #4 infected jurkat cells were not processed further. This data indicates that puromycin is effective to select shRNA infected jurkat cells. 
     As shown in  FIG.  29   , knockdown efficiency of tet2 depends on shRNAs. To determine mRNA expression level of tet1 and tet2 and tet3 in TET2 shRNAs infected jurkat cells, qRT-PCR experiment was performed. Compared to scramble shRNA, TET2 shRNA #1, #2, #8, and #9 knockdown tet2 gene at 35.6%, 22.7%, 21.6%, and 76.7% respectively. Surprisingly, while TET2 shRNA #9 knocks down tet2 efficiently, it also down-regulates tet1 and tet3 expression at 43.4% and 67.3% respectively. 3-actin serves as an internal control to quantify relative gene expression among samples tested. To increase reliability of qRT-PCR, two j-actin primers and one RPLP1 primer were used in this experiment. This data indicates that several TET2-targeting shRNA are capable of reducing mRNA levels of TET2, with shRNA #9 showing the most robust knockdown effect of tet2, while also affecting levels of TET1 and TET2. 
     As shown in  FIG.  30   , knockdown of TET2 protein in response to shRNAs correlates with knockdown of TET2 mRNA levels. To determine protein expression level of TET2 in TET2 shRNAs infected jurkat cells, a western blot experiment was performed. Similar to qRT-PCR data as shown in  FIG.  29   , TET2 shRNA #1, #2, #8, and #9 reduce TET2 protein level compared to scramble shRNA, while 3-actin is constitutively expressed in all samples tested. This data indicates that several TET2-targeting shRNA are capable of reducing TET2 protein levels in Jurkat cells, with shRNA #9 showing the most robust knockdown effect of Tet2. 
     Treatment of Primary T Cells with shRNA Tet2 Inhibitors 
     As shown in  FIG.  11   , TET2 shRNAs reduce 5-hmc levels in normal human T cells. TET2 and scramble control shRNA constructs expressing mCherry were introduced into normal human T cells. 5-hmc levels were determined by intracellular staining by FACS on day 6 following expansion with anti-CD3/CD28 beads. Knockdown of TET2 reduced overall 5-hmc levels. 
     As shown in  FIG.  12   , TET2 shRNAs expand Tscm T cells. TET2 and scramble control shRNA constructs expressing mCherry were introduced into normal human T cells. CD45RA+CD62L+CCR7+CD27+CD95+ Tscm T cells were determined by FACS staining on day 11 following expansion with anti-CD3/CD28 beads. Knockdown of TET2 promoted the expansion of T cells with a Tscm phenotype. 
     Tet2 Inhibition in CAR T Cells Using CRISPR/Cas Gene Editing Systems 
     In this example, inhibition of TET2 was explored in chimeric antigen receptor (CAR)-expressing T cells. 
     Methods 
     Guide RNA Molecules 
     gRNA molecules comprising the targeting sequences listed in Table 5 were used for the experiments described in this subexample. Unless otherwise indicated, all gRNA molecules were tested as dual gRNA molecules comprising the tracr and crRNA sequences described in this subexample. 
     
       
         
           
               
               
               
             
               
                 TABLE 5 
               
               
                   
               
               
                 Target 
                   
                   
               
               
                 Region 
                 TET2 gRNA targeting sequence 
                 guide reference 
               
               
                   
               
             
            
               
                 Exon 9 
                 CAGAGCACCAGAGUGCCGUC 
                 9_1 (also referred to as 
               
               
                   
                 (SEQ ID NO: 1273) 
                 Tet2 E9_1_Tet2) 
               
               
                   
               
               
                 Exon 9 
                 AGAGCACCAGAGUGCCGUCU 
                 9_2 (also referred to as 
               
               
                   
                 (SEQ ID NO: 1274) 
                 Tet2 E9_2_Tet2) 
               
               
                   
               
               
                 Exon 9 
                 UUCAGACCCAGACGGCACUC 
                 9_3 (also referred to as 
               
               
                   
                 (SEQ ID NO: 1275) 
                 Tet2 E9_3_Tet2) 
               
               
                   
               
               
                 Exon 9 
                 AUGGCAGCACAUUGGUAAGU 
                 9_4 (also referred to as 
               
               
                   
                 (SEQ ID NO: 1276) 
                 Tet2 E9_4_Tet2) 
               
               
                   
               
               
                 Exon 9 
                 CACAUUGGUAAGUUGGGCUG 
                 9_5 (also referred to as 
               
               
                   
                 (SEQ ID NO: 1277) 
                 Tet2 E9_5_Tet2) 
               
               
                   
               
               
                 Exon 9 
                 GACUUGCACAACAUGCAGAA 
                 9_6 (also referred to as 
               
               
                   
                 (SEQ ID NO: 1278) 
                 Tet2_E9_5_Tet2, Ex9-3 
               
               
                   
                   
                 or crEx9-3) 
               
               
                   
               
               
                 exon 7 
                 UCAUGGAGCAUGUACUACAA 
                 7_1 
               
               
                   
                 (SEQ ID NO: 1279) 
                   
               
               
                   
               
               
                 exon 7 
                 AACUUGCGCCUGUCAGGGGC 
                 7_2 
               
               
                   
                 (SEQ ID NO: 1280) 
                   
               
               
                   
               
               
                 exon 7 
                 CCAAGGAAGUUUAAGCUGCU 
                 7_3 
               
               
                   
                 (SEQ ID NO: 1281) 
                   
               
               
                   
               
               
                 exon 7 
                 CCAAGCAGCUUAAACUUCCU 
                 7_4 
               
               
                   
                 (SEQ ID NO: 1282) 
                   
               
               
                   
               
               
                 exon 8 
                 UUGGUGCCAUAAGAGUGGAC 
                 8_1 
               
               
                   
                 (SEQ ID NO: 1283) 
                   
               
               
                   
               
               
                 exon 8 
                 GCAAAACCUGUCCACUCUUA 
                 8_2 
               
               
                   
                 (SEQ ID NO: 1284) 
                   
               
               
                   
               
               
                 exon 8 
                 ALAUGUUGGUGCCALAAGAG 
                 8_3 
               
               
                   
                 (SEQ ID NO: 1285) 
                   
               
               
                   
               
               
                 exon 10 
                 AAAACGGAGUGGUGCCAUUC 
                 10_1 
               
               
                   
                 (SEQ ID NO: 1286) 
                   
               
               
                   
               
               
                 exon 10 
                 GUCUCUGACGUGGAUGAGUU 
                 10_2 
               
               
                   
                 (SEQ ID NO: 1287) 
                   
               
               
                   
               
               
                 exon 10 
                 UUUAUACAAAGUCUCUGACG 
                 10_3 
               
               
                   
                 (SEQ ID NO: 1288) 
                   
               
               
                   
               
               
                 exon 10 
                 AGAGAAGACAAUCGAGAAUU 
                 10_4 
               
               
                   
                 (SEQ ID NO: 1289) 
                   
               
               
                   
               
               
                 exon 10 
                 ACGUCAGAGACUUUGUAUAA 
                 10_5 
               
               
                   
                 (SEQ ID NO: 1290) 
                   
               
               
                   
               
               
                 exon 3 
                 GGAUAGAACCAACCAUGUUG 
                 3_1 (also referred to as 
               
               
                   
                 (SEQ ID NO: 1291) 
                 Tet2 E3_1_Tet2) 
               
               
                   
               
               
                 exon 3 
                 UUGUAGCCAGAGGUUCUGUC 
                 3_2 (also referred to as 
               
               
                   
                 (SEQ ID NO: 1292) 
                 Tet2 E3_2_Tet2) 
               
               
                   
               
               
                 exon 3 
                 UCUGUUGCCCUCAACAUGGU 
                 3_3_(also referred to as 
               
               
                   
                 (SEQ ID NO: 1293) 
                 Tet2_E3_3_Tet2, Ex3-3 
               
               
                   
                   
                 or crEx3-3) 
               
               
                   
               
               
                 exon 3 
                 GAUAGAACCAACCAUGUUGA 
                 3_4 (also referred to as 
               
               
                   
                 (SEQ ID NO: 1294) 
                 Tet2 E3_4_Tet2) 
               
               
                   
               
               
                 exon 3 
                 UUCUGGAGCUUUGUAGCCAG 
                 3_5 (also referred to as 
               
               
                   
                 (SEQ ID NO: 1295) 
                 Tet2 E3_5_Tet2) 
               
               
                   
               
            
           
         
       
     
     Generation of CRISPR CAR T Cells 
     Isolated and frozen Pan T cells were thawed and activated with CD3/CD28 beads (CD3/CD28 CTS Dynabeads® 43205D) on day 0. Activated T cells were transduced with lentivirus encoding either a BCMA CAR (BCMA-10 (139109) as described in WO2016/0046724; referred to herein as BCMA-10 CAR) or CD19 CAR (the CD19 CAR having the amino acid sequence of SEQ ID NO: 12 of WO2012/079000; referred to herein as CD19 CAR) on day 1. On day 3, transduced CAR T cells were electroporated to introduce CRISPR/Cas systems in the form of pre-complexed gRNA/Cas9 ribonuclear protein (“RNP”). To form RNP, all RNA samples were heated at 95C.  S. pyogenes  CAS9 Protein (NLS CAS9 iPROT106154, 37 μM) was diluted in buffer before tracrRNA (having the sequence: AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGC ACCGAGUCGGUGUUUUUUU (SEQ ID NO: 1296); AXO Labs) was added to it. After mixing CAS9 Protein with tracrRNA, the CRISPR RNA was added (in each case, each crRNA comprised the sequence nnnnnnnnnnnnnnnnnnnnGUUUUAGAGCUAUGCUGUUUUG (SEQ ID NO: 40), where the n residues represent the 20 ribonucleic acid residues of the indicated targeting sequence). The precomplexed RNPs were then added to a total of 1 million cells, RNP concentration was 3.2 μM. Electroporation was done by neon electroporator using  Neon® Transfection System  100  μL Kit  (MPKI0096) at 1600V, 10 ms, 3 pulses. The cells were kept in culture for 7 more days. Cells were then divided, with some used to perform flow cytometry: Staining for CAR (PE), CD3 (PerCP-Cy5.5), CD4 (V450) and CD8 (APC). Remaining T cells were frozen and used for functional assays and next generation sequencing (NGS) sample generation. 
     Next Generation Sequencing (NGS) Sample Generation 
     Frozen edited cell pellets (described above) were thawed and processed using DNeasy Blood &amp; Tissue Kit (Qiagen, 69506) to isolate genomic DNA. Eluted DNA was used to run PCR using Titanium Taq PCR kit (Clontech Laboratories, 639211) and TET2 primers (primers designed to flank the expected target site of the gRNA). PCR product was purified using QIAquickPCR Purification Kit (Qiagen, 28104). Purified PCR product was then used for T7E1 assay to detect base pair mismatches and confirm gene editing. PCR amplicons were subjected to standard Nextera NGS library prep (Illumina) and sequenced with paired-end reads on an Illumina MiSeq sequencer. Sequencing reads were aligned to the reference genome and variants were called. 
     Cytokine Production Assay 
     Effector cells (CAR T cells) are thawed on the day of the assay and counted on Cellometer (Nexelcom). These cells are then co cultured with different target cells at Effector:Target cell ratio of 2:1. 100ul of co-culture supernatant is harvested after 20h. These supernatants are then used measure the cytokines IL-2 and IFN-g released using Meso Scale Discovery, Proinflammatory Panel 1 catalog #N05049A-1 system according to the manufacturer&#39;s protocol. 
     T cell Proliferation Assay 
     CAR T cell proliferation in response to BCMA- or CD19-expressing target cells was evaluated. Target cell lines were: BCMA positive multiple myeloma cell lines, NCI-H929-luc, KMS-11-luc, and BCMA-negative Nalm6luc (CD19-positive cell line). CAR T cells were thawed incubated for 2 hours in T cell medium to recover. Cells were counted on a Cellometer. Target cells were irradiated at 10,000 rad. After irradiation, target cells were washed twice in complete T cell medium and counted. 30,000 Irradiated target cells were then co cultured with CART cells at 1:1 ratio. As a negative control, medium alone was added to CAR T cells. 
     The co-culture was incubated for 4 days at 37° C. On Day 4, coculture cells were stained for 20 mins on ice with CD3-percp cy5.5 (Ebioscience:45-0037), CD4-eflor450 (Ebioscience:48-0047), and CD8-APC(Ebioscience 17-0087 and measured by flow cytometry relative to CountBright Absolute Counting Beads (Life Technologies) to determine relative cell counts.). CAR expression was measured by two step incubation of 20 mins each on ice: Biotinylated-Protein L+Streptavidin-PE(Jackson immuno research). Flow cytometry data was acquired using BD 5 laser Fortessa and analyzed by FlowJo software. 
     Results 
       FIGS.  13 A and  13 B  show CAR expression in cells electroporated with and without Tet2 CRISPR.  FIG.  13 A  shows the gating strategy for determining CAR+ T cells. Lymphocytes were selected using forward scatter (FSC-A) and side scatter (SSC-A) as encircled. CD3-expressing cells were then selected (middle panel). CAR positive (PE positive cells using CAR detection by biotinylated protein and streptavitin-PE) cells indicated by the bar were then determined by gating relative to the negative control peak.  FIG.  13 B  shows the quantitation of the percentage of CAR positive cells. Cells were transduced with either the BCMA-10 CAR of the CD19 CAR as indicated. Cells were electroporated with RNP containing Cas9 protein, tracer RNA, and the indicated guide RNA targeting Tet2 (Ex3-3 targeting exon 3 or Ex9-6 targeting exon 9), or with no electroporation. CAR expression was determined 10 days after cell activation with beads. These data indicate that editing of Tet2 does not impact CAR expression in T cells. 
       FIG.  14    shows quantitation of CD4 and CD8 positive cells after CAR transduction and Tet2 editing. Cells were stained for CD3, CD4, CD8, and CAR expression at the end of the 10 day bead expansion. The left panel indicates the percentage of CD4 and CD8 positive cells in the total population of CD3+ cells. The right panel indicates the percentage of CD4 and CD8 positive cells in the population of CD3+ cells that are also CAR+. Cells were transduced with either the BCMA-10 CAR of the CD19 CAR or left untransduced (UTD) as indicated. Cells were electroporated with RNP containing Cas9 protein, tracer RNA, and the indicated guide RNA targeting Tet2 (Ex3-3 targeting exon 3 and Ex9-6 targeting exon 9), or with no electroporation. These data indicated that editing of Tet2 causes a small but consistent decrease in the percentage of CD8 cells and increase in the percentage of CD4 during the window of time of the bead-based expansion process. 
       FIG.  15    shows cell yield and viability after bead expansion for 10 days. The number of cells per mL (left panel) and the viability of cells (right panel) were measured by Cellometer after the 10 day bead expansion process. Cells were transduced with either the BCMA-10 CAR of the CD19 CAR or left untransduced (UTD) as indicated. Cells were electroporated with RNP containing Cas9 protein, tracer RNA, and the indicated guide RNA targeting Tet2 (Ex3-3 targeting exon 3 and Ex9-6 targeting exon 9), or with no electroporation. These data indicate that Tet2 editing causes an increase in cell number and viability relative to cells with no CRISPR/Cas9, with the Exon 9 targeting guide (ex9-6) showing the greatest impact in untransduced as well as CAR transduced cells. 
       FIG.  16    shows IL-2 production in response to cells either positive or negative for the antigen recognized by the CAR. CART cells or untransduced cells (UTD) were co-cultured with BCMA-positive/CD19-negative cells (KMS11 and NCIH929) or BCMA-negative/CD19-positive cells (NALM6) and cytokine secretion into the media was measured. IL-2 (pg/ml) levels are shown. Cells were prepared as described above. These data indicated that Tet2 editing causes an increase in IL-2 production by T cells in response to antigen with the Exon 9 targeting guide (Ex9-6) showing the greatest effect. 
       FIG.  17    shows interferon gamma production. CART cells or untransduced cells (UTD) were co-cultured with BCMA-positive/CD19-negative cells (KMS11 and NCIH929) or BCMA-negative/CD19-positive cells (NALM6) and cytokine secretion into the media was measured. Interferon gamma (IFN-g) (pg/ml) levels are shown. Cells were prepared as described above. These data indicated that Tet2 editing causes an increase in Interferon gamma production by T cells in response to antigen, with the Exon 9 targeting guide (Ex9-6) showing the greatest effect. 
       FIG.  18    shows antigen-driven CAR-T cell proliferation. CART cells or untransduced cells (UTD) were co-cultured with BCMA-positive/CD19-negative cells (KMS11 and NCIH929) or BCMA-negative/CD19-positive cells (NALM6) or no target cells (media) and proliferation of CAR positive T cells was measured. Cells were prepared as described above. These data indicated that Tet2 editing causes an increase in CAR+ T cell proliferation in response to antigen, with the Exon 9 targeting guide (CrEx9-6) showing the greatest effect. 
       FIG.  19    shows antigen-driven total T cell proliferation. CART cells or untransduced cells (UTD) were co-cultured with BCMA-positive/CD19-negative cells (KMS11 and NCIH929) or BCMA-negative/CD19-positive cells (NALM6) or no target cells (media) and proliferation of all CD3+ T cells was measured. Cells were prepared as described above. These data indicated that Tet2 editing causes an increase in CD3+ T cell proliferation in response to antigen, with the Exon 9 targeting guide (CrEx9-6) showing the greatest effect. 
       FIG.  20    shows antigen-driven CAR+ T cell proliferation. CART cells or untransduced cells (UTD) were co-cultured with BCMA-positive/CD19-negative cells (KMS11 and NCIH929) or BCMA-negative/CD19-positive cells (NALM6) or no target cells (media) and proliferation of all CD4+CD3+ T cells was measured. Cells were prepared as described above. These data indicated that Tet2 editing causes an increase in CD4+ T cell proliferation in response to antigen, with the Exon 9 targeting guide (CrEx9-6) showing the greatest effect. 
       FIG.  21    shows antigen-driven CAR+CD4+ T cell proliferation. CART cells or untransduced cells (UTD) were co-cultured with BCMA-positive/CD19-negative cells (KMS11 and NCIH929) or BCMA-negative/CD19-positive cells (NALM6) or no target cells (media) and proliferation of all CAR+CD4+CD3+ T cells was measured. Cells were prepared as described above. These data indicated that Tet2 editing causes an increase in CAR+CD4+ T cell proliferation in response to antigen, with the Exon 9 targeting guide (CrEx9-6) showing the greatest effect. 
       FIG.  22    shows antigen-driven CD8+ T cell proliferation. CART cells or untransduced cells (UTD) were co-cultured with BCMA-positive/CD19-negative cells (KMS11 and NCIH929) or BCMA-negative/CD19-positive cells (NALM6) or no target cells (media) and proliferation of all CD8+CD3+ T cells was measured. Cells were prepared as described above. These data indicated that Tet2 editing causes an increase in CD8+ T cell proliferation in response to antigen, with the Exon 9 targeting guide (CrEx9-6) showing the greatest effect. 
       FIG.  23    shows antigen-driven CAR+CD8+ T cell proliferation. CART cells or untransduced cells (UTD) were co-cultured with BCMA-positive/CD19-negative cells (KMS11 and NCIH929) or BCMA-negative/CD19-positive cells (NALM6) or no target cells (media) and proliferation of all CAR+CD8+CD3+ T cells was measured. Cells were prepared as described above. These data indicated that Tet2 editing causes an increase in CAR+CD8+ T cell proliferation in response to antigen, with the Exon 9 targeting guide (CrEx9-6) showing the greatest effect. 
       FIG.  24    shows % editing, and % frameshift edit by introduction of Tet2 targeting CRISPR/Cas systems. The level of editing in primary T cells after electroporation of RNP containing Cas9 protein, tracer RNA, and the indicated guide RNAs targeting either exon 3 or exon 9 of Tet2 is shown. The percentage of observed insertions or deletions of nucleotides relative to a reference genome is shown in the middle column (average % insertion/deletion). Editing that results in a shift in the open reading frame is shown in the far right column (average % frameshift). These data are the average of triplicates. These data indicate highly efficient genome editing in primary T cells with these guide RNA sequences. 
     The insertion and deletion pattern present at or near the target site for each gRNA was assessed by next generation sequencing. Briefly, T cells were electroporated with an RNP containing the indicated guide RNAs. After 48 hours, DNA was isolated and processed for sequencing.  FIG.  25    shows the 5 most common indels (insertions and/or deletions) observed in primary T cells for the guides RNAs targeting exon 3 of Tet2.  FIG.  26    shows the 5 most common indels (insertions and/or deletions) observed in primary T cells for the guides RNAs targeting exon 9 of Tet2. The percentages indicate the frequency with which a given editing pattern was observed. Insertions are shown by lowercase nucleotide letters (“a,” “g,” “c” or “t”), while deletions are shown by a dash (“—”). 
     ATAC-Seq Experiments 
     CD8+ T cells with and without the Tet2 insertion were expanded from a patient&#39;s post-infusion sample. Chromatin accessibility was assessed using Assay for Transposase-Accessible Chromatin with high throughput sequencing (ATAC-seq). This is essentially a technique for global chromatin mapping based on the transposition of “barcoded” DNA fragments. These DNA fragments get incorporated into regions of open chromatin, which allow for determination of which chromatin regions are opened versus closed. Based on the location of open or closed regions, pathway analyses can be done under the assumption that “open” equals to “expressed” and “closed” equals to “repressed.”  FIG.  30 A  shows Venn diagrams of ATAC peaks in the CAR+CD8+ T cells from a patient with a Tet2 disruption compared to CAR-CD8+ T cells from the same patient at the matched time point without the Tet2 disruption.  FIG.  28 B  show GO terms associated with ATAC peaks more closed in the cell population with the Tet2 disruption, compared to its counterpart. The significance of  FIG.  30 B  is, at least in part, that the chromatin landscape of the CD8+ T cells with the Tet2 disruption is possibly in line with a less differentiated cell that may be more “early memory-like” and less “effector-like.” These are the sort of cells that are thought to persist to provide robust and long-term anti-tumor activity. 
     ShRNA Studies 
     T cells from healthy donors were activated for 24 hours via CD3/CD28-coated beads, followed by lentiviral transduction with either the non-targeting (control) shRNA or the Tet2 shRNA. As shown in  FIG.  32 A , knock-down efficiency was assessed by qPCR and shown to be 50% (may mirror what happened in the patient in which Tet2 was disrupted via lentiviral integration). The differentiation phenotype was examined at day 14 by examining CCR7, CD45RO. Central memory cells are defined as CCR7+CD45RO+, whereas effector cells are CCR7-CD45RO−. To examine the differentiation phenotype specifically in cells with the 50% Tet2 knockdown, a GFP indicator was used in the shRNA constructs. The results are shown in  FIGS.  32 B and  32 C . 
     Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples specifically point out various aspects of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure. 
     EQUIVALENTS 
     The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific aspects, it is apparent that other aspects and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such aspects and equivalent variations.