Patent Publication Number: US-2021163624-A1

Title: T cell receptor-like antibodies specific for a wti peptide presented by hla-a2

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation application of U.S. application Ser. No. 16/055,535, filed on Aug. 6, 2018, which is a continuation application of U.S. application Ser. No. 15/364,953, filed on Nov. 30, 2016, now U.S. Pat. No. 10,040,865, which is a continuation of U.S. application Ser. No. 14/724,155, filed on May 28, 2015, now U.S. Pat. No. 9,540,448, which is a continuation application of U.S. application Ser. No. 14/008,447 filed Dec. 10, 2013, now U.S. Pat. No. 9,074,000, which is a National Phase filing under 35 U.S.C. § 371 of PCT International Application PCT/US2012/31892 filed Apr. 2, 2012, and published as WO2012/135854 on Oct. 4, 2012. This application also claims priority to U.S. Provisional Application No. 61/470,635, filed Apr. 1, 2011, and U.S. Provisional Application No. 61/491,392, filed May 31, 2011. These Provisional Applications are hereby incorporated by reference in their entirety into the present disclosure. 
    
    
     STATEMENT OF RIGHTS UNDER FEDERALLY-SPONSORED RESEARCH 
     This invention was made with government support under grants P01CA23766 and R01CA55349 awarded by the U.S. National Institutes of Health. The government has certain rights in the invention. 
    
    
     SEQUENCE LISTING 
     This application contains a Sequence Listing, created on May 28, 2015; the file, in ASCII format, is named 48071A_Seqlisting.txt, and is 80,348 bytes. The file is hereby incorporated by reference in its entirety into the application. 
     TECHNICAL FIELD 
     The present invention relates generally to antibodies against cytosolic proteins. More particularly, the invention relates to antibodies against Wilm&#39;s tumor oncogene protein (WT1), specifically antibodies that recognize a WT1 peptide in conjunction with a major histocompatability antigen. 
     BACKGROUND OF THE INVENTION 
     The Wilms&#39; tumor oncogene protein (WT1) is an attractive target for immunotherapy for most leukemias and a wide range of cancers. WT1 is a zinc finger transcription factor that is normally expressed in mesodermal tissues during embryogenesis. In normal adult tissue, WT1 expression is limited to low levels in CD34+ hematopoietic stem cells but is over-expressed in leukemias of multiple lineages and a wide range of solid tumors (1-2). More recently, WT1 expression has been reported to be a marker of minimal residual disease. Increasing transcript levels in patients with acute myeloid leukemia (AML) in morphologic remission have been predictive of overt clinical relapse (3, 4). Furthermore, antibodies to WT1 are detected in patients with hematopoietic malignancies and solid tumors, indicating that WT1 is a highly immunogenic antigen (7). 
     For the most part, clinically approved therapeutic monoclonal antibodies (mAbs) recognize structures of cell surface proteins. WT1, however, is a nuclear protein and, therefore, is inaccessible to classical antibody therapy. Up until now, immunotherapy targeting WT1 has been limited to cellular approaches, exclusively aimed at generating WT1-specific cytotoxic CD8 T cell (CTL) responses that recognize peptides presented on the cell surface by MHC class I molecules. 
     For induction of CTL responses, intracellular proteins are usually degraded by the proteasome or endo/lysosomes, and the resulting peptide fragments bind to MHC class I or II molecules. These peptide-MHC complexes are displayed at the cell surface where they provide targets for T cell recognition via a peptide-MHC (pMHC)-T cell receptor (TCR) interaction (8, 9). Vaccinations with peptides derived from the WT1 protein induce HLA-restricted cytotoxic CD8 T cells, which are capable of killing tumor cells. 
     To improve efficacy, cancer antigens can be targeted with monoclonal antibody therapy. Monoclonal antibody (mAb) therapy has been shown to exert powerful antitumor effects by multiple mechanisms, including complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) and direct cell inhibition or apoptosis-inducing effects on tumor cells that over-express the target molecules. Furthermore, mAb can be used as carriers to specifically deliver a cytotoxic moiety such as a radionuclide, cytotoxic drug or toxin to the tumor cells (18). 
     A tremendous benefit would exist if, in addition to a cellular immunotherapy approach, a humoral immunotherapy approach was available to target non-cell surface tumor antigens. Therefore, a monoclonal antibody (mAb) that mimics a T cell receptor in that it is specific for a target comprising a fragment of an intracellular protein in conjunction with an MHC molecule, for example, a WT1 peptide/HLA-A2 complex, would be a novel and effective therapeutic agent alone or as a vehicle capable of delivering potent anti-cancer reagents, such as drugs, toxins and radioactive elements. Such mAbs would also be useful as diagnostic or prognostic tools. 
     SUMMARY OF THE INVENTION 
     The present disclosure identifies and characterizes antigen-binding proteins, such as antibodies, that are able to target cytosolic/intracellular proteins, for example, the WT1 oncoprotein. The disclosed antibodies target a peptide/MHC complex as it would typically appear on the surface of a cell following antigen processing of WT1 protein and presentation by the cell. In that regard, the antibodies mimic T-cell receptors in that the antibodies have the ability to specifically recognize and bind to a peptide in an MHC-restricted fashion, that is, when the peptide is bound to an MHC antigen. The peptide/MHC complex recapitulates the antigen as it would typically appear on the surface of a cell following antigen processing and presentation of the WT1 protein to a T-cell. 
     The antibodies disclosed specifically recognize and bind to epitopes of a peptide/HLA-A2 complex, particularly a WT1/HLA-A0201 complex. Examples of peptides that are recognized by the antigen-binding proteins of the invention as part of an HLA-peptide complex include, but are not limited to, those shown in Table 7, for example, a peptide with the amino acid sequence RMFPNAPYL (SEQ ID NO: 1.) 
     In one aspect, therefore, the invention relates to an isolated antibody, or antigen-binding fragment thereof, that binds to a peptide with the amino acid sequence, RMFPNAPYL, when said peptide is bound to an MHC antigen, such as HLA-A2. 
     In another aspect, the invention relates to an isolated antigen-binding protein, antibody, or antigen-binding fragment thereof, comprising (A) (i) a heavy chain (HC) variable region comprising HC-CDR1, HC-CDR2 and HC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 2, 3, and 4; and alight chain (LC) variable region comprising LC-CDR1, LC-CDR2 and LC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 8, 9 and 10; (ii) a heavy chain (HC) variable region comprising HC-CDR1, HC-CDR2 and HC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 20, 21 and 22; and a light chain (LC) variable region comprising LC-CDR1, LC-CDR2 and LC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 26, 27 and 28; (iii) a heavy chain (HC) variable region comprising HC-CDR1, HC-CDR2 and HC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 38, 39 and 40; and alight chain (LC) variable region comprising LC-CDR1, LC-CDR2 and LC-CDR3 respectively, comprising amino acid sequences selected from SEQ ID NOS: 44, 45 and 46; (iv) a heavy chain (HC) variable region comprising HC-CDR1, HC-CDR2 and HC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 56, 57 and 58; and a light chain (LC) variable region comprising LC-CDR1, LC-CDR2 and LC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 62, 63 and 64; (v) a heavy chain (HC) variable region comprising HC-CDR1, HC-CDR2 and HC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 74, 75 and 76; and a light chain (LC) variable region comprising LC-CDR1, LC-CDR2 and LC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 80, 81 and 82; or (vi) a heavy chain (HC) variable region comprising HC-CDR1, HC-CDR2 and HC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 92, 93 and 94; and a light chain (LC) variable region comprising LC-CDR1, LC-CDR2 and LC-CDR3 respectively, comprising amino acid sequences SEQ ID NOS: 98, 99 and 100. 
     In another aspect, the invention relates to an isolated antigen-binding protein, antibody, or antigen-binding fragment thereof, comprising a V H  and V L  comprising first and second amino acid sequences, respectively, selected from SEQ ID NOS: 14 and 16; 32 and 34; 50 and 52; 68 and 70; 86 and 88; and 104 and 106. 
     In yet another aspect, the invention relates to an isolated antigen-binding protein, antibody, or antigen-binding fragment thereof, comprising an amino acid sequence selected from SEQ ID NOS: 18, 36, 54, 72, 90, and 108. 
     In a related aspect, the isolated antigen-binding protein comprises an antigen-binding region as disclosed in any of Tables 1-8. The antigen-binding protein may be a fusion protein. 
     In another aspect, the invention relates to an immunoconjugate comprising a first component which is an antigen-binding protein, antibody or antigen-binding fragment thereof as disclosed herein. The immunoconjugate comprises a second component that is a cytotoxin, a detectable label, a radioisotope, a therapeutic agent, a binding protein or a molecule having a second amino acid sequence. Where the second component is a binding protein or second antibody, the binding protein or second antibody has binding specificity for a target that is different from the HLA-peptide complex for which the first is specific. 
     In a related aspect, therefore, the present invention relates to bispecific antibody comprising an antigen-binding protein or functional fragment thereof as described herein. 
     In yet another aspect, the invention relates to nucleic acids that encode antigen binding proteins, including antibodies and chimeric antigen receptors specific for a WT1 peptide/HLA complex, in particular the complex of WT1 peptide RMFPNAPYL/HLA-A0201. 
     In another related aspect, the invention relates to cells comprising the nucleic acids or antigen binding proteins disclosed herein, including recombinant immune effector cells, such as, T-cells genetically modified to express a chimeric antigen receptor comprising an antigen binding region in accordance with the present disclosure. Cells which have been engineered to produce antibodies in accordance with the disclosure are also encompassed by the invention. 
     In a related aspect, the invention relates to vectors comprising the nucleic acids to encode the antigen binding proteins disclosed herein, including vectors to facilitate expression and/or secretion of an antigen binding protein such as an antibody or chimeric antigen receptor in accordance with the present disclosure. 
     In a related aspect, the invention relates to pharmaceutical compositions comprising the antigen binding proteins, antibodies, nucleic acids, vectors or cells comprising the nucleic acids or antigen binding proteins disclosed herein, together with a pharmaceutically acceptable carrier. 
     In another aspect, the invention relates to a method for detecting WT1 on the surface of cells or tissues using WT1 antibodies of the invention. 
     In yet another aspect, the invention relates to methods for treatment of a subject having a WT1-positive disease, comprising administering to the subject a therapeutically effective amount of an antigen binding protein, antibody or antigen binding fragment thereof, nucleic acid encoding the antigen binding protein or antibody or a cell comprising the nucleic acids or proteins as disclosed herein. The WT1-positive disease is a chronic leukemia, acute leukemia or WT1 +  cancer selected from the group consisting of chronic myelocytic leukemia, multiple myeloma (MM), acute lymphoblastic leukemia (ALL), acute myeloid/myelogenous leukemia (AML), myelodysplastic syndrome (MDS), mesothelioma, ovarian cancer, gastrointestinal cancers, breast cancer, prostate cancer and glioblastoma. In some embodiments, the antigen binding protein or antibody is a conjugate thereof having a cytotoxic moiety linked thereto. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows the amino acid sequence of Wilms tumor protein, (GenBank Accession No. P19544) with some HLA-restricted peptides bolded. The 121-140 peptide further encompasses a 9-mer (underlined), RMFPNAPYL (SEQ ID NO: 1), which, in addition to analogs thereof, has been shown to induce WT1-specific cytotoxic T-cell activity. 
         FIG. 2  is a graph showing that vaccination with WT1 peptides induces cytotoxic T cells against WT1+ leukemia cells. 
         FIG. 3  shows the results of a phage ELISA for specific binding of WT1/A2 (WA) versus PBS control or R3/HLAA0201 (R3). 
         FIG. 4  shows specific binding of only WT1 phage antibodies that bind to T2 cells pulsed with WT1A peptide were selected. 
         FIG. 5  shows the binding affinity of a full-length IgG1 of a WT1 antibody to RMF/A0201 complex tested by titration of the antibody at various concentrations. Results are shown for T2 cells pulsed with 50 ug/ml RMF (upper panel). Control antibody is shown in the lower panel. 
         FIG. 6  shows the dependence on density of RMF/HLA-A0201 complex recognized by WT1 antibody on T2 cells pulsed with RMF (upper panel) or control, RHAMM-R3 (lower panel). 
         FIG. 7  shows an expression vector for expression of human antibodies. 
         FIG. 8  shows the results of SDS-PAGE analysis of WT1/A2 antibodies under reducing and non-reducing conditions. 
         FIG. 9  shows the results of kinetic binding analysis of an WT1/A2 antibody demonstrating affinity of the antibody toward WT1/A2. 
         FIG. 10  shows the affinity (K D ) of antibody binding to WT1/A2 complex. 
         FIG. 11  shows the mean fluorescence intensity (MFI) by flow cytometry of peptide titration on binding of some embodiments, mAb clone 5 (upper panel), clone 15 (middle panel) and control (lower panel) to live T2 cells pulsed with varying concentrations of peptide, WT1-A, WT1-A1 or control. 
         FIG. 12  shows the results of peptide titration on binding of a WT1 antibody, mAb 5 (upper panel), mAb 15 (lower panel) to live T2 cells pulsed with varying concentrations of WT1A peptide. 
         FIG. 13  shows the binding specificity of one embodiment, mAb 5, at different concentrations (50 μg/ml upper; 25 μg/ml middle; and 12.5 μg/ml lower) of peptide (R3, WT1-A1, WT1-A or no peptide.) 
         FIG. 14  shows the binding specificity of one embodiment, mAb 15, at different concentrations (50 μg/ml upper; 25 μg/ml lower) of peptide (R3, WT1-A1, WT1-A or no peptide). 
         FIG. 15  shows dose-dependent binding of mAbs 5 (upper panel) and 15 (lower panel) to T2 cells pulsed with WT1-A, WT1-A1, or RHAMM-R3 peptide. 
         FIG. 16  shows binding of mAbs 5 and 15 to U266, a myeloma cell line. 
         FIG. 17  shows binding of mAb 15 to BV173, a cell line derived from an individual with (Ph1)-positive acute leukemia. 
         FIG. 18  shows the specific binding of ESK1 (#13) to WT1/A2 complex on the surface of T2 cells pulsed with WT1 peptide. 
         FIG. 19  and  FIG. 20  show that WT1 antibody is able to recognize RMF peptide in which substitution of different positions of the RMF peptide with alanine is made (see also Table 10) and that the loss of binding seen with substitution of position 1 by either alanine (WT1-A1-B) or tyrosine (WT1-A1), was not due to the reduction of peptide binding affinity to the HLA-A2 molecule, as both peptides showed the strongest binding in T2 stabilization assay using the mAb specific for the HLA-A2 molecule, clone BB7. 
         FIG. 21  shows recognition by WT1 antibody of naturally presented RMF/HLA-A0201 complex on the cell surface of human mesothelioma cell lines, JMN (WT1 + /A0201 + ) but not MSTO (WT1 + /HLA-A0201 − ). 
         FIG. 22  shows binding of WT1 antibodies to human CML-derived cell line BV173. 
         FIG. 23  is a Scatchard analysis based on binding of WT1 antibody to JMN cells and shows an avidity constant of about 0.2 nM. 
         FIG. 24  shows WT1 antibody binding to a panel of mesothelioma and leukemia cells. 
         FIG. 25  shows the results of flow cytometric analyses gated on CD33 and CD34 double positive AML blast cells from an HLA-A2 positive patient. ESK1 binds to the leukemia blasts. 
         FIG. 26  shows the results of flow cytometric analyses gated on CD33 and CD34 double positive AML blast cells from an HLA-A2 negative patient. WT1mAb ESK1 did not bind to the blasts. 
         FIG. 27  shows WT1mAb ESK1 mediated ADCC against T2 cells pulsed with RMF peptide. 
         FIG. 28  shows the ability of WT1 antibody to mediate ADCC with human effectors in JMN and leukemia cell line BV173 (lower panel) but not MSTO cells. 
         FIG. 29  shows that WT1 mAbs are effective against human leukemia cell line BV173 but not HL60 cells, which are not HLA-A2+. 
         FIG. 30  shows that WT1 antibody induces ADCC against primary AML blasts from an HLA-A2 positive patient. 
         FIG. 31  shows the results of treatment of human BV173 in NSG mice using antibodies of the invention. 
         FIG. 32  shows that at later time points, mice treated with WT1 antibody only began to relapse, while antibody with effectors cured 2 of 5 mice. 
         FIG. 33  shows that WT1 antibody significantly reduces tumor burden in a dose-dependent manner. 
         FIG. 34  shows that antibody with altered carbohydrate in Fc (MAGE) is more active in ADCC than original antibody. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     All publications, patents and other references cited herein are incorporated by reference in their entirety into the present disclosure. 
     In practicing the present invention, many conventional techniques in molecular biology, microbiology, cell biology, biochemistry, and immunology are used, which are within the skill of the art. These techniques are described in greater detail in, for example, Molecular Cloning: a Laboratory Manual 3rd edition, J. F. Sambrook and D. W. Russell, ed. Cold Spring Harbor Laboratory Press 2001; Recombinant Antibodies for Immunotherapy, Melvyn Little, ed. Cambridge University Press 2009; “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal Cell Culture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (Academic Press, Inc.); “Current Protocols in Molecular Biology” (F. M. Ausubel et al., eds., 1987, and periodic updates); “PCR: The Polymerase Chain Reaction”, (Mullis et al., ed., 1994); “A Practical Guide to Molecular Cloning” (Perbal Bernard V., 1988); “Phage Display: A Laboratory Manual” (Barbas et al., 2001). The contents of these references and other references containing standard protocols, widely known to and relied upon by those of skill in the art, including manufacturers&#39; instructions are hereby incorporated by reference as part of the present disclosure. The following abbreviations are used throughout the application: 
     Ab: Antibody 
     ADCC: Antibody-dependent cellular cytotoxicity 
     ALL: Acute lymphocytic leukemia 
     AML: Acute myeloid leukemia 
     APC: Antigen presenting cell 
     β2M: Beta-2-microglobulin 
     BiTE: Bi-specific T cell engaging antibody 
     CAR: Chimeric antigen receptor 
     CDC: Complement dependent cytotoxicity 
     CMC: Complement mediated cytotoxicity 
     CDR: Complementarity determining region (see also HVR below) 
     C L : Constant domain of the light chain 
     CH 1 : 1 st  constant domain of the heavy chain 
     CH 1, 2, 3 : 1 st , 2 nd  and 3 rd  constant domains of the heavy chain 
     CH 2, 3 : 2 nd  and 3 rd  constant domains of the heavy chain 
     CHO: Chinese hamster ovary 
     CTL: Cytotoxic T cell 
     E:T Ratio: Effector:Target ratio 
     Fab: Antibody binding fragment 
     FACS: Flow assisted cytometric cell sorting 
     FBS: Fetal bovine serum 
     FR: Framework region 
     HC: Heavy chain 
     HLA: Human leukocyte antigen 
     HVR-H: Hypervariable region-heavy chain (see also CDR) 
     HVR-L: Hypervariable region-light chain (see also CDR) 
     Ig: Immunoglobulin 
     IRES: Internal ribosome entry site 
     K D : Dissociation constant 
     k off : Dissociation rate 
     k on : Association rate 
     MHC: Major histocompatibility complex 
     MM: Multiple myeloma 
     scFv: Single-chain variable fragment 
     TCR: T cell receptor 
     V H : Variable heavy chain includes heavy chain hypervariable region and heavy chain variable framework region 
     V L : Variable light chain includes light chain hypervariable region and light chain variable framework region 
     WT1: Wilms tumor protein 1 
     In the description that follows, certain conventions will be followed as regards the usage of terminology. Generally, terms used herein are intended to be interpreted consistently with the meaning of those terms as they are known to those of skill in the art. 
     An “antigen-binding protein” is a protein or polypeptide that comprises an antigen-binding region or antigen-binding portion, that is, has a strong affinity to another molecule to which it binds. Antigen-binding proteins encompass antibodies, chimeric antigen receptors (CARs) and fusion proteins. 
     “Antibody” and “antibodies” as those terms are known in the art refer to antigen binding proteins of the immune system. The term “antibody” as referred to herein includes whole, full length antibodies having an antigen-binding region, and any fragment thereof in which the “antigen-binding portion” or “antigen-binding region” is retained, or single chains, for example, single chain variable fragment (scFv), thereof. A naturally occurring “antibody” is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as V H ) and a heavy chain constant (CH) region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as V L ) and alight chain constant C L  region. The light chain constant region is comprised of one domain, C L . The V H  and V L  regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each V H  and V L  is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. 
     The term “antigen-binding portion” or “antigen-binding region” of an antibody, as used herein, refers to that region or portion of the antibody that binds to the antigen and which confers antigen specificity to the antibody; fragments of antigen-binding proteins, for example, antibodies includes one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., an peptide/HLA complex). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of antigen-binding fragments encompassed within the term “antibody fragments” of an antibody include a Fab fragment, a monovalent fragment consisting of the V L , V H , C L  and CH1 domains; a F(ab) 2  fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the V H  and CH1 domains; a Fv fragment consisting of the V L  and V H  domains of a single arm of an antibody; a dAb fragment (Ward et al., 1989 Nature 341:544-546), which consists of a V H  domain; and an isolated complementarity determining region (CDR). 
     Furthermore, although the two domains of the Fv fragment, V L  and V H , are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V L  and V H  regions pair to form monovalent molecules. These are known as single chain Fv (scFv); see e.g., Bird et al., 1988 Science 242:423-426; and Huston et al., 1988 Proc. Natl. Acad. Sci. 85:5879-5883. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. 
     An “isolated antibody” or “isolated antigen-binding protein” is one which has been identified and separated and/or recovered from a component of its natural environment. “Synthetic antibodies” or “recombinant antibodies” are generally generated using recombinant technology or using peptide synthetic techniques known to those of skill in the art. 
     Traditionally, the MHC-peptide complex could only be recognized by a T-cell receptor (TCR), limiting our ability to detect an epitope of interest using T cell-based readout assays. In the present disclosure, antigen binding proteins, including antibodies, having an antigen-binding region based on scFvs that are selected from human scFv phage display libraries using recombinant HLA-peptide complexes are described. These molecules demonstrated exquisite specificity, for example as shown with anti-WT1 antibodies that recognize only HLA-A2-RMFPNAPYL complexes. In addition, along with their inability to bind to HLA-complexes containing other peptides, the molecules were also unable to bind to the peptides themselves, further demonstrating their TCR-like specificity. 
     The scFvs of the disclosure selected by phage display were initially tested for their ability to bind to peptide presented on the surface of HLA-positive cells. After T2 cells were incubated in the presence of peptide, fluorescently labeled antibodies could be used to selectively recognize the antigen pulsed cells using flow cytometry. 
     In some embodiments, the invention includes antibodies that have the scFv sequence fused to one or more constant domains of the heavy to form an antibody with an Fc region of a human immunoglobulin to yield a bivalent protein, increasing the overall avidity and stability of the antibody. In addition, the Fc portion allows the direct conjugation of other molecules, including but not limited to fluorescent dyes, cytotoxins, radioisotopes etc. to the antibody for example, for use in antigen quantitation studies, to immobilize the antibody for affinity measurements, for targeted delivery of a therapeutic agent, to test for Fc-mediated cytotoxicity using immune effector cells and many other applications. 
     The results presented here highlight the specificity, sensitivity and utility of the antibodies of the invention in targeting MHC-peptide complexes. 
     The molecules of the invention are based on the identification and selection of single chain variable fragments (scFv) using phage display, the amino acid sequence of which confers the molecules&#39; specificity for the MHC restricted peptide of interest and forms the basis of all antigen binding proteins of the disclosure. The scFv, therefore, can be used to design a diverse array of “antibody” molecules, including, for example, full length antibodies, fragments thereof, such as Fab and F(ab′) 2 , minibodies, fusion proteins, including scFv-Fc fusions, multivalent antibodies, that is, antibodies that have more than one specificity for the same antigen or different antigens, for example, bispecific T-cell engaging antibodies (BiTe), tribodies, etc. (see Cuesta et al.,  Multivalent antibodies: when design surpasses evolution. Trends in Biotechnology  28:355-362 2010). 
     In an embodiment in which the antigen-binding protein is a full length antibody, the heavy and light chains of an antibody of the invention may be full-length (e.g., an antibody can include at least one, and preferably two, complete heavy chains, and at least one, and preferably two, complete light chains) or may include an antigen-binding portion (a Fab, F(ab′) 2 , Fv or a single chain Fv fragment (“scFv”)). In other embodiments, the antibody heavy chain constant region is chosen from, e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE. In some embodiments, the immunoglobulin isotype is selected from IgG1, IgG2, IgG3, and IgG4, more particularly, IgG1 (e.g., human IgG1). The choice of antibody type will depend on the immune effector function that the antibody is designed to elicit. 
     In constructing a recombinant immunoglobulin, appropriate amino acid sequences for constant regions of various immunoglobulin isotypes and methods for the production of a wide array of antibodies are known to those of skill in the art. 
     In one embodiment, the antibody or other antigen binding protein is an anti-WT1/HLA-A2 scFv or or antigen-binding fragment thereof having an antigen binding region that comprises the amino acid sequence of SEQ ID NO: 18 and specifically binds to a peptide with the amino acid sequence RMFPNAPYL (SEQ ID NO: 1) in conjunction with HLA-A0201. In some embodiments, the anti-WT1 antibody is a scFv-Fc fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 1. 
     
       
         
           
               
               
             
               
                 TABLE 1 
               
               
                   
               
               
                 Antigen 
                 WT1 (Ext002 #3) 
               
               
                 Peptide 
                 RMFPNAPYL (SEQ ID NO: 1) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 CDRs: 
                 1 
                 2 
                 3 
               
               
                   
               
               
                 VH 
                 GGTFSSYAIS 
                 GIIPIFGTANYAQKFQG 
                 RIPPYYGMDV 
               
               
                   
                 (SEQ ID NO: 2) 
                 (SEQ ID NO: 3) 
                 (SEQ ID NO: 4) 
               
               
                   
               
               
                 DNA 
                 ggaggcaccttcagcag 
                 gggatcatccctatctttggtac 
                 cggattcccccgtactacggtat 
               
               
                   
                 ctatgctatcagc 
                 agcaaactacgcacagaagtt 
                 ggacgtc (SEQ ID NO: 7) 
               
               
                   
                 (SEQ ID NO: 5) 
                 ccagggc (SEQ ID NO: 6) 
                   
               
               
                   
               
               
                 VL 
                 SGSSSNIGSNYVY 
                 RSNQRPS 
                 AAWDDSLNGVV 
               
               
                   
                 (SEQ ID NO: 8) 
                 (SEQ ID NO: 9) 
                 (SEQ ID NO: 10) 
               
               
                   
               
               
                 DNA 
                 tctggaagcagctccaac 
                 aggagtaatcagcggccctca 
                 gcagcatgggatgacagcctg 
               
               
                   
                 atcggaagtaattatgtat 
                 (SEQ ID NO: 12) 
                 aatggtgtggta 
               
               
                   
                 ac (SEQ ID NO: 11) 
                   
                 (SEQ ID NO: 13) 
               
               
                   
               
            
           
           
               
               
            
               
                 Full 
                 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLE 
               
               
                 VH 
                 WMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYY 
               
               
                   
                 CARRIPPYYGMDVWGQGTTVTVSS (SEQ ID NO: 14) 
               
               
                   
               
               
                 DNA 
                 caggtgcagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgc 
               
               
                   
                 aaggcttctggaggcaccttcagcagctatgctatcagctgggtgcgacaggcccctggacaagg 
               
               
                   
                 gcttgagtggatgggagggatcatccctatctttggtacagcaaactacgcacagaagttccaggg 
               
               
                   
                 cagagtcacgattaccgcggacgaatccacgagcacagcctacatggagctgagcagcctgag 
               
               
                   
                 atctgaggacacggccgtgtattactgtgcgagacggattcccccgtactacggtatggacgtctgg 
               
               
                   
                 ggccaagggaccacggtcaccgtctcctca (SEQ ID NO: 15) 
               
               
                   
               
               
                 Full 
                 QTVVTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKL 
               
               
                 VL 
                 LIYRSNQRPSGVPDRFSGSKSGTSASLAISGPRSVDEADYYCAAWDD 
               
               
                   
                 SLNGVVFGGGTKLTVLG (SEQ ID NO: 16) 
               
               
                   
               
               
                 DNA 
                 cagactgtggtgactcagccaccctcagcgtctgggacccccgggcagagggtcaccatctcttgtt 
               
               
                   
                 ctggaagcagctccaacatcggaagtaattatgtatactggtaccaacagctcccaggaacggcc 
               
               
                   
                 cccaaactcctcatctataggagtaatcagcggccctcaggggtccctgaccgattctctggctcca 
               
               
                   
                 agtctggcacctcagcctccctggccatcagtgggccccggtccgtggatgaggctgattattactgt 
               
               
                   
                 gcagcatgggatgacagcctgaatggtgtggtattcggcggagggaccaagctgaccgtcctagg 
               
               
                   
                 t (SEQ ID NO: 17) 
               
               
                   
               
               
                 scFv 
                 QTVVTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKL 
               
               
                   
                 LIYRSNQRPSGVPDRFSGSKSGTSASLAISGPRSVDEADYYCAAWDD 
               
               
                   
                 SLNGVVFGGGTKLTVLG SRGGGGSGGGGSGGGSLEMA QVQLVQSG 
               
               
                   
                 AEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFG 
               
               
                   
                 TANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARRIPPYY 
               
               
                   
                 GMDVWGQGTTVTVSS (SEQ ID NO: 18) 
               
               
                   
               
               
                 DNA 
                 cagactgtggtgactcagccaccctcagcgtctgggacccccgggcagagggtcaccatctcttgtt 
               
               
                   
                 ctggaagcagctccaacatcggaagtaattatgtatactggtaccaacagctcccaggaacggcc 
               
               
                   
                 cccaaactcctcatctataggagtaatcagcggccctcaggggtccctgaccgattctctggctcca 
               
               
                   
                 agtctggcacctcagcctccctggccatcagtgggccccggtccgtggatgaggctgattattactgt 
               
               
                   
                 gcagcatgggatgacagcctgaatggtgtggtattcggcggagggaccaagctgaccgtcctagg 
               
               
                   
                 t tctagaggtggtggtggtagcggcggcggcggctctggtggtggatccctcgagatggc   
               
               
                   
                   c caggtgcagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctg 
               
               
                   
                 caaggcttctggaggcaccttcagcagctatgctatcagctgggtgcgacaggcccctggacaag 
               
               
                   
                 ggcttgagtggatgggagggatcatccctatctttggtacagcaaactacgcacagaagttccagg 
               
               
                   
                 gcagagtcacgattaccgcggacgaatccacgagcacagcctacatggagctgagcagcctga 
               
               
                   
                 gatctgaggacacggccgtgtattactgtgcgagacggattcccccgtactacggtatggacgtctg 
               
               
                   
                 gggccaagggaccacggtcaccgtctcctca (SEQ ID NO: 19) 
               
               
                   
               
            
           
         
       
     
     In another embodiment, the antibody or antigen binding protein is an anti-WT1 scFv or antigen-binding fragment thereof that has an antigen binding region that comprises the amino acid sequence of SEQ ID NO: 36 and specifically binds to a peptide with the amino acid sequence RMFPNAPYL (SEQ ID NO: 1) in conjunction with HLA-A0201. In other embodiments, the anti-WT-1 antibody is a scFv-Fc fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 2. 
     
       
         
           
               
               
             
               
                 TABLE 2 
               
               
                   
               
               
                 Antigen 
                 WT1 (Ext002 #5) 
               
               
                 Peptide 
                 RMFPNAPYL (SEQ ID NO: 1) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 CDRs: 
                 1 
                 2 
                 3 
               
               
                   
               
               
                 VH 
                 GDSVSSNSAAWN 
                 RTYYGSKWYNDYAVS 
                 GRLGDAFDI 
               
               
                   
                 (SEQ ID NO: 20) 
                 VKS (SEQ ID NO: 21) 
                 (SEQ ID NO: 22) 
               
               
                   
               
               
                 DNA 
                 ggggacagtgtctctagc 
                 aggacatactacgggtccaag 
                 ggtcgcttaggggatgcttttga 
               
               
                   
                 aacagtgctgcttggaac 
                 tggtataatgattatgcagtatct 
                 tatc (SEQ ID NO: 25) 
               
               
                   
                 (SEQ ID NO: 23) 
                 gtgaaaagt (SEQ ID NO: 24) 
                   
               
               
                   
               
               
                 VL 
                 RASQSISSYLN 
                 AASSLQS 
                 QQSYSTPLT 
               
               
                   
                 (SEQ ID NO: 26) 
                 (SEQ ID NO: 27) 
                 (SEQ ID NO: 28) 
               
               
                   
               
               
                 DNA 
                 cgggcaagtcagagcatt 
                 gctgcatccagtttgcaaagt 
                 caacagagttacagtacccct 
               
               
                   
                 agcagctatttaaat 
                 (SEQ ID NO: 30) 
                 ctcact (SEQ ID NO: 31) 
               
               
                   
                 (SEQ ID NO: 29) 
                   
                   
               
               
                   
               
            
           
           
               
               
            
               
                 Full 
                 QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGL 
               
               
                 VH 
                 EWLGRTYYGSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTA 
               
               
                   
                 VYYCARGRLGDAFDIWGQGTMVTVSS (SEQ ID NO: 32) 
               
               
                   
               
               
                 DNA 
                 caggtacagctgcagcagtcaggtccaggactggtgaagccctcgcagaccctctcactcacctgt 
               
               
                   
                 gccatctccggggacagtgtctctagcaacagtgctgcttggaactggatcaggcagtccccatcg 
               
               
                   
                 agaggccttgagtggctgggaaggacatactacgggtccaagtggtataatgattatgcagtatctg 
               
               
                   
                 tgaaaagtcgaataaccatcaacccagacacatccaagaaccagttctccctgcagctgaactct 
               
               
                   
                 gtgactcccgaggacacggctgtgtattactgtgcaagaggtcgcttaggggatgcttttgatatctgg 
               
               
                   
                 ggccaagggacaatggtcaccgtctcttca (SEQ ID NO: 33) 
               
               
                   
               
               
                 Full 
                 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIY 
               
               
                 VL 
                 AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLT 
               
               
                   
                 FGGGTKVDIKR (SEQ ID NO: 34) 
               
               
                   
               
               
                 DNA 
                 gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttg 
               
               
                   
                 ccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagccccta 
               
               
                   
                 agctcctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagtggcagtggatct 
               
               
                   
                 gggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaaca 
               
               
                   
                 gagttacagtacccctctcactttcggcggagggaccaaagtggatatcaaacgt (SEQ ID 
               
               
                   
                 NO: 35) 
               
               
                   
               
               
                 scFv 
                 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIY 
               
               
                   
                 AASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLT 
               
               
                   
                 FGGGTKVDIKR SRGGGGSGGGGSGGGGSLEMA QVQLQQSGPGLVK 
               
               
                   
                 PSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYGSKWY 
               
               
                   
                 NDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARGRLGDAF 
               
               
                   
                 DIWGQGTMVTVSS (SEQ ID NO: 36) 
               
               
                   
               
               
                 DNA 
                 gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttg 
               
               
                   
                 ccgggcaagtcagagcattagcagctatttaaattggtatcagcagaaaccagggaaagccccta 
               
               
                   
                 agctcctgatctatgctgcatccagtttgcaaagtggggtcccatcaaggttcagtggcagtggatct 
               
               
                   
                 gggacagatttcactctcaccatcagcagtctgcaacctgaagattttgcaacttactactgtcaaca 
               
               
                   
                 gagttacagtacccctctcactttcggcggagggaccaaagtggatatcaaacgt tctagaggtg   
               
               
                   
                   gtggtggtagcggcggcggcggctctggtggtggtggatccctcgagatggcc caggtac 
               
               
                   
                 agctgcagcagtcaggtccaggactggtgaagccctcgcagaccctctcactcacctgtgccatct 
               
               
                   
                 ccggggacagtgtctctagcaacagtgctgcttggaactggatcaggcagtccccatcgagaggc 
               
               
                   
                 cttgagtggctgggaaggacatactacgggtccaagtggtataatgattatgcagtatctgtgaaaa 
               
               
                   
                 gtcgaataaccatcaacccagacacatccaagaaccagttctccctgcagctgaactctgtgactc 
               
               
                   
                 ccgaggacacggctgtgtattactgtgcaagaggtcgcttaggggatgcttttgatatctggggccaa 
               
               
                   
                 gggacaatggtcaccgtctcttca (SEQ ID NO: 37) 
               
               
                   
               
            
           
         
       
     
     In another embodiment, the antibody or antigen binding protein is an anti-WT1 scFv or antigen binding fragment thereof that has an antigen binding region that comprises the amino acid sequence of SEQ ID NO: 54 and specifically binds to a peptide with the amino acid sequence RMFPNAPYL (SEQ ID NO: 1) in conjunction with HLA-A0201. In other embodiments, the anti-WT-1 antibody is a scFv-Fc fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 3. 
     
       
         
           
               
               
             
               
                 TABLE 3 
               
               
                   
               
               
                 Antigen 
                 WT1 (Ext002 #13) 
               
               
                 Peptide 
                 RMFPNAPYL (SEQ ID NO: 1) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 CDRs: 
                 1 
                 2 
                 3 
               
               
                   
               
               
                 VH 
                 GYSFTNFWIS 
                 RVDPGYSYSTYSPSF 
                 VQYSGYYDWFDP 
               
               
                   
                 (SEQ ID NO: 38) 
                 QG (SEQ ID NO: 39) 
                 (SEQ ID NO: 40) 
               
               
                   
               
               
                 DNA 
                 ggatacagcttcaccaact 
                 agggttgatcctggctactctta 
                 gtacaatatagtggctactatg 
               
               
                   
                 tctggatcagc 
                 tagcacctacagcccgtccttc 
                 actggttcgacccc 
               
               
                   
                 (SEQ ID NO: 41) 
                 caaggc (SEQ ID NO: 42) 
                 (SEQ ID NO: 43) 
               
               
                   
               
               
                 VL 
                 SGSSSNIGSNTVN 
                 SNNQRPS 
                 AAWDDSLNGWV 
               
               
                   
                 (SEQ ID NO: 44) 
                 (SEQ ID NO: 45) 
                 (SEQ ID NO: 46) 
               
               
                   
               
               
                 DNA 
                 tctggaagcagctccaac 
                 agtaataatcagcggccctca 
                 gcagcatgggatgacagcct 
               
               
                   
                 atcggaagtaatactgtaa 
                 (SEQ ID NO: 48) 
                 gaatggttgggtg 
               
               
                   
                 ac (SEQ ID NO: 47) 
                   
                 (SEQ ID NO: 49) 
               
               
                   
               
            
           
           
               
               
            
               
                 Full VH 
                 QMQLVQSGAEVKEPGESLRISCKGSGYSFTNFWISWVRQMPGKGLE 
               
               
                   
                 WMGRVDPGYSYSTYSPSFQGHVTISADKSTSTAYLQWNSLKASDTA 
               
               
                   
                 MYYCARVQYSGYYDWFDPWGQGTLVTVSS (SEQ ID NO: 50) 
               
               
                   
               
               
                 DNA 
                 cagatgcagctggtgcagtccggagcagaggtgaaagagcccggggagtctctgaggatctcct 
               
               
                   
                 gtaagggttctggatacagcttcaccaacttctggatcagctgggtgcgccagatgcccgggaaa 
               
               
                   
                 ggcctggagtggatggggagggttgatcctggctactcttatagcacctacagcccgtccttccaag 
               
               
                   
                 gccacgtcaccatctcagctgacaagtctaccagcactgcctacctgcagtggaacagcctgaag 
               
               
                   
                 gcctcggacaccgccatgtattactgtgcgagagtacaatatagtggctactatgactggttcgacc 
               
               
                   
                 cctggggccagggaaccctggtcaccgtctcctca (SEQ ID NO: 51) 
               
               
                   
               
               
                 Full 
                 QAVVTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQVPGTAPK 
               
               
                 VL 
                 LLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWD 
               
               
                   
                 DSLNGWVFGGGTKLTVLG (SEQ ID NO: 52) 
               
               
                   
               
               
                 DNA 
                 caggctgtggtgactcagccaccctcagcgtctgggacccccgggcagagggtcaccatctcttgt 
               
               
                   
                 tctggaagcagctccaacatcggaagtaatactgtaaactggtaccagcaggtcccaggaacgg 
               
               
                   
                 cccccaaactcctcatctatagtaataatcagcggccctcaggggtccctgaccgattctctggctc 
               
               
                   
                 caagtctggcacctcagcctccctggccatcagtgggctccagtctgaggatgaggctgattattac 
               
               
                   
                 tgtgcagcatgggatgacagcctgaatggttgggtgttcggcggagggaccaagctgaccgtcct 
               
               
                   
                 aggt (SEQ ID NO: 53) 
               
               
                   
               
               
                 scFv 
                 QAVVTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQVPGTAPK 
               
               
                   
                 LLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWD 
               
               
                   
                 DSLNGWVFGGGTKLTVLG SRGGGGSGGGGSGGGGSLEMA QMQLV 
               
               
                   
                 QSGAEVKEPGESLRISCKGSGYSFTNFWISWVRQMPGKGLEWMGR 
               
               
                   
                 VDPGYSYSTYSPSFQGHVTISADKSTSTAYLQWNSLKASDTAMYYCA 
               
               
                   
                 RVQYSGYYDWFDPWGQGTLVTVSS (SEQ ID NO: 54) 
               
               
                   
               
               
                 DNA 
                 caggctgtggtgactcagccaccctcagcgtctgggacccccgggcagagggtcaccatctcttgt 
               
               
                   
                 tctggaagcagctccaacatcggaagtaatactgtaaactggtaccagcaggtcccaggaacgg 
               
               
                   
                 cccccaaactcctcatctatagtaataatcagcggccctcaggggtccctgaccgattctctggctc 
               
               
                   
                 caagtctggcacctcagcctccctggccatcagtgggctccagtctgaggatgaggctgattattac 
               
               
                   
                 tgtgcagcatgggatgacagcctgaatggttgggtgttcggcggagggaccaagctgaccgtcct 
               
               
                   
                 aggttctagaggtggtggtggtagcggcggcggcggctctggtggtggtggatccctcgagatgg 
               
               
                   
                 cccagatgcagctggtgcagtccggagcagaggtgaaagagcccggggagtctctgaggatct 
               
               
                   
                 cctgtaagggttctggatacagcttcaccaacttctggatcagctgggtgcgccagatgcccggga 
               
               
                   
                 aaggcctggagtggatggggagggttgatcctggctactcttatagcacctacagcccgtccttcca 
               
               
                   
                 aggccacgtcaccatctcagctgacaagtctaccagcactgcctacctgcagtggaacagcctga 
               
               
                   
                 aggcctcggacaccgccatgtattactgtgcgagagtacaatatagtggctactatgactggttcga 
               
               
                   
                 cccctggggccagggaaccctggtcaccgtctcctca (SEQ ID NO: 55) 
               
               
                   
               
            
           
         
       
     
     In another embodiment, the antibody or antigen binding protein is an anti-WT1 scFv or antigen binding fragment thereof that has an antigen binding region that comprises the amino acid sequence of SEQ ID NO: 72 and specifically binds to a peptide with the amino acid sequence RMFPNAPYL (SEQ ID NO:1) in conjunction with HLA-A0201. In other embodiments, the anti-WT-1 antibody is a scFv-Fc fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 4. 
     
       
         
           
               
               
             
               
                 TABLE 4 
               
               
                   
               
               
                 Antigen 
                 WT1 (Ext002 #15) 
               
               
                 Peptide 
                 RMFPNAPYL (SEQ ID NO: 1) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 CDRs: 
                 1 
                 2 
                 3 
               
               
                   
               
               
                 VH 
                 GYNFSNKWIG 
                 IIYPGYSDITYSPSFQG 
                 HTALAGFDY 
               
               
                   
                 (SEQ ID NO: 56) 
                 (SEQ ID NO: 57) 
                 (SEQ ID NO: 58) 
               
               
                   
               
               
                 DNA 
                 ggctacaactttagcaaca 
                 atcatctatcccggttactcgga 
                 cacacagctttggccggctttg 
               
               
                   
                 agtggatcggc 
                 catcacctacagcccgtccttc 
                 actac (SEQ ID NO: 61) 
               
               
                   
                 (SEQ ID NO: 59) 
                 caaggc (SEQ ID NO: 60) 
                   
               
               
                   
               
               
                 VL 
                 RASQNINKWLA 
                 KASSLES 
                 QQYNSYAT 
               
               
                   
                 (SEQ ID NO: 62) 
                 (SEQ ID NO: 63) 
                 (SEQ ID NO: 64) 
               
               
                   
               
               
                 DNA 
                 Cgggccagtcagaatatc 
                 aaggcgtctagtttagaaagt 
                 caacaatataatagttatgcga 
               
               
                   
                 aataagtggctggcc 
                 (SEQ ID NO: 66) 
                 cg (SEQ ID NO: 67) 
               
               
                   
                 (SEQ ID NO: 65) 
                   
                   
               
               
                   
               
            
           
           
               
               
            
               
                 Full 
                 QVQLVQSGAEVKKPGESLKISCKGSGYNFSNKWIGWVRQLPGRGLE 
               
               
                 VH 
                 WIAIIYPGYSDITYSPSFQGRVTISADTSINTAYLHWHSLKASDTAMYYC 
               
               
                   
                 VRHTALAGFDYWGLGTLVTVSS (SEQ ID NO: 68) 
               
               
                   
               
               
                 DNA 
                 caggtgcagctggtgcagtctggagcagaggtgaaaaagcccggagagtctctgaagatctcctg 
               
               
                   
                 taagggttctggctacaactttagcaacaagtggatcggctgggtgcgccaattgcccgggagagg 
               
               
                   
                 cctggagtggatagcaatcatctatcccggttactcggacatcacctacagcccgtccttccaaggc 
               
               
                   
                 cgcgtcaccatctccgccgacacgtccattaacaccgcctacctgcactggcacagcctgaaggc 
               
               
                   
                 ctcggacaccgccatgtattattgtgtgcgacacacagctttggccggctttgactactggggcctgg 
               
               
                   
                 gcaccctggtcaccgtctcctca (SEQ ID NO: 69) 
               
               
                   
               
               
                 Full 
                 DIQMTQSPSTLSASVGDRVTITCRASQNINKWLAWYQQRPGKAPQLLI 
               
               
                 VL 
                 YKASSLESGVPSRFSGSGSGTEYTLTISSLQPDDFATYYCQQYNSYAT 
               
               
                   
                 FGQGTKVEIKR (SEQ ID NO: 70) 
               
               
                   
               
               
                 DNA 
                 gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcacaatcacttg 
               
               
                   
                 ccgggccagtcagaatatcaataagtggctggcctggtatcagcagagaccagggaaagcccct 
               
               
                   
                 cagctcctgatctataaggcgtctagtttagaaagtggggtcccatctaggttcagcggcagtggatc 
               
               
                   
                 tgggacagaatacactctcaccatcagcagcctgcagcctgatgattttgcaacttattactgccaac 
               
               
                   
                 aatataatagttatgcgacgttcggccaagggaccaaggtggaaatcaaacgt (SEQ ID NO: 71) 
               
               
                   
               
               
                 scFv 
                 DIQMTQSPSTLSASVGDRVTITCRASQNINKWLAWYQQRPGKAPQLLI 
               
               
                   
                 YKASSLESGVPSRFSGSGSGTEYTLTISSLQPDDFATYYCQQYNSYAT 
               
               
                   
                 FGQGTKVEIKR SRGGGGSGGGGSGGGGSLEMA QVQLVQSGAEVKK 
               
               
                   
                 PGESLKISCKGSGYNFSNKWIGWVRQLPGRGLEWIAI IYPGYSDITYSP 
               
               
                   
                 SFQGRVTISADTSINTAYLHWHSLKASDTAMYYCVRHTALAG FDYWGL 
               
               
                   
                 GTLVTVSS (SEQ ID NO: 72) 
               
               
                   
               
               
                 DNA 
                 gacatccagatgacccagtctccttccaccctgtctgcatctgtaggagacagagtcacaatcacttg 
               
               
                   
                 ccgggccagtcagaatatcaataagtggctggcctggtatcagcagagaccagggaaagcccct 
               
               
                   
                 cagctcctgatctataaggcgtctagtttagaaagtggggtcccatctaggttcagcggcagtggatc 
               
               
                   
                 tgggacagaatacactctcaccatcagcagcctgcagcctgatgattttgcaacttattactgccaac 
               
               
                   
                 aatataatagttatgcgacgttcggccaagggaccaaggtggaaatcaaacgt tctagaggtggt   
               
               
                   
                   ggtggtagcggcggcggcggctctggtggtggtggatccctcgagatggcc caggtgcag 
               
               
                   
                 ctggtgcagtctggagcagaggtgaaaaagcccggagagtctctgaagatctcctgtaagggttct 
               
               
                   
                 ggctacaactttagcaacaagtggatcggctgggtgcgccaattgcccgggagaggcctggagtg 
               
               
                   
                 gatagcaatcatctatcccggttactcggacatcacctacagcccgtccttccaaggccgcgtcacc 
               
               
                   
                 atctccgccgacacgtccattaacaccgcctacctgcactggcacagcctgaaggcctcggacac 
               
               
                   
                 cgccatgtattattgtgtgcgacacacagctttggccggctttgactactggggcctgggcaccctggt 
               
               
                   
                 caccgtctcctca (SEQ ID NO: 73) 
               
               
                   
               
            
           
         
       
     
     In another embodiment, the antibody or antigen binding protein is an anti-WT1 scFv or antigen binding fragment thereof that has an antigen binding region that comprises the amino acid sequence of SEQ ID NO: 90 and specifically binds to a peptide with the amino acid sequence RMFPNAPYL (SEQ ID NO: 1) in conjunction with HLA-A0201. In other embodiments, the anti-WT-1 antibody is a scFv-Fc fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 5. 
     
       
         
           
               
               
             
               
                 TABLE 5 
               
               
                   
               
               
                 Antigen 
                 WT1 (Ext002 #18) 
               
               
                 Peptide 
                 RMFPNAPYL (SEQ ID NO: 1) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 CDRs: 
                 1 
                 2 
                 3 
               
               
                   
               
               
                 VH 
                 GFTFDDYGMS 
                 GINWNGGSTGYADS 
                 ERGYGYHDPHDY 
               
               
                   
                 (SEQ ID NO: 74) 
                 VRG (SEQ ID NO: 75) 
                 (SEQ ID NO: 76) 
               
               
                   
               
               
                 DNA 
                 gggttcacctttgatgattat 
                 ggtattaattggaatggtggt 
                 gagcgtggctacgggtacca 
               
               
                   
                 ggcatgagc 
                 agcacaggttatgcagactc 
                 tgatccccatgactac 
               
               
                   
                 (SEQ ID NO: 77) 
                 tgtgaggggc (SEQ ID 
                 (SEQ ID NO: 79) 
               
               
                   
                   
                 NO: 78) 
                   
               
               
                   
               
               
                 VL 
                 GRNNIGSKSVH 
                 DDSDRPS 
                 QVWDSSSDHVV 
               
               
                   
                 (SEQ ID NO: 80) 
                 (SEQ ID NO: 81) 
                 (SEQ ID NO: 82) 
               
               
                   
               
               
                 DNA 
                 gggagaaacaacattgg 
                 gatgatagcgaccggccctc 
                 caggtgtgggatagtagtagt 
               
               
                   
                 aagtaaaagtgtgcac 
                 a (SEQ ID NO: 84) 
                 gatcatgtggta 
               
               
                   
                 (SEQ ID NO: 83) 
                   
                 (SEQ ID NO: 85) 
               
               
                   
               
            
           
           
               
               
            
               
                 Full 
                 EVQLVQSGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKG 
               
               
                 VH 
                 LEWVSGINWNGGSTGYADSVRGRFTISRDNAKNSLYLQMNSLRAE 
               
               
                   
                 DTALYYCARERGYGYHDPHDYWGQGTLVTVSS (SEQ ID NO: 86) 
               
               
                   
               
               
                 DNA 
                 gaagtgcagctggtgcagtctgggggaggtgtggtacggcctggggggtccctgagactctcct 
               
               
                   
                 gtgcagcctctgggttcacctttgatgattatggcatgagctgggtccgccaagctccagggaag 
               
               
                   
                 gggctggagtgggtctctggtattaattggaatggtggtagcacaggttatgcagactctgtgagg 
               
               
                   
                 ggccgattcaccatctccagagacaacgccaagaactccctgtatctgcaaatgaacagtctg 
               
               
                   
                 agagccgaggacacggccttgtattactgtgcgagagagcgtggctacgggtaccatgatccc 
               
               
                   
                 catgactactggggccaaggcaccctggtgaccgtctcctca (SEQ ID NO: 87) 
               
               
                   
               
               
                 Full 
                 QSVVTQPPSVSVAPGKTARITCGRNNIGSKSVHWYQQKPGQAPVL 
               
               
                 VL 
                 VVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVW 
               
               
                   
                 DSSSDHVVFGGGTKLTVLG (SEQ ID NO: 88) 
               
               
                   
               
               
                 DNA 
                 cagtctgtcgtgacgcagccgccctcggtgtcagtggccccaggaaagacggccaggattac 
               
               
                   
                 ctgtgggagaaacaacattggaagtaaaagtgtgcactggtaccagcagaagccaggccag 
               
               
                   
                 gcccctgtgctggtcgtctatgatgatagcgaccggccctcagggatccctgagcgattctctgg 
               
               
                   
                 ctccaactctgggaacacggccaccctgaccatcagcagggtcgaagccggggatgaggcc 
               
               
                   
                 gactattactgtcaggtgtgggatagtagtagtgatcatgtggtattcggcggagggaccaagct 
               
               
                   
                 gaccgtcctaggt (SEQ ID NO: 89) 
               
               
                   
               
               
                 scFv 
                 QSVVTQPPSVSVAPGKTARITCGRNNIGSKSVHWYQQKPGQAPVL 
               
               
                   
                 VVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVW 
               
               
                   
                 DSSSDHVVFGGGTKLTVLG SRGGGGSGGGGSGGSLEMA EVQLVQ 
               
               
                   
                 SGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVS 
               
               
                   
                 GINWNGGSTGYADSVRGRFTISRDNAKNSLYLQMNSLRAEDTALYY 
               
               
                   
                 CARERGYGYHDPHDYWGQGTLVTVSS (SEQ ID NO: 90) 
               
               
                   
               
               
                 DNA 
                 cagtctgtcgtgacgcagccgccctcggtgtcagtggccccaggaaagacggccaggattac 
               
               
                   
                 ctgtgggagaaacaacattggaagtaaaagtgtgcactggtaccagcagaagccaggccag 
               
               
                   
                 gcccctgtgctggtcgtctatgatgatagcgaccggccctcagggatccctgagcgattctctgg 
               
               
                   
                 ctccaactctgggaacacggccaccctgaccatcagcagggtcgaagccggggatgaggcc 
               
               
                   
                 gactattactgtcaggtgtgggatagtagtagtgatcatgtggtattcggcggagggaccaagct 
               
               
                   
                 gaccgtcctaggt tctagaggtggtggtggtagcggcggcggcggctctggtggatccc   
               
               
                   
                   tcgagatggcc gaagtgcagctggtgcagtctgggggaggtgtggtacggcctggggggtcc 
               
               
                   
                 ctgagactctcctgtgcagcctctgggttcacctttgatgattatggcatgagctgggtccgccaag 
               
               
                   
                 ctccagggaaggggctggagtgggtctctggtattaattggaatggtggtagcacaggttatgca 
               
               
                   
                 gactctgtgaggggccgattcaccatctccagagacaacgccaagaactccctgtatctgcaa 
               
               
                   
                 atgaacagtctgagagccgaggacacggccttgtattactgtgcgagagagcgtggctacggg 
               
               
                   
                 taccatgatccccatgactactggggccaaggcaccctggtgaccgtctcctca (SEQ ID 
               
               
                   
                 NO: 91) 
               
               
                   
               
            
           
         
       
     
     In another embodiment, the antibody or antigen binding protein is an anti-WT1 scFv or antigen binding fragment thereof that has an antigen binding region that comprises the amino acid sequence of SEQ ID NO: 108 and specifically binds to a peptide with the amino acid sequence RMFPNAPYL (SEQ ID NO: 1) in conjunction with HLA-A0201. In other embodiments, the anti-WT-1 antibody is a scFv-Fc fusion protein or full length human IgG with VH and VL regions or CDRs selected from Table 6. 
     
       
         
           
               
               
             
               
                 TABLE 6 
               
               
                   
               
               
                 Antigen 
                 WT1 (Ext002 #23) 
               
               
                 Peptide 
                 RMFPNAPYL (SEQ ID NO. 1) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
            
               
                 CDRs: 
                 1 
                 2 
                 3 
               
               
                   
               
               
                 VH 
                 GFSVSGTYMG 
                 LLYSGGGTYHPASLQ 
                 GGAGGGHFDS 
               
               
                   
                 (SEQ ID NO. 92) 
                 G (SEQ ID NO. 93) 
                 (SEQ ID NO. 94) 
               
               
                   
               
               
                 DNA 
                 gggttctccgtcagtggcac 
                 cttctttatagtggtggcggcac 
                 gaggggcaggaggtggcc 
               
               
                   
                 ctacatgggc (SEQ ID 
                 ataccacccagcgtccctgca 
                 actttgactcc (SEQ ID 
               
               
                   
                 NO. 95) 
                 gggc (SEQ ID NO. 96) 
                 NO. 97) 
               
               
                   
               
               
                 VL 
                 TGSSSNIGAGYDVH 
                 GNSNRPS 
                 AAWDDSLNGYV 
               
               
                   
                 (SEQ ID NO. 98) 
                 (SEQ ID NO. 99) 
                 (SEQ ID NO. 100) 
               
               
                   
               
               
                 DNA 
                 actgggagcagctccaac 
                 ggtaacagcaatcggccctca 
                 gcagcatgggatgacagcct 
               
               
                   
                 atcggggcaggttatgatgt 
                 (SEQ ID NO. 102) 
                 gaatggttatgtc 
               
               
                   
                 acac (SEQ ID NO. 101) 
                   
                 (SEQ ID NO. 103) 
               
               
                   
               
            
           
           
               
               
            
               
                 Full 
                 EVQLVETGGGLLQPGGSLRLSCAASGFSVSGTYMGWVRQAPGKGLE 
               
               
                 VH 
                 WVALLYSGGGTYHPASLQGRFIVSRDSSKNMVYLQMNSLKAEDTAVY 
               
               
                   
                 YCAKGGAGGGHFDSWGQGTLVTVSS (SEQ ID NO. 104) 
               
               
                   
               
               
                 DNA 
                 gaggtgcagctggtggagaccggaggaggcttgctccagccgggggggtccctcagactctcctg 
               
               
                   
                 tgcagcctctgggttctccgtcagtggcacctacatgggctgggtccgccaggctccagggaaggg 
               
               
                   
                 actggagtgggtcgcacttctttatagtggtggcggcacataccacccagcgtccctgcagggccg 
               
               
                   
                 attcatcgtctccagagacagctccaagaatatggtctatcttcaaatgaatagcctgaaagccgag 
               
               
                   
                 gacacggccgtctattactgtgcgaaaggaggggcaggaggtggccactttgactcctggggcca 
               
               
                   
                 aggcaccctggtgaccgtctcctca (SEQ ID NO. 105) 
               
               
                   
               
               
                 Full 
                 QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPK 
               
               
                 VL 
                 LLIYGNSNRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWD 
               
               
                   
                 DSLNGYVFGTGTKLTVLG (SEQ ID NO. 106) 
               
               
                   
               
               
                 DNA 
                 cagtctgtgttgacgcagccgccctcagtgtctggggccccagggcagagggtcaccatctcctgc 
               
               
                   
                 actgggagcagctccaacatcggggcaggttatgatgtacactggtaccagcagcttccaggaac 
               
               
                   
                 agcccccaaactcctcatctatggtaacagcaatcggccctcaggggtccctgaccgattctctggc 
               
               
                   
                 tccaagtctggcacctcagcctccctggccatcagtgggctccagtctgaggatgaggctgattatta 
               
               
                   
                 ctgtgcagcatgggatgacagcctgaatggttatgtcttcggaactgggaccaagctgaccgtccta 
               
               
                   
                 ggt (SEQ ID NO. 107) 
               
               
                   
               
               
                 scFv 
                 QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPK 
               
               
                   
                 LLIYGNSNRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWD 
               
               
                   
                 DSLNGYVFGTGTKLTVLG SRGGGGSGGGGSGGGGSLEMA EVQLVETG 
               
               
                   
                 GGLLQPGGSLRLSCAASGFSVSGTYMGWVRQAPGKGLEWVALLYSGG 
               
               
                   
                 GTYHPASLQGRFIVSRDSSKNMVYLQMNSLKAEDTAVYYCAKGGAGG 
               
               
                   
                 GHFDSWGQGTLVTVSS (SEQ ID NO. 108) 
               
               
                   
               
               
                 DNA 
                 cagtctgtgttgacgcagccgccctcagtgtctggggccccagggcagagggtcaccatctcctgc 
               
               
                   
                 actgggagcagctccaacatcggggcaggttatgatgtacactggtaccagcagcttccaggaac 
               
               
                   
                 agcccccaaactcctcatctatggtaacagcaatcggccctcaggggtccctgaccgattctctggc 
               
               
                   
                 tccaagtctggcacctcagcctccctggccatcagtgggctccagtctgaggatgaggctgattatta 
               
               
                   
                 ctgtgcagcatgggatgacagcctgaatggttatgtcttcggaactgggaccaagctgaccgtccta 
               
               
                   
                 ggt tctagaggtggtggtggtagcggcggcggcggctctggtggtggtggatccctcgag   
               
               
                   
                   atggcc gaggtgcagctggtggagaccggaggaggcttgctccagccgggggggtccctcaga 
               
               
                   
                 ctctcctgtgcagcctctgggttctccgtcagtggcacctacatgggctgggtccgccaggctccagg 
               
               
                   
                 gaagggactggagtgggtcgcacttctttatagtggtggcggcacataccacccagcgtccctgca 
               
               
                   
                 gggccgattcatcgtctccagagacagctccaagaatatggtctatcttcaaatgaatagcctgaaa 
               
               
                   
                 gccgaggacacggccgtctattactgtgcgaaaggaggggcaggaggtggccactttgactcctg 
               
               
                   
                 gggccaaggcaccctggtgaccgtctcctca (SEQ ID NO. 109) 
               
               
                   
               
            
           
         
       
     
     Other embodiments of the disclosed antibodies and antigen binding proteins encompass those comprising light and heavy hypervariable regions and constant regions, for example as shown in Tables 7 (heavy chain), 8 (light chain) and 9 (constant regions). 
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 7 
               
               
                   
               
               
                   
                 CDR-H1 
                 CDR-H2 
                 CDR-H3 
                 SEQ ID NO. 
               
               
                   
               
             
            
               
                 Group I 
                   
                   
                   
                   
               
               
                 EXT002-12(166) 
                 SNAVAWN 
                 RTYRGSTYY---ALSV 
                 G-SNSAFDF 
                 119-121 
               
               
                   
               
               
                 EXT002-5(184) 
                 SNSAAWN 
                 RTYYGSKWYNDYAVSV 
                 GRLGDAFDI 
                 122-124 
               
               
                   
               
               
                 EXT002-8(184) 
                 SDGAAWN 
                 RTYYRSKWYNDYAVSV 
                 GDYYYGMDV 
                 125-127 
               
               
                   
               
               
                 Consensus(191) 
                 SNAAAWN 
                 RTYYGSKWYNDYAVSV 
                 GAFDI 
                 128-130 
               
               
                   
               
               
                 Group II 
                   
                   
                   
                   
               
               
                 EXT002-14(163) 
                 SYWIS 
                 RIDPSDSYTNYSPSFQG 
                 GD------YDFYLDP-- 
                 131-133 
               
               
                   
               
               
                 EXT002-25(163) 
                 SYGIS 
                 WISAYNGNTNYAQKLQG 
                 DLYSSGWYESYYYGMDV 
                 134-136 
               
               
                   
               
               
                 EXT002-3(186) 
                 SYAIS 
                 GIIPIFGTANYAQKFQG 
                 RIP-P------YYGMDV 
                 137-139 
               
               
                   
               
               
                 EXT002-30(163) 
                 SYGIS 
                 WISAHNGNTNYAQKLQG 
                 DR-------VWFGDLSD 
                 134, 140, 141 
               
               
                   
               
               
                 EXT002-33(163) 
                 SYAIS 
                 GIIPIFGTANYAQKEQG 
                 NYDFWSG-----DAFDI 
                 137, 142, 143 
               
               
                   
               
               
                 Consensus(188) 
                 SYAIS 
                 IPGTNYAQKFQG 
                 FYGMDV 
                 137, 144, 145 
               
               
                   
               
               
                 Group III 
                   
                   
                   
                   
               
               
                 EXT002-34(161) 
                 DYGMS 
                 GINWNGGSTGYADSV 
                 ERGY-GYHDPHDY 
                 146-148 
               
               
                   
               
               
                 EXT002-40(157) 
                 NYTMN 
                 SISLSGAYIYYADSL 
                 EGYSSSVYDAFDL 
                 149-151 
               
               
                   
               
               
                 EXT002-45(165) 
                 SYGMH 
                 GILSDGGKDYYVDSV 
                 CSSN-YGNDAFDI 
                 152-154 
               
               
                   
               
               
                 EXT002-48(165) 
                 TYSMN 
                 SISSGAYSIFYADSV 
                 DQYYGDKWDAFDI 
                 155-157 
               
               
                   
               
               
                 Consensus(170) 
                 SYGMN 
                 SISSGGSIYYADSV 
                 EYYWDAFDI 
                 158-160 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
               
               
             
               
                 TABLE 8 
               
               
                   
               
               
                   
                 CDR-L1 
                 CDR-L2 
                 CDR-L3 
                 SEQ ID NOS. 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Group I 
                   
                   
                   
                   
               
               
                 EXT002-1(46) 
                 CSGSSSNIGS-NTVN 
                 SNNQRPSG 
                 AAWDDSLNG--WVFG 
                 161-163 
               
               
                   
               
               
                 EXT002-10(46) 
                 CSGSSSNIGS-NTVN 
                 SNNQRPSG 
                 EAWDDSLKG--PVFG 
                 161, 162, 164 
               
               
                   
               
               
                 EXT002-12(22) 
                 CTGSSSNIGAGYDVH 
                 GNSNRPSG 
                 QSYDSSLSADNYVFG 
                 165-167 
               
               
                   
               
               
                 EXT002-13(46) 
                 CSGSSSNIGS-NTVN 
                 SNNQRPSG 
                 AAWDDSLNG--WVFG 
                 161-163 
               
               
                   
               
               
                 EXT002-2(46) 
                 CSGSSSNIGR-NIVN 
                 SNIERPSG 
                 ASWDDSLNG--VLFG 
                 168-170 
               
               
                   
               
               
                 EXT002-20(46) 
                 CSGSRSNIAS-NGVG 
                 KNDQRPSG 
                 SAWDDSLDGH-VVFG 
                 171-173 
               
               
                   
               
               
                 EXT002-23(46) 
                 CTGSSSNIGAGYDVH 
                 GNSNRPSG 
                 AAWDDSLNG--YVFG 
                 165, 166, 174 
               
               
                   
               
               
                 EXT002-25(22) 
                 CSGSSSNIGS-STVN 
                 SNSQRPSG 
                 AAWDDSLNG--VVFG 
                 175-177 
               
               
                   
               
               
                 EXT002-3(46) 
                 CSGSSSNIGS-NYVY 
                 RSNQRPSG 
                 AAWDDSLNG--VVFG 
                 178, 179, 177 
               
               
                   
               
               
                 EXT002-30(22) 
                 CSGSSSNIGR-NTVN 
                 SNNQRPSG 
                 AAWDDSLNG--YVFG 
                 180, 162, 174 
               
               
                   
               
               
                 EXT002-33(22) 
                 CSGSSSNIGN-DYVS 
                 DNNKRPSG 
                 GTWDNSLSA--WVFG 
                 181-183 
               
               
                   
               
               
                 EXT002-36(22) 
                 CSGSSSNIGS-NSVY 
                 NNNQRPSG 
                 ATWDDSLSG--WVFG 
                 184-186 
               
               
                   
               
               
                 EXT002-40(22) 
                 CSGSSSNIGS-NYVY 
                 RNNQRPSG 
                 AAWDDSLSA--WVFG 
                 178, 187, 188 
               
               
                   
               
               
                 EXT002-42(46) 
                 CSGSTSNIGS-YYVS 
                 DNNNRPSG 
                 GTWDSSLSA--WVFG 
                 189-191 
               
               
                   
               
               
                 EXT002-45(22) 
                 CSGSSSNIGN-NYVS 
                 DNNKRPSG 
                 GTWDSSLSA--WVFG 
                 192, 182, 191 
               
               
                   
               
               
                 EXT002-48(22) 
                 CSGSNSNIGT-NTVT 
                 SNFERPSG 
                 SAWDDSFNG--PVFG 
                 193-195 
               
               
                   
               
               
                 EXT002-6(46) 
                 CSGSSSNIGS-NYVS 
                 RNNQRPSG 
                 AAWDDGLRG--YVFG 
                 196, 187, 197 
               
               
                   
               
               
                 EXT002-9(22) 
                 CSGSSSNIGS-NTVN 
                 SNNQRPSG 
                 EAWDDSLKG--PVFG 
                 161, 162, 164 
               
               
                   
               
               
                 Consensus(46) 
                 CSGSSSNIGSNV 
                 NNQRPSG 
                 AAWDDSLGWVFG 
                 161-163 
               
               
                   
               
               
                 Group II 
                   
                   
                   
                   
               
               
                 EXT002-24(24) 
                 RASQSISSYLN 
                 AASSLQS 
                 QQSYSTP--T 
                 198-200 
               
               
                   
               
               
                 EXT002-31(24) 
                 RASQGISNYLA 
                 AASTLQS 
                 QKYNSAPGVT 
                 201-203 
               
               
                   
               
               
                 EXT002-35(24) 
                 RASQSINGWLA 
                 RASTLQS 
                 QQSSSLP-FT 
                 204-206 
               
               
                   
               
               
                 EXT002-5(48) 
                 RASQSISSYLN 
                 AASSLQS 
                 QQSYSTP-LT 
                 198-200 
               
               
                   
               
               
                 EXT002-7(48) 
                 RASQGISYYLA 
                 AASTLKS 
                 QQLNSYP-LT 
                 207-209 
               
               
                   
               
               
                 EXT002-B(48) 
                 RASQSISSYLN 
                 AASSLQS 
                 QQSYSTP-WT 
                 198-200 
               
               
                   
               
               
                 Consensus(48) 
                 RASQSISSYLN 
                 AASSLQS 
                 QQSYSTPLT 
                 198-200 
               
               
                   
               
               
                 Group III 
                   
                   
                   
                   
               
               
                 EXT002-16(23) 
                 GGNNIGSKSVH 
                 DDSDRPS 
                 QVWDSSSDHPV 
                 210-212 
               
               
                   
               
               
                 EXT002-17(47) 
                 GGNNIGSKSVH 
                 DDSDRPS 
                 QVWDSSGDHPV 
                 210, 211, 213 
               
               
                   
               
               
                 EXT002-19(47) 
                 GGNNIGSKSVH 
                 YDSDRPS 
                 QVWDSSSDHPV 
                 210, 214, 212 
               
               
                   
               
               
                 EXT002-21(19) 
                 GGTNIGSRFVH 
                 DDSDRPS 
                 QVWDSSGDHPV 
                 215, 211, 213 
               
               
                   
               
               
                 EXT002-22(47) 
                 GGNNVESKSVH 
                 YDRDRPS 
                 EVWDSGSDHPV 
                 216-218 
               
               
                   
               
               
                 EXT002-32(23) 
                 GGKNIGSKSVH 
                 YDSDRPS 
                 QVWDSGSDHYV 
                 219, 214, 220 
               
               
                   
               
               
                 EXT002-34(23) 
                 GGNNIGSKSVH 
                 DDSDRPS 
                 QVWISSGDRVI 
                 210, 211, 221 
               
               
                   
               
               
                 EXT002-43(23) 
                 GGDNIGSQGVH 
                 YDTDRPS 
                 QVWGASSDHPV 
                 222-224 
               
               
                   
               
               
                 Consensus(47) 
                 GGNNIGSKSVH 
                 YDSDRPS 
                 QVWDSSSDHPV 
                 210, 214, 212 
               
               
                   
               
               
                 Group IV 
                   
                   
                   
                   
               
               
                 EXT002-11(47) 
                 TGTSSDVGGYNYVS 
                 DVSKRPS 
                 GIYTYSDSW--V 
                 225-227 
               
               
                   
               
               
                 EXT002-14(23) 
                 TGTSSDVGGYNYVS 
                 DVGNRPS 
                 SSYTSSSTR--V 
                 225, 228, 229 
               
               
                   
               
               
                 EXT002-26(23) 
                 TGTRSDVGLYNYVA 
                 DVIYRPG 
                 SSYTNIGTV--L 
                 230-232 
               
               
                   
               
               
                 EXT002-4(47) 
                 TGTSSDFGDYDYVS 
                 DVSDRPS 
                 QSYDSSLSGSGV 
                 233-235 
               
               
                   
               
               
                 Consensus(47) 
                 TGTSSDVGGYNYVS 
                 DVSRPS 
                 SSYTSSSV 
                 225, 234, 229 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 9 
               
               
                   
               
               
                 Constant Regions 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                 Human heavy chain 
                 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL 
               
               
                 constant region 
                 TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT 
               
               
                 and IgG1 Fc domain 
                 KVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR 
               
               
                 sequence 
                 TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR 
               
               
                   
                 VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ 
               
               
                   
                 VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT 
               
               
                   
                 TPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK 
               
               
                   
                 SLSLSPGK (SEQ ID NO. 236) 
               
               
                   
               
               
                 Human light chain 
                 TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL 
               
               
                 (kappa) 
                 QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG 
               
               
                   
                 LSSPVTKSFNRGEC (SEQ ID NO. 237) 
               
               
                   
               
               
                 Human light chain 
                 QPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGS 
               
               
                 (lambda) 
                 PVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEG 
               
               
                   
                 STVEKTVAPTECS (SEQ ID NO. 238) 
               
               
                   
               
            
           
         
       
     
     The invention relates to recombinant antigen-binding proteins, antibodies and antigen binding fragments thereof that specifically recognize epitopes of a complex of a peptide/protein fragment derived from an intracellular protein, and an MHC class I molecule, for example, as the complex might be displayed at the cell surface during antigen presentation. The heavy and light chains of an antibody of the invention may be full-length (e.g., an antibody can include at least one, and preferably two, complete heavy chains, and at least one, and preferably two, complete light chains) or may include an antigen-binding portion (a Fab, F(ab′) 2 , Fv or a single chain Fv fragment (“scFv”)). In other embodiments, the antibody heavy chain constant region is chosen from, e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE, particularly chosen from, e.g., IgG1, IgG2, IgG3, and IgG4, more particularly, IgG1 (e.g., human IgG1). In another embodiment, the antibody light chain constant region is chosen from, e.g., kappa or lambda, particularly kappa. 
     The antibodies and antigen binding proteins of the present invention are intended to encompass bispecific antibodies, including bispecific T-cell engaging antibodies, that is, antibodies comprising two antibody variable domains on a single polypeptide chain that are able to bind two separate antigens. Where a first portion of a bispecific antibody binds an antigen on a tumor cell for example and a second portion of a bispecific antibody recognizes an antigen on the surface of a human immune effector cell, the antibody is capable of recruiting the activity of that effector cell by specifically binding to the effector antigen on the human immune effector cell. In some instances, bispecific antibodies, therefore, are able to form a link between effector cells, for example, T cells and tumor cells, thereby enhancing effector function. 
     In one embodiment, the constant region/framework region is altered, for example, by amino acid substitution, to modify the properties of the antibody (e.g., to increase or decrease one or more of: antigen binding affinity, Fc receptor binding, antibody carbohydrate, for example, glycosylation, fucosylation etc., the number of cysteine residues, effector cell function, effector cell function, complement function or introduction of a conjugation site). Furthermore, conjugation of the antibody to a drug, toxin, radioisotope, cytokine, inflammatory peptide or cytotoxic agent is also contemplated. 
     In one embodiment, the antibody is an anti-WT1/A2 antibody and comprises the human IgG1 constant region and Fc domain shown in Table 9. In one embodiment, the anti-WT1/A2 antibody comprises a human kappa sequence, or a human lambda sequence having the sequence set forth in Table 9. The amino acid sequences for some complementarity determining regions (CDRs) of antibodies of the invention are shown in Tables 1-8. 
     The present invention is based on the identification of antigen-specific binding sequences from which a variety of antigen-binding proteins can be produced. In addition to antibodies specific for an antigen that represents a protein fragment (peptide)/HLA complex similar to that typically recognized by a T-cell receptor following antigen processing and presentation of the protein to the T-cell, identification of amino acid and nucleic sequences as disclosed herein for the preparation of antibodies can also be used to generate other antigen-binding molecules including chimeric antigen receptors (CARs), with specificity for the protein fragment (peptide)/HLA complex. These can be incorporated into cells to make them specifically ctyotoxic to the antigen expressing cell. 
     The present invention employs a novel approach to obtaining therapeutic antibodies to any protein, including those proteins that are inaccessible because they are not expressed on the cell surface. Nearly any intracytoplasmic or intranuclear protein (in addition to cell surface proteins) is a potential target for the approach described herein. This includes, but is not limited to, oncogenic proteins, transcription factors, enzymes, etc. 
     In order to target tumor antigens derived from intracellular or nuclear proteins, development of a therapeutic antibody an uncommon approach was required. This approach is to generate recombinant mAbs that recognize the peptide/MHC complex expressed on the cell surface, with the same specificity as a T-cell receptor (TCR). Such mAbs share functional homology with TCRs regarding target recognition, but confer higher affinity and capabilities of arming with potent cytotoxic agents that antibodies feature. Technically, TCR-like mAbs may be generated by conventional hybridoma techniques known to those of skill in the art, to produce human, humanized or chimeric antibodies. 
     Furthermore, fully-human mAbs are preferred for therapeutic use in humans because murine antibodies cause an immunogenicity reaction, known as the HAMA (human anti-mouse antibodies) response (24, 25), when administered to humans, causing serious side effects, including anaphylaxis and hypersensitivity reactions. This immunogenicity reaction is triggered by the human immune system recognizing the murine antibodies as foreign because of slightly different amino acid sequences from natural human antibodies. Humanization methods known in the art (26, 27) can be employed to reduce the immunogenicity of murine-derived antibodies (28). 
     Recently, the use of phage display libraries has made it possible to select large numbers of Ab repertoires for unique and rare Abs against very defined epitopes (for more details on phage display see McCafferty et al., Phage antibodies: filamentous phage displaying antibody variable domains. Nature, 348: 552-554.) The rapid identification of human Fab or single chain Fv (ScFV) fragments highly specific for tumor antigen-derived peptide-MHC complex molecules has thus become possible (19-22). More recently, immuno-toxins, generated by fusing TCR-like Fab specific for melanoma Ag MART-1 26-35/A2 or gp100 280-288/A2 to a truncated form of  Pseudomonas  endotoxin, have been shown to inhibit human melanoma growth both in vitro and in vivo (23). In addition, by engineering full-length mAb using the Fab fragments, it is possible to directly generate a therapeutic human mAb, by-passing months of time-consuming work, normally needed for developing therapeutic mAbs. The present invention involves the development of a TCR-like, fully human mAb that recognizes, for example, the WT1 peptide/HLA-A2 complex (RMFPNAPYL, SEQ ID NO: 1) for cancer therapy. 
     Recombinant antibodies with TCR-like specificity represent a new and valuable tool for research and therapeutic applications in tumor immunology and immunotherapy. WT1 is a well-established and validated tumor antigen that has been investigated throughout the world as a marker, prognostic factor and therapeutic target. It was recently prioritized as the top priority tumor antigen by an NCI task force (29). 
     Identification of Peptides with High Predictive Binding to HLA Molecules 
     In one embodiment, the present invention relates to a method for the generation of antibodies that specifically bind to HLA-restricted peptides, which, when presented as part of a peptide/MHC complex are able to elicit a specific cytotoxic T-cell response. HLA class I molecules present endogenous derived peptides of about 8-12 amino acids in length to CD8+ cytotoxic T lymphocytes. Peptides to be used in the method of the present invention are generally about 6-22 amino acids in length, and in some embodiments, between about 9 and 20 amino acids and comprise an amino acid sequence derived from a protein of interest, for example, human WT1 protein (Genbank accession no. P19544) or an analog thereof. 
     Peptides suitable for use in generating antibodies in accordance with the method of the present invention can be determined based on the presence of HLA-A0201-binding motifs and the cleavage sites for proteasomes and immune-proteasomes using computer prediction models known to those of skill in the art. For predicting MHC class I binding sites, such models include, but are not limited to, ProPred1 (described in more detail in Singh and Raghava,  ProPred: prediction of HLA - DR binding sites. BIOINFORMATICS  17(12):1236-1237 2001), and SYFPEITHI (see Schuler et al.  SYFPEITHI, Database for Searching and T - Cell Epitope Prediction. in Immunoinformatics Methods in Molecular Biology , vol 409(1): 75-93 2007) 
     HLA-A*0201 is expressed in 39-46% of all caucasians and therefore, represents a suitable choice of MHC antigen for use in the present method. For preparation of one embodiment of a WT1 peptide antigen, amino acid sequences and predicted binding of putative CD84+ epitopes to HLA-A0201 molecules were identified using the predictive algorithm of the SYFPEITHI database (see Schuler et al.  SYFPEITHI, Database for Searching and T - Cell Epitope Prediction . in  Immunoinformatics Methods in Molecular Biology , vol 409(1): 75-93 2007). 
     Once appropriate peptides have been identified, peptide synthesis may be done in accordance with protocols well known to those of skill in the art. Because of their relatively small size, the peptides of the invention may be directly synthesized in solution or on a solid support in accordance with conventional peptide synthesis techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. The synthesis of peptides in solution phase has become a well-established procedure for large-scale production of synthetic peptides and as such is a suitable alternative method for preparing the peptides of the invention. (See for example,  Solid Phase Peptide Synthesis  by John Morrow Stewart and Martin et al.  Application of Almez - mediated Amidation Reactions to Solution Phase Peptide Synthesis , Tetrahedron Letters Vol. 39, pages 1517-1520 1998.) 
     Each of the peptides used in the protocols described herein was purchased and synthesized by Genemed Synthesis, Inc. (San Antonio, Tex.) using fluorenylmethoxycarbonyl chemistry and solid-phase synthesis and purified by high-pressure liquid chromatography. The quality of the peptides was assessed by high-performance liquid chromatography analysis, and the expected molecular weight was observed using matrix-assisted laser desorption mass spectrometry. Peptides were sterile and 70% to 90% pure. The peptides were dissolved in DMSO and diluted in PBS (pH 7.4) or saline at 5 mg/mL and stored at −80° C. 
     Subsequent to peptide selection, binding activity of selected peptides is tested using the antigen-processing-deficient T2 cell line, which increases expression of HLA-A when stabilized by a peptide in the antigen-presenting groove. Briefly, T2 cells are pulsed with peptide for a time sufficient to induce HLA-A expression. HLA-A expression of T2 cells is then measured by immunostaining with a fluorescently labeled monoclonal antibody specific for HLA-A (for example, BB7.2) and flow cytometry. Fluorescence index (FI) is calculated as the mean fluorescence intensity (MFI) of HLA-A0201 on T2 cells as determined by fluorescence-activated cell-sorting analysis, using the formula FI=(MFI[T2 cells with peptide]/MFI [T2 cells without peptide]-1. 
     Fully human T-cell receptor (TCR)-like antibodies to Wilm&#39;s tumor oncognene protein (WT1) were produced using the method disclosed herein. TCR-like anti-WT1 antibodies generated by phage display technology are specific for a WT1 peptide/HLA complex similar to that which induces HLA-restricted cytotoxic CD8 T-cells. 
     The WT1 protein sequence was screened using the SYFPEITHI algorithm and WT1 peptides (for example, peptides designated 428, 328, and 122) were identified that had predicted high-affinity binding to multiple HLA molecules that are highly expressed in the Caucasian population. Peptide 428 spans WT1 amino acids 428-459, peptide 328 spans WT1 amino acids 328-349, and peptide 122 spans WT1 amino acids 122-140 (see  FIG. 1 ) 
     Heteroclitic peptides can also be designed by conservative amino acid substitutions of MHC-binding residues expected to enhance the affinity toward the MHC class 1 allele, as predicted by the prediction algorithm. WT1 peptide 122 comprises within it a known CD8+ epitope (126-134). In one embodiment, therefore, a modified peptide of the peptide that spans the WT1 amino acid residues 126-134 and contains a modified amino acid at positions may be used. Peptides used for alanine mutagenesis of WT1A (otherwise designated RFM) were named based on the position where the substitution was made. Examples of WT1 peptides which may be used are shown in Table 10 along with irrelevant peptides, RHAMM-R3 and EW. 
     
       
         
           
               
               
               
               
             
               
                   
                 TABLE 10 
               
               
                   
                   
               
             
            
               
                   
                 WT1A(RMF) 
                 RMFPNAPYL 
                 SEQ ID NO.: 1 
               
               
                   
                   
               
               
                   
                 WT1A1-B 
                 AMFPNAPYL 
                 SEQ ID NO.: 110 
               
               
                   
                   
               
               
                   
                 WT1A-3 
                 RMAPNAPYL 
                 SEQ ID NO.: 111 
               
               
                   
                   
               
               
                   
                 WT1A-4 
                 RMFANAPYL 
                 SEQ ID NO.: 112 
               
               
                   
                   
               
               
                   
                 WT1A-5 
                 RMFPAAPYL 
                 SEQ ID NO.: 113 
               
               
                   
                   
               
               
                   
                 WT1A-7 
                 RMFPNAAYL 
                 SEQ ID NO.: 114 
               
               
                   
                   
               
               
                   
                 WT1A-8 
                 RMFPNAPAL 
                 SEQ ID NO.: 115 
               
               
                   
                   
               
               
                   
                 RHAMM-R3 
                 ILSLELMKL 
                 SEQ ID NO.: 116 
               
               
                   
                   
               
               
                   
                 EW 
                 QLQNPSYDK 
                 SEQ ID NO.: 117 
               
               
                   
                   
               
            
           
           
               
               
               
            
               
                   
                 RSDELVRHHNMHQRNMTKL 
                 SEQ ID NO.: 118 
               
               
                   
                   
               
               
                   
                 PGCNKRYFKLSHLQMHSRKHTG 
                 SEQ ID NO.: 119 
               
               
                   
                   
               
               
                   
                 SGQA R MFPNAPYLPSCLES 
                 SEQ ID NO.: 120 
               
               
                   
                   
               
               
                   
                 SGQA Y MFPNAPYLPSCLES 
                 SEQ ID NO.: 121 
               
               
                   
                   
               
            
           
         
       
     
     Once a suitable peptide has been identified, the target antigen to be used for phage display library screening, that is, a peptide/HLA complex (for example, WT1 peptide/HLA-A0201) is prepared by bringing the peptide and the histocompatibility antigen together in solution to form the complex. 
     Selecting a High Affinity ScFV Against a WT1 Peptide 
     The next step is the selection of phage that bind to the target antigen of interest with high affinity, from phage in a human phage display library that either do not bind or that bind with lower affinity. This is accomplished by iterative binding of phage to the antigen, which is bound to a solid support, for example, beads or mammalian cells followed by removal of non-bound phage and by elution of specifically bound phage. In one embodiment, antigens are first biotinylated for immobilization to, for example, streptavidin-conjugated Dynabeads M-280. The phage library is incubated with the cells, beads or other solid support and non binding phage is removed by washing. Clones that bind are selected and tested. 
     Once selected, positive scFv clones are tested for their binding to HLA-A2/peptide complexes on live T2 cell surfaces by indirect flow cytometry. Briefly, phage clones are incubated with T2 cells that have been pulsed with Wt1-A peptide, or an irrelevant peptide (control). The cells are washed and then with a mouse anti-M13 coat protein mAb. Cells are washed again and labeled with a FITC-goat (Fab) 2  anti-mouse Ig prior to flow cytometry. 
     In other embodiments, the anti-WT1/A antibodies may comprise one or more framework region amino acid substitutions designed to improve protein stability, antibody binding, expression levels or to introduce a site for conjugation of therapeutic agents. These scFv are then used to produce recombinant human monoclonal Igs in accordance with methods known to those of skill in the art. 
     Methods for reducing the proliferation of leukemia cells is also included, comprising contacting leukemia cells with a WT1 antibody of the invention. In a related aspect, the antibodies of the invention can be used for the prevention or treatment of leukemia. Administration of therapeutic antibodies is known in the art. 
     Antibody Conjugates with Anti-Cancer Agents 
     Monoclonal antibodies represent the preferred vehicle for the targeted delivery of bioactive agents to cancer sites, including antibody-based delivery of cytotoxics, radionuclides or immunomodulatory cytokines. Conjugates of the antibodies of the invention with therapeutic agents, including without limitation, drugs (such as calecheamicin, aureastatin, doxorubicin), or toxins (such as ricin, diphtheria, gelonin) or radioisotopes emitting alpha or beta particles (such as,  90 I,  131 I,  225 Ac,  213 Bi,  223 Ra and  227 Th), inflammatory peptides (such as IL2, TNF, IFN-γ) are encompassed by the invention. 
     Pharmaceutical Compositions and Methods of Treatment 
     WT1 antibodies of the present invention can be administered for therapeutic treatments to a patient suffering from a tumor or WT1-associated pathologic condition in an amount sufficient to prevent, inhibit, or reduce the progression of the tumor or pathologic condition. Progression includes, e.g, the growth, invasiveness, metastases and/or recurrence of the tumor or pathologic condition. Amounts effective for this use will depend upon the severity of the disease and the general state of the patient&#39;s own immune system. Dosing schedules will also vary with the disease state and status of the patient, and will typically range from a single bolus dosage or continuous infusion to multiple administrations per day (e.g., every 4-6 hours), or as indicated by the treating physician and the patient&#39;s condition. 
     The identification of medical conditions treatable by WT1 antibodies of the present invention is well within the ability and knowledge of one skilled in the art. For example, human individuals who are either suffering from a clinically significant leukemic disease or who are at risk of developing clinically significant symptoms are suitable for administration of the present WT1 antibodies. A clinician skilled in the art can readily determine, for example, by the use of clinical tests, physical examination and medical/family history, if an individual is a candidate for such treatment. 
     Non-limiting examples of pathological conditions characterized by WT1 expression include chronic myelocytic leukemia, acute lymphoblastic leukemia (ALL), acute myeloid/myelogenous leukemia (AML) and myelodysplastic syndrome (MDS). Additionally, solid tumors, in general and in particular, tumors associated with mesothelioma, ovarian cancer, gastrointestinal cancers, breast cancer, prostate cancer and glioblastoma are amenable to treatment using WT1 antibodies. 
     In another embodiment, therefore, the present invention provides a method of treating a medical condition by administering a WT1 antibody of the present invention in combination with one or more other agents. For example, an embodiment of the present invention provides a method of treating a medical condition by administering a WT1 antibody of the present invention with an antineoplastic or antiangiogenie agent. The WT1 antibody can be chemically or biosynthetically linked to one or more of the antineoplastic or antiangiogenic agents. 
     Any suitable method or route can be used to administer a WT1 antibody of the present invention, and optionally, to coadminister antineoplastic agents and/or antagonists of other receptors. Routes of administration include, for example, oral, intravenous, intraperitoneal, subcutaneous, or intramuscular administration. It should be emphasized, however, that the present invention is not limited to any particular method or route of administration. 
     It is noted that a WT1 antibody of the present invention can be administered as a conjugate, which binds specifically to the receptor and delivers a toxic, lethal payload following ligand-toxin internalization. 
     It is understood that WT1 antibodies of the invention will be administered in the form of a composition additionally comprising a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the binding proteins. The compositions of the injection may, as is well known in the art, be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the mammal. 
     Other aspects of the invention include without limitation, the use of antibodies and nucleic acids that encode them for treatment of WT1 associated disease, for diagnostic and prognostic applications as well as use as research tools for the detection of WT1 in cells and tissues. Pharmaceutical compositions comprising the disclosed antibodies and nucleic acids are encompassed by the invention. Vectors comprising the nucleic acids of the invention for antibody-based treatment by vectored immunotherapy are also contemplated by the present invention. Vectors include expression vectors which enable the expression and secretion of antibodies, as well as vectors which are directed to cell surface expression of the antigen binding proteins, such as chimeric antigen receptors. 
     Cells comprising the nucleic acids, for example cells that have been transfected with the vectors of the invention are also encompassed by the disclosure. 
     For use in diagnostic and research applications, kits are also provided that contain a WT1 antibody or nucleic acids of the invention, assay reagents, buffers, and the like. 
     The method of the present invention will now be described in more detail with respect to representative embodiments. 
     Materials 
     Cell samples, cell lines and antibodies. After informed consent on Memorial Sloan-Kettering Cancer Center Institutional Review Board approved protocols, peripheral blood mononuclear cells (PBMC) from HLA-typed healthy donors and patients were obtained by Ficoll density centrifugation. The sources for obtaining human mesothelioma cell lines are described previously (29). The cell lines include: H-Meso1A, JMN, VAMT, H2452, H2373, H28, MSTO, Meso 11, Meso 34, Meso 37, Meso 47, and Meso 56. All cells were HLA typed by the Department of Cellular Immunology at Memorial Sloan-Kettering Cancer Center. Leukemia cell lines LAMA81, BV173, and 697, (WT1+, A0201+) were kindly provided by Dr. H. J. Stauss (University College London, London, United Kingdom). Melanoma cell line MeWo (WT1-, A201+), SKLY16 (B-cell lymphoma; WT1-, A0201+); K562, RwLeu4, and HL60, all WT1+ leukemias, and a TAP-deficient T2 cell line were obtained from the American Type Culture Collection. The cell lines were cultured in RPMI 1640 supplemented with 5% FCS, penicillin, streptomycin, 2 mmol/L glutamine, and 2-mercaptoethanol at 37 C/5% CO2. 
     Monoclonal Ab against human HLA-A2 (clone BB7.2) conjugated to FITC or APC, and its isotype control mouse IgG2b/FITC or APC, to human or mouse CD3, CD19, CD56, CD33, CD34 (BD Biosciences, San Diego), goat F(ab)2 anti-human IgG conjugated with PE or FITC and goat F(ab)2 anti-mouse Ig&#39;s conjugated to fluorescent (In Vitrogen, City) were purchased. Mouse mAb to HLA-class I (W6/32) was obtained from the MSKCC Monoclonal antibody Core Facility. 
     Peptides. All peptides were purchased and synthesized by Genemed Synthesis, Inc. (San Antonio, Tex.). Peptides were &gt;90% pure. (Table 1.) The peptides were dissolved in DMSO and diluted in saline at 5 mg/mL and frozen at −180 C. Biotinylated single chain WT1 peptide/HLA-A0201 and RHAMM-3/HLA-A0201 complexes were synthesized by refolding the peptides with recombinant HLA-A2 and beta2 microglobulen (β2M) at the Tetramer facility at MSKCC. 
     Animals. Eight to 10 week-old NOD.Cg-Prkdc scid IL2rgtm1Wjl/SzJ mice, known as NOD scid gamma (NSG), were purchased from the Jackson Laboratory (Bar Harbor, Me.) or obtained from MSKCC animal breeding facility. 
     Methods 
     Flow cytometry analysis. For cell surface staining, cells were incubated with appropriate mAbs for 30 minutes on ice, washed, and incubated with secondary antibody reagents when necessary. Flow cytometry data were collected on a FACS Calibur (Becton Dickinson) and analyzed with FlowJo V8.7.1 and 9.4.8 software. 
     Selection and characterization of scFv specific for WT1 peptide/HLA-A0201 complexes. A human scFv antibody phage display library was used for the selection of mAb clones. In order to reduce the conformational change of MHC1 complex introduces by immobilizing onto plastic surfaces, a solution panning method was used in place of conventional plate panning. In brief, biotinylated antigens were first mixed with the human scFv phage library, then the antigen-scFv antibody complexes were pulled down by streptavidin-conjugated Dynabeads M-280 through a magnetic rack. Bound clones were then eluted and were used to infect  E. coli  XL1-Blue. The scFv phage clones expressed in the bacteria were purified (35, 36). Panning was performed for 3-4 cycles to enrich scFv phage clones binding to HLA-A0201/WT1 complex specifically. Positive clones were determined by standard ELISA method against biotinylated single chain HLA-A0201/WT1 peptide complexes. Positive clones were further tested for their binding to HLA-A2/peptide complexes on live cell surfaces by flow cytometry, using a TAP-deficient, HLA-A0201+ cell line, T2. T2 cells were pulsed with peptides (50 ug/ml) in the serum-free RPM11640 medium, in the presence of 20 μg/ml β2 M ON. The cells were washed, and the staining was performed in following steps. 
     The cells were first stained with purified scFv phage clones, and followed by staining with a mouse anti-M13 mAb, and finally the goat F(ab)2 anti-mouse Ig&#39;s conjugate to FITC. Each step of the staining was done between 30-60 minutes on ice and the cells were washed twice between each step of the staining. 
     Engineering full length mAb using the selected ScFv fragments. Full-length human IgG1 of the selected phage clones were produced in HEK293 and Chinese hamster ovary (CHO) cell lines, as described (37). In brief, antibody variable regions were subcloned into mammalian expression vectors, with matching human lambda or kappa light chain constant region and human IgG1 constant region sequences. Molecular weight of the purified full length IgG antibodies were measured under both reducing and non-reducing conditions by electrophoresis. 
     Engineering chimeric antigen receptors and immune effector cells. Nucleic acids that encode antibodies and antigen binding proteins identified herein can be used engineer recombinant immune effector cells. Methods and vectors to generate genetically modified T-cells, for example, are known in the art (See Brentjens et al.,  Safety and persistence of adoptively transferred autologous CD 19- targeted T cells in patients with relapsed or chemotherapy refractory B - cell leukemias  in Blood 118(18):4817-4828, November 2011). 
     Characterization of the full-length human IgG1 for the WT1p/A2 complex. Initially, specificities of the fully human IgG1 mAbs for the WT1 peptide/A2 complex were determined by staining T2 cells pulsed with or without RMF or RHAMM-R3 control peptides, followed by secondary goat F(ab)2 anti-human IgG mAb conjugate to PE or FITC. The fluorescence intensity was measured by flow cytometry. The same method was used to determine the binding of the mAbs to fresh tumor cells and cell lines. 
     Radioimmunoassays. WT1 ab1 was labeled with 125-I (PerkinElmer) using the chloramine-T method (38). 100 μg antibody was reacted with 1 mCi 125-I and 20 μg chloramine-T, quenched with 200 μg Na metabisulfite, then separated from free 125-I using a 10DG column (company) equilibrated with 2% bovine serum albumin in PBS. Specific activities of products were in the range of 7-8 mCi/mg. 
     Hematopoietic cell lines, adherent cell lines (harvested with a non-enzymatic cell stripper (name)), PBMCs from normal donors and AML patients were obtained as described. Cells were washed once with PBS and re-suspended in 2% human serum in PBS at 10 7  cells/mL at 0°. Cells (10 6  tube in duplicate) were incubated with 125-I-labeled WT1 AB1 (1 μg/mL) for 45 minutes on ice, then washed extensively with 1% bovine serum albumin in PBS at 0°. To determine specific binding, a duplicate set of cells was assayed after pre-incubation in the presence of 50-fold excess unlabeled WT1 AB1 for 20 minutes on ice. Bound radioactivity was measured by a gamma counter, specific binding was determined, and the number of bound antibodies per cell was calculated from specific activity. 
     Antibody-dependent cellular cytotoxicity (ADCC). Target cells used for ADCC were T2 cells pulsed with or without WT1 or RHAAM-3 peptides, and tumor cell lines without peptide pulsing. WT1 ab1 or its isotype control human IgG1 at various concentrations were incubated with target cells and fresh PBMCs at different effector: target (E:T) ratio for 16 hrs. The supernatant were harvested and the cytotoxicity was measured by LDH release assay using Cytotox 96 non-radioreactive kit from Promega following their instruction. Cytotoxicity is also measured by standard 4 hours 51Cr-release assay. 
     Transduction and selection of luciferase/GFP positive cells. BV173 and JMN cells were engineered to express high level of GFP-luciferase fusion protein, using lentiviral vectors containing a plasmid encoding the luc/GFP (39). Using single cell cloning, only the cells showing high level GFP expression were selected by flow cytometry analysis and were maintained and used for the animal study. 
     Therapeutic trials of the WT1 ab1 in a human leukemia xenograft NSG model. Two million BV173 human leukemia cells were injected IV into NSG mice. On day 5, tumor engraftment was confirmed by firefly luciferase imaging in all mice that were to be treated; mice were then randomly divided into different treatment groups. On day 6 and day 10, mAb WT1 ab1 or the isotype control mAb were injected IV. In animals that also received human effector cells with or without mAb, cells (CD34 and CD3-depleted healthy donor human PBMCs) were injected IV into mice (10 cells/mouse) 4 hr before the mAb injections. Tumor growth was assessed by luminescence imaging once to twice a week, and clinical activity was assessed daily. 
     Selection and characterization of scFv specific for WT peptide/HLA-A0201 complexes. Selection of an WT1-specific scFV is achieved using a 9-mer T1-derived peptide comprising amino acids 126-134 (RMFPNAPYL, SEQ ID NO: 1) of WT1. This peptide has been shown to be processed and presented by HLA-A0201 to induce cytotoxic CD8 +  T cells that are capable of killing WT1-positive tumor cells. 
     Representative data from a patient with AML after 6 vaccinations with a WT1 RMF peptide are shown in  FIG. 2  as evidence that the WT1 peptides are immunogenic in humans. CD3 T cells were stimulated with WT1-A peptide (amino acids 126-134) and cytotoxicity was measured using a standard Cr release assay against 697 (A0201A WT1+) or SKLY-16 (A0201+WT1-) cell lines. The SKLY-16 cells pulsed with WT1-A or irrelevant peptide EW were used as positive and negative controls for the specificity of the killing. Effector: Target (E:T) ratios are indicated on the X-axis. Data demonstrates that T cells killed WT1+ tumor cells in a HLA-A0201-restricted manner. 
     Well established phage display libraries and screening methods known to those of skill in the art were used to select scFv fragments highly specific for an WT1-A peptide/HLA-A2 complex. In one embodiment, a human scFv antibody phage display library (7×10 10  clones) was used for the selection of mAb clones. In order to reduce the conformational change of MHC1 complex introduced by immobilizing onto plastic surfaces, a solution panning method was used in place of conventional plate panning. In brief, biotinylated antigens were first mixed with the human scFv phage library, then the antigen-scFv phage antibody complexes were pulled down by streptavidin-conjugated Dynabeads M-280 through a magnetic rack. 
     Bound clones were then eluted and were used to infect  E. coli  XL1-Blue. The scFv phage clones expressed in the bacteria were purified (35, 36). Panning was performed for 3-4 cycles to enrich scFv phage clones binding to HLA-A0201/WT1 complex specifically. Positive clones were determined by standard ELISA method against biotinylated single chain HLA-A0201/WT1 peptide complexes ( FIG. 3 ). Positive clones were further tested for their binding to HLA-A2/peptide complexes on live cell surfaces by flow cytometry, using a TAP-deficient, HLA-A0201 +  cell line, T2. T2 cells were pulsed with peptides (50 μg/ml) in serum-free RPM11640 medium, in the presence of 20 μg/m β2 M overnight. The cells were washed, and staining was performed as follows. 
     The cells were first stained with purified scFv phage clones, followed by staining with a mouse anti-M13 mAb, and finally, a goat F(ab)2 anti-mouse Ig conjugated to FITC. Each step of the staining was done for 30-60 minutes on ice. The cells were washed twice between each staining step. Results are shown in  FIG. 4 . The phage clone of WT1 ab1 was shown to bind to T2 cells pulsed with only WT1-A peptide (RMFPNAPYL: abbreviated hereinafter as RMF), but not to T2 cells alone, T2 cells pulsed with control EW peptide, or heteroclitic peptide WT1-A. 
     Binding affinity of the full-length IgG1 of WT1 ab1 to the peptide/A0201 complex was tested by titration of WT1 ab1 at indicated concentrations. T2 cells were pulsed with 50 μg/ml or 10 μg/ml, followed by secondary goat F(ab) anti-human IgG/PE. Results are shown in  FIG. 5 . 
       FIG. 6  shows the density of peptide/HLA-A0201 complex recognized by WT1 ab. T2 cells were pulsed overnight (ON) with RMF (upper panel) or RHAMM-R3 (lower panel) peptides at indicated concentrations, and binding of WT1 ab1, WT1 ab3 and WT1 ab5 at a concentration of 1 μg/ml was analyzed by flow cytometry. 
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Summary of phage panning for WTI/A2 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 Number 
                 Solution 
                   
               
               
                   
                   
                 of single 
                 ELISA 
                 Number 
               
               
                   
                 Rounds of 
                 clone 
                 positive 
                 of Unique 
               
               
                 Phage libraries 
                 panning 
                 screened 
                 Rate 
                 Clones 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 scFv-spleen A 
                 4 
                 72 
                 41/96  
                 13 
               
               
                 scFv-spleen B 
                 4 
                 47 
                 3/47 
                 2 
               
               
                 scFv-spleen C 
                 3 
                 58 
                 0/58 
                 0 
               
               
                 scFv-PBMC A 
                 4 
                 68 
                 34/68  
                 10 
               
               
                 scFv-PBMC B 
                 3 
                 90 
                 19/90  
                 7 
               
               
                 Fab-spleen A 
                 4 
                 12 
                 2/12 
                 0 
               
               
                 Fab-spleen B 
                 4 
                 36 
                 0/36 
                 0 
               
               
                 Fab-spleen C 
                 4 
                 24 
                 2/24 
                 1 
               
               
                 Fab-spleen C 
                 3 
                 72 
                 38/72  
                 5 
               
               
                 Fab-spleen D 
                 4 
                 72 
                 4/72 
                 1 
               
               
                 Fab-spleen D 
                 4 
                 72 
                 4/72 
                 3 
               
               
                   
               
            
           
         
       
     
     The positive scFv clones were tested for their binding to HLA-A2/peptide complexes on live cell surfaces by indirect flow cytometry on: (i) a TAP deficient HLA-A0201 +  T2 cells pulsed with WT1 peptide or irrelevant peptide; (ii) a WT1+ HLAA0201 +  cell lines such as BV173, U266 and control WTI HLA-A0201 +  cell line SKLY16, or WT1 +  HLA-A0201 cell line, K562, without pulsing with the peptide. The latter determine the recognition and binding affinity of the scFv to the naturally processed WT1p/A2 complex on tumor cells. 
     A total of 28 phage clones were screened for their ability to produce mAb specific for the WTI-A peptide/A2 complex. The recognition of the WT1 p/A2 complex on live cells was measured by the binding of the phage scFv to T2 cells pulsed with the WTI-A peptide and the other HLA-A2-binding peptides (50 μg/ml). These include: T2 cells alone; T2 cells pulsed with WTI-A peptide; T2 cells pulsed with heteroclitic peptide WT1-A1; T2 cells pulsed with irrelevant EW peptide (HLA-A0201-binding 9-mer peptide, derived from Ewing sarcoma) or RHAMM-R3 ( FIG. 4 ). 
     
       
         
           
               
               
               
             
               
                 TABLE 12 
               
               
                   
               
               
                   
                 Positive for binding toT2 
                 Selected for Construction 
               
               
                 Clone # 
                 pulsed with WT1A 
                 of full-length IgG1 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 1 
                 + 
                   
               
               
                 2 
                 + 
               
               
                 3 
                 + 
                 + 
               
               
                 4 
                 + 
               
               
                 5 
                 + 
                 + 
               
               
                 6 
                 + 
               
               
                 7 
               
               
                 8 
                 + 
               
               
                 9 
               
               
                 10 
               
               
                 11 
               
               
                 12 
               
               
                 13 
                 + 
                 + 
               
               
                 14 
               
               
                 15 
                 + 
                 + 
               
               
                 16 
               
               
                 17 
                 + 
               
               
                 18 
                 + 
                 + 
               
               
                 19 
                 + 
               
               
                 20 
                 + 
               
               
                 21 
               
               
                 22 
                 + 
               
               
                 23 
                 + 
                 + 
               
               
                 24-28 
               
               
                   
               
            
           
         
       
     
     Engineering Full Length mAb Using the Selected ScFv Fragments. 
     Phage display technology allows for the rapid selection and production of antigen-specific scFv and Fab fragments, which are useful in and of themselves, or which can be further developed to provide complete antibodies, antigen binding proteins or antigen binding fragments thereof. Complete mAbs with Fc domains have a number of advantages over the scFv and Fab antibodies. First, only full length Abs exert immunological function such as CDC and ADCC mediated via Fc domain. Second, bivalent mAbs offer stronger antigen-binding affinity than monomeric Fab Abs. Third, plasma half-life and renal clearance will be different with the Fab and bivalent mAb. The particular features and advantages of each can be matched to the planned effector strategy. Fourth, bivalent mAb may be internalized at different rates than scFv and Fab, altering immune function or carrier function. Alpha emitters, for example, do not need to be internalized to kill the targets, but many drugs and toxins will benefit from internalization of the immune complex. In one embodiment, therefore, once scFv clones specific for WT1p/A2 were obtained from phage display libraries, a full length IgG mAb using the scFv fragments was produced. 
     To produce recombinant human monoclonal IgG in Chinese hamster ovary (CHO) cells, a full length IgG mAb was engineered based on a method known to those of skill in the art (Tomomatsu et al., Production of human monoclonal antibodies against FceRIa by a method combining in vitro immunization with phage display. Biosci Biotechnol Biochem 73(7): 1465-1469 2009). Briefly, antibody variable regions were subcloned into mammalian expression vectors ( FIG. 7 ), with matching Lambda or Kappalight chain constant sequences and IgG1 subclass Fc (for example, see Table 9) (33,34). Purified full length IgG antibodies showed expected molecular weight under both reducing and non-reducing conditions ( FIG. 8 ). Kinetic binding analysis (35) confirmed specific binding of full length IgG to WT1/A2, with a K D  in nanomolar range ( FIGS. 9 and 10 .) 
     Example 1 
     Selection of ScFv Specific for WT1p/A2 Complex Using a Fully Human Phage Display Library. 
     Phage display against HLA-A0201/WT1 peptide complex was performed for 3-4 panning rounds to enrich the scFv phage clones binding to HLA-A0201/WT1 peptide complex specifically. Individual scFv phage clones positive for the WT1 peptide/A2 complex were determined by ELISA and the clones that possessed unique DNA coding sequences were subjected to further characterization. To test if the ScFv bound to the WT1p/A2 complex on live cells, the positive phage clones were tested for binding to a TAP deficient, HLA-A0201-positive cell line, T2. T2 cells can only present the exogenous peptides and therefore have been widely used for detection of specific epitopes presented by HLA-A2 molecules. A total 35 phage clones were screened on T2 cells and 15 clones showed specific binding to T2 cells pulsed with only WT1 RMF peptide, but not to T2 cells alone or pulsed with control RHAMM-3 peptide ( FIG. 4 ). The scFv phage clones were unable to bind to several tumor cell lines that are WT1- and HLA-A2 positive suggesting the affinity of the ScFv was weak, compared to full-length bivalent mAb. 
     Example 2 
     Generation of Full-Length Human IgG1. 
     Immunological function such as CDC and ADCC depend on the Fc domain of bivalent IgG. In addition, bivalent mAbs offer stronger antigen-binding avidity than monomeric scFv Abs. Therefore, 6 ScFv phage clones among 15 positive phage clones were selected to produce the full-length human monoclonal IgG1 in HEK293 and Chinese hamster ovary (CHO) cells. In brief, variable regions of the mAbs were subcloned into mammalian expression vectors with matching human lambda or kappa light chain constant region and human IgG1 constant region sequences. Purified full length IgG antibodies showed expected molecular weight under both reducing and non-reducing conditions ( FIG. 8 ). Five clones were successfully engineered into human IgG1. 
     Example 3 
     Specificity and Binding Avidity of the IgG1 mAb 
     Binding to Human Cell Lines. 
     T2 cells, pulsed with or without RMF or RHAMM-3 peptides initially were used to determine the binding specificity of the mAb. Three out of five human IgG1, including WT1 ab1, showed specific binding to the T2 cells that were pulsed only with WT1 peptide, but not to T2 alone or T2 pulsed with control peptide RHAMM-R3. The binding avidity of the mAb were substantially enhanced (50 to 100 fold), compared to their parental scFv phage clones. Two mAbs among the five showed binding to T2 cells alone or pulsed with the control peptide RHAMM-R3, although the binding was greatly enhanced by pulsing the cells with RMF peptide. This suggested that these two mAb also had high avidity for epitopes on the HLA-A2 molecule alone and therefore were excluded from further investigation. This was not unexpected, as it has been a common problem for producing such mAb against peptide/MHC complexes, given the predominance of the MHC class I molecule epitopes within the complexes. It also suggests that the precise specificity of the mAb for the complexes might not be determined easily at the scFv stage, due to the lower affinity compared to the bivalent IgG1 mAb. 
     The binding affinity of the three remaining mAb specific for the WT1p/A2 complex first was investigated on T2 cells pulsed with or without RMF and control RHAMM-R3 peptides (50 ug/ml) by titration of the mAbs. Mab WT1 ab1 showed the strongest binding, down to a concentration of 0.01 ug/ml. Isotype control human IgG1 showed no binding at any concentrations tested ( FIG. 5 ). In addition to WT1 ab1, the two other mAb, WT1 ab3 and WT1 ab5, showed specific binding at a range of &lt;1 ug/ml of the mAb concentrations used. The specific recognition of the mAb also depended on the antigenic density on the cell surface. T2 cells were pulsed with RMF or R3 peptides at 50, 25, 12.5, 6.25, 3.13 and 1.6 ug/ml; the test mAb were used at 1 ug/ml for the T2 binding assay. WT1 ab1 could detect the RMF peptide/A2 complex on T2 cells in a concentration-dependent manner at concentrations as low as 1.6 ug/ml, with significantly higher fluorescence intensity than the other 2 mAb ( FIG. 6 ). These results further confirmed that the WT1 ab1 possessed the highest avidity for the RMFp/A0201 complex. 
     Example 4 
     Epitope Mapping. 
     To investigate with more precision the epitope for WT1 ab1 recognition, RMF peptides were substituted at positions 1, 3, 4, 5, 6, 7 and 8 with alanine and pulsed onto T2 cells and were tested for binding of WT1 ab1. Positions 2 and 9 of the RMF were left intact, as these are the anchor residues for peptide binding to the HLA-A0201 molecule. Except for position 1, alanine substitutions at other positions did not greatly affect the binding of the WT1 ab1, as compared to the native RMF peptide ( FIG. 19 ). However, substitution of position 1 by either alanine (WT1-A1-B) or tyrosine (WT1-A1), completely abrogated the binding of WT1 ab1. The loss of binding was not due to the reduction of peptide binding affinity to the HLA-A2 molecule, as both peptides showed the strongest binding in T2 stabilization assay using the mAb specific for the HLA-A2 molecule, clone BB7 ( FIG. 20 ). These results show that the arginine at position 1 of the RMF peptide is one of the most crucial for the WT1 ab1 recognition. The role of the residues at positions #2 and 9, could not be assessed. 
     The next important question was whether WT1 ab1 was able to recognize naturally processed WT1 epitope RMF presented by HLA-A0201 molecules on the cell surface. A panel of cell lines was selected based on the expression of WT1 mRNA and HLA genotyping (Table 12). 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 12 
               
               
                   
                   
               
               
                   
                   
                   
                   
                 Ratio of 
               
               
                   
                 HLA-A2 
                 WT1 
                 WT1 AB 
                 BB7.2: 
               
               
                   
                 genotype 
                 mRNA 
                 binding 
                 Isotype 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Mesothelioma/solid tumor 
                   
                   
                   
                   
               
               
                 JMN 
                 + 
                 + 
                 + 
                 248 
               
               
                 Meso 37 
                 + 
                 + 
                 + 
                 68 
               
               
                 Meso 47 
                 + (02xx) 
                 + 
                 + 
                 17 
               
               
                 H2452 
                 + 
                 + 
                 + 
                 20 
               
               
                 Meso34 
                 + 
                 + 
                 + 
                 37.3 
               
               
                 Meso-56 
                 + (02xx) 
                 + 
                 + 
                 23 
               
               
                 H2373 
                 + 
                 + 
                 − 
                 1.6 
               
               
                 MSTO 
                 − 
                 + 
                 − 
                 1.4 
               
               
                 VAMT 
                 − 
                 3+ 
                 − 
                 NT 
               
               
                 Mewo 
                 + 
                 − 
                 − 
                 3 
               
               
                 Leukemias and other 
               
               
                 hematopoietic cell lines 
               
               
                 BV173 
                 + 
                 ++ 
                 + 
                 196 
               
               
                 BA25 
                 + 
                 ? 
                 + 
                 117.5 
               
               
                 ALL-3 
                 + 
                 + 
                 + 
                 60 
               
               
                 U266 
                 + 
                 + 
                 − 
                 1.8 
               
               
                 697 
                 + 
                 5+ 
                 − 
                 4.1 
               
               
                 LAMA 
                 + 
                 2+ 
                 − 
                 6 
               
               
                 SKLY-16 
                 + 
                 − 
                 − 
                 1.9 
               
               
                 HL-60 
                 − 
                 3+ 
                 − 
                 0.4 
               
               
                 K562 
                 − 
                 2+ 
                 − 
                 1.5 
               
               
                 T2 
                 + 
                 NT 
                 − 
                 &gt;20 
               
               
                   
               
            
           
         
       
     
     WT1 mRNA expression level was estimated according to a previous study (Rena), by quantitative RT-PCR. 
     Among 7 human mesothelioma cell lines that are positive for both HLA-A0201 and WT1 mRNA, WT1 ab1 bound to 6 out of 7 cell lines, but not to the cells that were either HLA-A0201 negative (MSTO and VAMT) or WT1 mRNA negative, such as melanoma cell line, Mewo ( FIG. 21 ). 
     Similarly, among 9 leukemia cell lines tested, WT1 ab1 bound to 3 cell lines BV173 ( FIG. 22 ), BA25 and ALL-3, that are positive for both WT1 mRNA and HLA-A0201, but not to HLA-A2-negative cell lines HL60 and K562, that have been demonstrated to express a high level of WT1 transcripts in numerous studies. 
     As expected, intensity of binding of the WT1 AB1 also appeared to be directly associated with the expression level of HLA-A0201 molecule, as shown in mesothelioma cells H2373, leukemia cell lines 697 and LAMA, and myeloma cell line U266. Although these cell lines were positive for both WT1 transcripts and HLA-A2, the expression level of the HLA-A2 was low (Table 12) and the mAb did not show binding. On the other hand, the results obtained with T2 cells argue against the possibility of WT1 ab1 binding to HLA-A0201 alone as T2 cells expressed a high level of HLA-A2 molecule. Notably, WT1 ab1 did not bind to T2 cells alone or pulsed with R3 and other HLA-A0201-binding peptides such as Ewing sarcoma-derived (EW) or the heteroclitic peptide for the RMF peptide, WT1-A1; these two peptides have been shown to have higher affinity for the HLA-A0201 molecule in T2 stabilization assay (28). These results provided strong evidences that WT1 ab1 recognition was specific for epitopes jointly composed of the RMF peptide and the A0201 molecule in a complex. The binding of the other two mAb, WT1 ab3 and WT1 ab5, to the BV173 and JMN cells was also weaker than WT1 ab1. 
     Example 5 
     Quantitation of WT1 ab1 Binding Sites on Cells. 
     A radioimmunoassay using 1251-labeled WT1 ab1 was used to confirm the specificity of the antibody for WT1+ HLA-A0201+ cell lines, to determine an affinity constant and to assess the number of antibody binding sites per cell on a panel of cell lines. Scatchard analysis based on binding to JMN cells showed an avidity constant of about 0.2 nM ( FIG. 23 ). This number was confirmed by interferometry using a Forte Bio device. 125-I-labeled WT1 ab1 was used to confirm the specificity of the antibody for WT1+ HLA-A0201+ cell lines, and to assess the number of antibody binding sites on a panel of cell lines ( FIG. 24 ). Because we cannot determine whether the bivalent mAb is binding to 1 or 2 complexes on the surface, total epitopes per cell could be as high as twice the number of mAb binding sites. Again, WT1 ab1 bound to JMN, ALL-3, BA25, BV173, which are positive for both HLA-A0201 and WT1 mRNA, but not HLA-A0201 negative (HL60) or WT1 mRNA negative (SKLY-16) cells. WT1 ab1 did not bind to 697 cells, which are both HLA-A0201 and WT1 positive, but contain low levels of HLA-A0201 (Table 12), confirming that a certain level of total MHC complex is needed to present sufficient WT1 peptide for WT1 ab1 binding. T2 pulsed with RMF bound the highest number of mAb (50,000 per cell), followed by JMN cells which bound 6×103 WT1 ab1 molecules per cell, translating to between 6×103 and 1.2×104 RMF peptide/A2 complexes per cell assuming monovalent or bivalent antibody binding, respectively. The three positive leukemia cell lines bound between 1×103 and 2×103 WT1 ab1 molecules, or 2×103-4×103 binding sites ( FIG. 24 ). These results were confirmed by quantitative flow cytometry. 
     Example 6 
     WT1 Ab1 Binding to Leukemic Patient Samples. 
     We next investigated if WT1 ab1 is able to detect the RMF epitope on primary AML cells. Radioimmunoassay showed significant binding of the WT1 AB1 to AML blasts of patient 1, who is HLA-A2 positive and WT1 mRNA + . WT1 ab1 bound to CD33+ and CD34+ double positive cells that account for more than 83% of the whole cell populations ( FIG. 25 ). WT1 ab1 did not bind to the cells of 3 other patients who are either HLA-A2 positive but mRNA negative or HLA-A2 negative. WT1 ab1 did not bind to PBMCs from either HLA-A2 positive or negative healthy donors. The results were confirmed by flow cytometry analysis. WT1 AB1 did not show significant binding to the blasts from the patients who were A0201 negative ( FIG. 26 ). The results were consistent with the results obtained with mRNA expression of the cells. These data confirm that the level of RMFp/HLA-A0201 on the surface of leukemia cells is adequate to allow reactivity with the WT1 ab1 and the levels on WT1 negative healthy cells is not significant. 
     Example 7 
     WT1 AB1 Mediates ADCC Against Tumor Cells 
     ADCC is considered to be one of the major effector mechanisms of therapeutic mAb in humans. In the presence of human PBMC, WT1 ab1 mediated dose-dependent PBMC ADCC against the T2 cells loaded with RMF peptide, but not T2 cells alone or T2 cells pulsed with control R3 peptide ( FIG. 27 ). Importantly, WT1 ab1 was able to mediate ADCC against naturally presented RMF epitope by HLA-A0201 molecule on tumor cells, such as the mesothelioma cell line, JMN ( FIG. 33 ), and the leukemia cell line BV173 ( FIG. 34 ), but not the HLA-A2 negative cells MSTO ( FIG. 28 ) or HL-60 ( FIG. 29 ). The killing was consistently observed at 1 μg/ml or below of WT1 ab1 using PBMCs as effector cells from multiple healthy donors. Importantly, WT1 ab1 also killed primary A0201-positive AML blasts that were positive for the WT1 ab1 binding, but not the blasts that were HLA-A0201 negative ( FIG. 30 ). These results demonstrated that WT1 ab1 mediates specific ADCC against cells that naturally express RMF and HLA-A0201 complex at physiologic levels as well as on cell lines. 
     Example 8 
     WT1 AB1 Eliminates Human Leukemia Cells in NSG Mice 
     The efficacy of WT1 ab1 in vivo was tested in NOD SCID gamma (NSG) mice xenografted intravenously 6 days previously with BV173 bcr/abl positive acute lymphoblastic leukemia. At the time of treatment, mice had leukemia in their liver, spleen, and BM visible by luciferase imaging. NSG mice lack mature B-, T- and NK-cells, and we hypothesized that introducing human effector cells (CD3-, CD34-, PBMCs) along with WT1 ab1 treatment would recapitulate in vivo the ADCC-mediated anti-tumor effects observed in vitro. Injection of effectors along with two 100 μg doses of WT1 ab1 nearly ablated tumor growth compared to controls ( FIG. 31 ). This effect was durable over the course of the experiment ( FIG. 32 ). Interestingly, early on in the trials, effector cells alone or combined with control IgG appeared to promote more rapid growth of leukemia relative to mice injected with leukemia alone, demonstrating that the anti-tumor effect was unrelated to the effectors by themselves. Several of the mice given effectors (with or without control mAb) died early in the experiment with massive infiltration of the BV173. 
     Surprisingly, WT1 ab1 treatment without human effectors also dramatically reduced tumor burden as well as the WT1 ab1 combined with effectors for approximately 30 days ( FIG. 32 ), though tumors eventually relapsed far more quickly in the WT1 ab1 alone group, when compared to WT1 ab1 combined with effectors group ( FIG. 32 ). We confirmed the effect of WT1 ab1 alone and titrated the dosage to evaluate potency. WT1 ab1 alone produced a marked reduction in tumor burden at early time points at all doses tested (25-100 μg times 2 doses). Tumors in all treatment groups relapsed slowly after antibody therapy was stopped; and by day 23 (13 days after the last antibody injection), significantly more tumor relapse could be observed in the 25 μg group compared to the 100 μg dose group, indicating a dose-response to WT1 ab1 therapy ( FIG. 33 ). Before treatment, mice displayed the largest tumor burden in the liver, which was quickly cleared by WT1 ab1. Upon relapse, tumor in the highest dose group appeared to develop mainly in bone marrow, while tumor returned more quickly to the liver in mice treated with the lowest dose. 
     Example 9 
     Engineering Antibodies to Enhance their Cytotoxic Abilities. 
     Bispecific antibodies are constructed that recognize both WT1/A2 complex and CD3 on immune T cells as described (43,44) with a human IgG1 Fc. Bispecific antibodies are expected to recruit and target cytotoxic T cells to WT1/A2 positive cancer cells, while maintaining Fc effector functions and long half life in vivo. Three mechanisms are involved in the specific killing of cancer cells mediated by bispecific antibodies: i) killing by activated T cells; ii) ADCC activity; iii) CDC activity. Other formats of bispecific antibodies can be constructed, such tandem scFv molecules (taFv), diabodies (Db), or single chain diabodies (scDb), and fusion protein with human serum albumin (45, 46, 47, 48), but are devoid of Fc effector functions with distinct pharmacokinetic profiles. 
     WT1/A2 target specific-ADCC activity is enhanced by expressing antibodies recombinantly in glycol-engineered CHO cells as described in U.S. Pat. Nos. 8,025,879; 8,080,415; and 8,084,022. The modified oligosaccharide N-glycan on Asn297 alters effector functions as follows: 1) higher affinity binding to CD16/FcRIIIa for improved ADCC activity mediated by human Natural Killer cells; 2) reduced binding affinity to CD32b/FcRIIb, an inhibitory receptor expressed in multiple types of immune cells (except NK cells), for improved ADCC activity mediated by effector cells such as neutrophils and antigen presentation by macrophage and DC cells (50, 51, 52). Enhanced antibodies are expected to achieve better efficacy for cancer treatment in vivo. 
     Glycosylation (specifically fucosylation) variants of IgG Fc can be produced using host expression cells and methods described in U.S. Pat. Nos. 8,025,879; 8,080,415; and 8,084,022, the contents of which are incorporated by reference. Briefly, messenger RNA (mRNA) coding for heavy or light chain of the antibodies disclosed herein, is obtained by employing standard techniques of RNA isolation purification and optionally size based isolation. cDNAs corresponding to mRNAs coding for heavy or light chain are then produced and isolated using techniques known in the art, such as cDNA library construction, phage library construction and screening or RT-PCR using specific relevant primers. In some embodiments, the cDNA sequence may be one that is wholly or partially manufactured using known in vitro DNA manipulation techniques to produce a specific desired cDNA. The cDNA sequence can then be positioned in a vector which contains a promoter in reading frame with the gene and compatible with the low fucose-modified host cell. 
     Numerous plasmids that contain appropriate promoters, control sequences, ribosome binding sites, and transcription termination sites, and optionally convenient markers are known in the art, these include but are not limited to, vectors described in U.S. Pat. Nos. 4,663,283 and 4,456,748. In one embodiment, the cDNA coding for the light chain and that coding for the heavy chain may be inserted into separate expression plasmids. In an alternative embodiment, the cDNA coding for the light chain and that coding for the heavy chain may be inserted together in the same plasmid, so long as each is under suitable promoter and translation control. Results are shown in  FIG. 34 . 
     REFERENCES 
     
         
         1. Mundlos S, et al. Nuclear localization of the protein encoded by the Wilms&#39; tumor gene WT1 in embryonic and adult tissues.  Development  1993; 119: 1329-41. 
         2. Keilholz U, et al. Wilms&#39; tumor gene 1 (WT1) in human neoplasia.  Leukemia  2005; 19: 1318-1323. 
         3. Inoue K, et al. WT1 as a new prognostic factor and a new marker for the detection of minimal residual disease in acute leukemia. Blood 1994; 84 (9): 3071-3079. 
         4. Ogawa H, et al. The usefulness of monitoring WT1 gene transcripts for the prediction and management of relapse following allogeneic stem cell transplantation in acute type leukemia. Blood 2003; 101 (5): 1698-1704. 
         5. Yarnagarni T, et al. Growth Inhibition of Human Leukemic Cells by WT1 (Wilms Tumor Gene) Antisense Oligodeoxynucleotides: Implications for the Involvement of WT1 in Leukemogenesis.  Blood  1996; 87: 2878-2884. 
         6. Bellantuono I, et al. Two distinct HLA-A0201-presented epitopes of th Wilms tumor antigen 1 can function as targets for leukemia-reactive CTL.  Blood  2002; 100 (10): 3835-3837. 
         7. Gaiger A, et al. WT1-specific serum antibodies in patients with leukemia.  Clin. Cancer Res.  2001; 7 (suppl 3): 761-765. 
         8. Oka Y, et al. WT1 peptide cancer vaccine for patients with hematopoietic malignancies and solid cancers.  The Scientific World Journal  2007; 7: 649-665. 
         9. Kobayashi H, et al. Defining MHC class II T helper epitopes from WT1 antigen.  Cancer Immunol. Immunother.  2006; 55 (7): 850-860. 
         10. Pinilla-Ibarz J, et al. Improved human T-cell responses against synthetic HLA-A0201 analog peptides derived from the WT1 oncoprotein.  Leukemia  2006; 20 (11): 2025-2033. 
         11. May R J, et al. Peptide epitopes from the Wilms tumor 1 oncoprotein stimulate CD4+ and CD8+ T cells that recognize and kill human malignant mesothelioma tumor cells.  Clin Cancer Res.  2007; 13:4547-4555. 
         12. Keiholz U, et al. A clinical and immunologic phase 2 trial of Wils tumor gene product (WT1) peptide vaccination in patients with AML and MDS. Blood 2009; 113: 6541-6548. 
         13. Rezwani K, et al. Leukemia-associated antigen-specific T-cell responses following combined PR1 and WT1 peptide vaccination in patients with myeloid malignancies. Blood 2008; 111 (1): 236-242. 
         14. Maslak P, et al., Vaccination with synthetic analog peptides derived from WT1 oncoprotein induces T cell responses in patients with complete remission from acute myeloid leukemia.  Blood  2010; Accpt Minor rev. 
         15. Krug L M, et al. WT1 peptide vaccinations induce CD4 and CD8 T cell immune responses in patients with mesothelioma and non-small cell lung cancer.  Cancer Immunol Immunother  2010; in revision. 
         16. Morris E, et al. Generation of tumor-specific T-cell therapies.  Blood Reviews  2006; 20: 61-69. 
         17. Houghton A N et al. Monoclonal antibody therapies—a “constant” threat to cancer.  Nat Med  2000; 6:373-374. 
         18. Miederer M, et al. Realizing the potential of the Actinium-225 radionuclide generator in targeted alpha particle therapy applications.  Adv Drug Deliv Rev  2008; 60 (12): 1371-1382. 
         19. Noy R, T-cell-receptor-like antibodies: novel reagents for clinical cancer immunology and immunotherapy.  Expert Rev Anticancer Ther  2005: 5 (3): 523-536. 
         20. Chames P, et al. Direct selection of a human antibody fragment directed against the tumor T-cell epitope HLA-A1-MAGE-A1 from a nonimmunized phage-Fab library. Proc Natl Acad Sci USA 2000; 97: 7969-7974. 
         21. Held G, et al. Dissecting cytotoxic T cell responses towards the NY-ESO-1 protein by peptide/MHC-specific antibody fragments.  Eur J Immunol.  2004: 34:2919-2929. 
         22. Lev A, et al. Isolation and characterization of human recombinant antibodies endowed with the antigen-specific, major histocompatibility complex-restricted specificity of T cells directed toward the widely expressed tumor T cell-epitopes of the telomerase catalytic subunit.  Cancer Res  2002; 62: 3184-3194. 
         23. Klechevsky E, et al. Antitumor activity of immunotoxins with T-cell receptor-like specificity against human melanoma xenografts.  Cancer Res  2008; 68 (15): 6360-6367. 
         24. Azinovic I, et al. Survival benefit associated with human anti-mouse antibody (HAMA) in patients with B-cell malignancies.  Cancer Immunol Immunother  2006; 55(12):1451-8. 
         25. Tjandra J J, et al. Development of human anti-murine antibody (HAMA) response in patients.  Immunol Cell Biol  1990; 68(6):367-76. 
         26. Riechmann L, et al. Reshaping human antibodies for therapy.  Nature  1988; 332 (6162): 332:323. 
         27. Queen C, et al. A humanized antibody that binds to the interleukin 2 receptor.  Proc Natl Acad Sci USA  1989; 86 (24): 10029-33. 
         28. Gerd R, et al. Serological Analysis of Human Anti-Human Antibody Responses in Colon Cancer Patients Treated with Repeated Doses of Humanized Monoclonal Antibody A33 . Cancer Res  2001; 61, 6851-6859. 
         29. Cheever M A, et al. The prioritization of cancer antigens: A national Cancer Institute pilot project for the acceleration of translational research.  Clin Cancer Res  2009; 15 (17): 5323-5337. 
         30. Drakos E, et al. Differentiual expression of WT1 gene product in non-Hodgkin lymphomas.  Appl Immunohistochem Mol Morphol  2005; 13 (2):132-137. 
         31. Asemissen A M, et al. Identification of a highly immunogenic HLA-A*01-binding T cell epitope of WT1 . Clin Cancer Res  2006; 12 (24):7476-7482. 
         32. Tomimatsu K, et al. Production of human monoclonal antibodies against FceRIa by a method combining in vitro immunization with phage display.  Biosci Biotechnol Biochem  2009; 73 (7): 1465-1469. 
         33. Lidija P, et al. An integrated vector system for the eukaryotic expression of antibodies or their fragments after selection from phage display libraries.  Gene  1997; 187(1): 9-18. 
         34. Lisa J H, et al. Crystallographic structure of an intact IgG1 monoclonal antibody.  Journal of Molecular Biology  1998; 275 (5): 861-872. 
         35. Yasmina N A, et al. Probing the binding mechanism and affinity of tanezumab, a recombinant humanized anti-NGF monoclonal antibody, using a repertoire of biosensors.  Protein Science  2008; 17(8): 1326-1335. 
         36. Roberts W K, et al. Vaccination with CD20 peptides induces a biologically active, specific immune response in mice.  Blood  2002: 99 (10): 3748-3755. 
         37. Caron P C, Class K, Laird W, Co M S, Queen C, Scheinberg D A. Engineered humanized dimeric forms of IgG are more effective antibodies.  J Exp Med  176:1191-1195. 1992. 
         38. McDevitt M, et al. Tumor targeting with antibody-functionalized, radiolabeled carbon nanotubes.  J. Nuclear Med  2207; 48 (7))1180-1189. 
         39. Xue S A, et al. Development of a Wilms&#39; tumor-specific T-cell receptor for clinical trials: engineered patient&#39;s T cells can eliminate autologous leukemia blasts in NOD/SCID mice.  Haematologica  2010; 95 (1): 126-134. 
         40. McDevitt M R, et al. Tumor therapy with targeted atomic nanogenerators.  Science  2001; 294 (5546):1537-1540. 
         41. Borchardt P E, et al. Targeted Actinium-225 in vivo generators for therapy of ovarian cancer.  Cancer Res  2003; 63: 5084-5090. 
         42. Singh Jaggi J, et al. Selective alpha-particle mediated depletion of tumor vasculature with vascular normalization.  Plos One  2007; 2 (3): e267. 
         43. Yan W, et al. Enhancing antibody Fc heterodimer formation through electrostatic steering effects.  J. Biol. Chem.  2010; 285: 19637-19646. 
         44. Rossi E A, et al. Stably tethered multi-functional structures of defined composition made by the dock and lock method for use in cancer targeting.  Proc Natl Aca Sci USA  2006; 103:6841-6. 
         45. Ryutaro A, et al. Cytotoxic enhancement of a bispecific diabody by format conversion to tandem single-chain variable fragment (taFv).  J Biol Chem  2011; 286: 1812-1818. 
         46. Anja L, et al. A recombinant bispecific single-chain antibody, CD19×CD3, induces rapid and high lymphoma-directed cytotoxicity by unstimulated T lymphocytes.  Blood  2000; 95(6):2098-2103. 
         47. Weiner G J, et al. The role of T cell activation in anti-CD3× antitumor bispecific antibody therapy.  J. Immunology  1994; 152(5): 2385-2392. 
         48. Dafne M, et al. Improved pharmacokinetics of recombinant bispecific antibody molecules by fusion to human serum albumin.  J Biol Chem  2007; 282: 12650-12660. 
         49. Liu C, et al. Modified host cells and uses thereof, PCT/US2010/0081195. 
         50. Francisco J, et al. Neutrophils Contribute to the Biological Antitumor Activity of Rituximab in a Non-Hodgkin&#39;s Lymphoma Severe Combined Immunodeficiency Mouse Model.  Clin Cancer Res  2003; 9: 5866. 
         51. Kavita M, et al. Selective blockade of inhibitory Fc receptor enables human dendritic cell maturation with IL-12p70 production and immunity to antibody-coated tumor cells.  Proc natl Aca Sci USA  2005; 102(8): 2910-2915. 
         52. Raphael A, et al. Inhibitory Fc receptors modulate in vivo cytoxicity against tumor targets.  Nature Medicine  2000; 6:443-446. 
         53. Milenic E D. Monoclonal antibody-based therapy strategies: providing options for the cancer patient.  Curr Pharm Des.  2002; 8: 1794-1764. 
         54. Grillo-Lopez A J. Anti-CD20 mAbs: modifying therapeutic strategies and outcomes in the treatment of lymphoma patients.  Expert Rev Anticancer Ther.  2002: 2 (3): 323-329. 
         55. Jones K L &amp; Buzdar A U. Evolving novel anti-Her2 strategies.  Lancet Oncol.  2009: 10 (12): 1179-1187. 
         56. Reddy M M, Deshpande A &amp; Sattler M. targeting JAK2 in the therapy of myeloproliferative neoplasms. Exper Opin Ther targets 2012: 3: 313-324. 
         57. Takeuchi K &amp; Ito F. Receptor tyrosine kinases and targeted cancer therapeutics. Biol Pharm Bull. 2011; 34 (12) 1774-1780. 
         58. Roychowdhury S &amp; Talpaz M. Managing resistance in chronic myeloid leukemia. Blood Rev. 2011; (6): 279-290. 
         59. Konnig R. Interactions between MHC molecules and co-receptors of the TCR.  Curr Opin Immunol  2002:14 (1) 75-83. 
         60. Sergeeva A, Alatrash G, He H, Ruisaard K, Lu S, Wygant J, McIntyre B W, Ma Q, Li D, St John L, Clise-Dwyer K &amp; Molldrem J J. An anti-PR1/HLA-A2 T-cell receptor-like antibody mediated complement-dependent cytotoxicity against acute myeloid leukemia progenitor cells.  Blood  2011; 117 (16): 4262-4272). 
         61. Takigawa N, Kiura K &amp; Kishimoto T. Medical Treatment of Mesothelioma: Anything New?  Curr Oncol Rep  2011; DOI 10.1007/s11912-011-0172-1. 
         62. Raja S, Murthy S C &amp; Mason D P. Malignant Pleural Mesothelioma.  Curr Oncol Rep  2011; DOI 10. 1007/s11912-0177-9. 
         63. Gerber J M, Qin L, Kowalski J, Smith D, Griffin C A, Vala M S, Collector M I, Perkins B, Zahurak M, Matsui W, Gocke C D, Sharkis S, Levitsky H &amp; Jones R J. Characterization of chronic myeloid leukemia stem cells. 2011 ; Am J Hematol.  86: 31-37. 
         64. Rezwani K, Yong A S, Savani B N, Mielke S, Keyvanfar K, Gostick E, Price D A, Douek D C &amp; Barrett A J. Graft-versus-leukemia effects associated with detectable Wilms tumor-1 specific T lymphocytes after allogeneic stem-cell transplantation for acute lymphoblastic leukemia.  Blood  2007: 110 (6): 1924-1932. 
         65. Persic L, Roberts A, Wilton J et al. An integrated vector system for the eukaryotic expression of antibodies or their fragments after selection from phage display libraries.  Gene  1997; 187(1): 9-18. 
         66. Cheng L, Xiang J Y, Yan S et al. Modified host cells and uses thereof. PCT/US2010/0081195. 
         67. Lindmo T, Boven E, Cuttitta F, Fedorko J &amp; Bunn P A Jr. Determination of the immunoreactive fraction of radiolabeled monoclonal antibodies by linear extrapolation to binding at infinite antigen excess.  J Immunol Methods.  1984; 72 (1): 77-89. 
         68. Feng M, Zhang J L, Anver M, Hassan R &amp; Ho M. In vivo imaging of human malignant mesothelioma growth orthotopically in the peritoneal cavity of nude mice.  J Cancer  2011; 2: 123-131.