Office Action 1: Non-Final Rejection (2024-06-21)
Rejection types: 112 - Written Description/Enablement

CLAIMS:
Amendments to the Claims: The following listing of claims will replace all prior versions and listings of claims in the case.

1\. (Original) A vector, comprising:
a polynucleotide comprising one or more transcriptional units, wherein each of the one or more transcriptional units is operatively linked to a promoter active in T cells, and wherein the one or more transcriptional units encode:
i. a first engineered signaling polypeptide, wherein said first engineered signaling polypeptide is a chimeric polypeptide comprising an extracellular domain, a transmembrane domain, and a first intracellular signaling domain capable of activating a cellular signaling pathway, wherein said transmembrane domain is covalently attached to said extracellular domain and said first intracellular signaling domain, wherein said transmembrane domain is between 15 and 100 amino acids, wherein said first intracellular signaling domain comprises an intracellular signaling domain that comprises a first ITAM motif, wherein said first intracellular signaling domain is selected from CD79A having at least 90% sequence identity to the sequence of SEQ ID NO:210 and CD79B having at least 90% sequence identity to the sequence of SEQ ID NO:211, and
wherein said extracellular domain comprises a leucine zipper dimerizing motif, and
ii. a second engineered signaling polypeptide comprising a chimeric antigen receptor (CAR) or a recombinant TCR.

2\. (Original) The vector of claim 1, wherein the leucine zipper dimerizing motif is from a jun polypeptide.

3\. (Original) The vector of claim 1, wherein a linker of between 1 and 4 alanine residues is present between the extracellular domain and the transmembrane domain.

4\. (Original) The vector of claim 1, wherein the first intracellular signaling domain comprises a second ITAM motif.

5\. (Currently Amended) The vector of claim 114\]\]. w herein:
the first intracellular signaling domain comprises the intracellular signaling domain from CD79A and the transmembrane domain does not comprise a transmembrane domain from CD79A, or
the first intracellular signaling domain comprises the intracellular signaling domain from CD79B and the transmembrane domain does not comprise a transmembrane domain from CD79B .

6\. (Currently Amended) The vector of claim 1, wherein:
the first intracellular signaling domain comprises the intracellular signaling domain from CD79A and comprises the tvrosine residue corresponding to Y4 and/or Y15 of SEQ ID NO:42. or the first intracellular signaling domain comprises the I\[an \]\]intracellular signaling domain from \[\[of \]\]CD79B y and comprises the tvrosine residue corresponding to Y16 and/or Y27 of SEQ ID NO:21 1.

7-10. (Canceled) 89

11\. (Currently Amended) The vector of claim 6, wherein the intracellular signaling domain from CD79A is at least 95% identical to the sequence of SEQ ID NO:210, or the intracellular signaling domain \[\[of \]\]from CD79B is at least 95% identical to the sequence of SEQ ID NO:211.

12\. (Canceled)

13\. (Currently Amended) The vector of claim 111611, wherein the first engineered signaling polypeptide comprises a second intracellular signaling domain, and wherein:
the first intracellular signaling domain comprises the intracellular signaling domain from CD79A and the second intracellular signaling domain does not comprise an intracellular signaling domain from CD79A, or
the first intracellular signaling domain comprises the intracellular signaling domain from CD79B and the second intracellular signaling domain does not comprise an intracellular signaling domain from CD79B. , , , , .

14\. (Currently Amended) The vector of claim 13, wherein the first intracellular signaling domain comprises the intracellular signaling domain from CD79A, and wherein the second intracellular signaling domain is capable of activating a cellular signaling pathway and is 90% identical to an intracellular signaling domain from a gene selected from CSF3R. IL3RA, IL10RB, ILl i RA, IL17RB, ILI7RC, and IL23R. .

. . . , . , , , , , . .

. . . . . . . . . . . .

. , . . . . . . . . .

. . . .

15\. (Currently Amended) The vector of claim 13. wherein the first intracellular signaling domain comprises the intracellular signaling domain from CD79B. and wherein the second intracellular signaling domain is capable of activating a cellular signaling pathway and is 90% identical to an intracellular signaling domain from a gene selected from CSF2RA, CSF3R. EPOR. IFNAR2, ILiRi, IL6ST. IL9R. ILI2RB2. IL23R.

MPL, MvD88, and OSM, , , y.

16\. (Currently Amended) The vector of claim 1, wherein the first engineered signaling polypeptide is a fusion polypeptide, wherein the fusion polypeptide further comprises a recognition domain that is recognized by a monoclonal antibody approved biologic.
17\. (Original) The vector of claim \[, wherein the vector is a replication incompetent recombinant retroviral particle.

18\. (Original) The replication incompetent recombinant retroviral particle of claim 17, wherein the replication incompetent recombinant retroviral particle is a lentivirus.

19\. (Original) The replication incompetent recombinant retroviral particle of claim 17, wherein the replication incompetent recombinant retroviral particle further comprises a membrane-bound T cell activation element, and wherein the membrane-bound T cell activation element comprises a means for binding to CD3.

20\. (Original) The replication incompetent recombinant retroviral particle of claim 19, wherein the means for binding to CD3 is anti-CD3 antibody, wherein the membrane-bound T cell activation element is fused to a heterologous membrane attachment sequence, and wherein the heterologous membrane attachment sequence is a GPI anchor attachment sequence.

21\. (Currently Amended) The replication incompetent recombinant retroviral particle of claim 17, wherein the replication incompetent recombinant retroviral particle further comprises a heterologous viral envelope protein , , , , , , , .

22\. (Original) The replication incompetent recombinant retroviral particle of claim 17, wherein the replication incompetent recombinant retroviral particle further comprises a heterologous viral envelope protein fused to an anti-CD3 antibody.

23\. (Original) The replication incompetent recombinant retroviral particle of claim 17, wherein the first engineered signaling polypeptide is encoded in a reverse orientation with respect to a cis-acting RNA packaging element of the vector.

24\. (Original) The replication incompetent recombinant retroviral particle of claim 17, wherein the first engineered signaling polypeptide possesses the property of:
a) improving expansion of pre-activated PBMCs transduced with a retroviral particle comprising a nucleic acid construct encoding the first engineered signaling polypeptide when transduced along with a nucleic acid encoding an anti-CD 19 CAR comprising a CD3 zeta intracellular activating domain but no co- stimulatory domain, between day 7 and day 21 of in vitro culturing in the absence of exogenously added cytokines, compared to a control construct identical to the nucleic acid construct comprising the first engineered signaling polypeptide but without the first engineered signaling polypeptide, under identical conditions; and/or
b) expanding pre-activated PBMCs transduced with a nucleic acid construct encoding the first engineered signaling polypeptide at least 2-fold between day 7 and day 21 of in vitro culturing in the absence of exogenously added cytokines, when transduced along with a nucleic acid encoding an anti- CD 19 CAR comprising a CD3 zeta intracellular activating domain but no costimulatory domain.

25\. (Currently Amended) The replication incompetent recombinant retroviral particle of claim 17, wherein the replication incompetent recombinant retroviral particle further comprises a membrane-bound cytokine on the surface of the replication incompetent recombinant retroviral particle, wherein the membrane-bound cytokine comprises a fusion polypeptide of IL-7 and DAF, and wherein the fusion polypeptide comprises the amino acid sequence of SEO ID NO:462.

26\. (Original) A method for genetically modifying a T cell of a subject, wherein the method comprises:
contacting a population of T cells comprising the T cell ex vivo, with a population of replication incompetent recombinant retroviral particles according to claim
19, wherein said contacting is performed for less than 12 hours to facilitate membrane fusion of the T cell to the replication incompetent recombinant retroviral particle, thereby genetically modifying the T cell.

27\. (Original) The method of claim 26, wherein the method further comprises: collecting blood comprising the population of T cells from a subject before contacting the population of T cells ex vivo with the population of replication incompetent recombinant retroviral particles; and
reintroducing the genetically modified T cell into the subject.

28\. (Canceled)

29\. (Original) The method of claim 26, wherein the population of T cells comprises between 90% and 100% resting T cells.

30\. (Original) The method of claim 27, wherein the population of T cells undergoes 4 or fewer cell divisions ex vivo prior to being reintroduced into the subject.

31\. (Canceled) 3 4 5 6 7 8

OFFICE ACTION:
DETAILED ACTION
Notice of Pre-AIA  or AIA  Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Priority
Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. Applicant has not complied with one or more conditions for receiving the benefit of an earlier filing date under 35 U.S.C. 120 as follows:
The later-filed application must be an application for a patent for an invention which is also disclosed in the prior application (the parent or original nonprovisional application or provisional application). The disclosure of the invention in the parent application and in the later-filed application must be sufficient to comply with the requirements of 35 U.S.C. 112(a) or the first paragraph of pre-AIA  35 U.S.C. 112, except for the best mode requirement.  See Transco Products, Inc. v. Performance Contracting, Inc., 38 F.3d 551, 32 USPQ2d 1077 (Fed. Cir. 1994)
The disclosure of the prior-filed application, Application Nos. 62/564,991; ,62/564,253; 62/560,176; 15/644,778; 15/462,855; 62/467,039; 62/360,041 and 62/390,093 fail to provide adequate support or enablement in the manner provided by 35 U.S.C. 112(a) or pre-AIA  35 U.S.C. 112, first paragraph for one or more claims of this application.  The first disclosure of instant SEQ ID NOs: 210 and 211 is found in PCT/US2018/020818 (as SEQ ID NOs 418 and 419, respectively), filed 3/3/2018.  Thus, the instant claims are afforded a priority dated of 3/3/2018.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA  35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-6, 11, 13-27, 29 and 30 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA  35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
The claims encompass vectors and retroviral particles comprising CD79 intracellular signaling domains “with 90% sequence identity” to SEQ ID NOs 210 or 211, e.g. as recited in claim 1.  According to the definitions in the specification, this element can be any domain with the recited functions of intracellular signaling and activating a T cell.  Thus, the claims encompass a very broad genus of substitutions, mutations, deletions, and insertions of the CD79 protein domains.
However, applicant was not in possession of the claimed genera of CD79 intracellular signaling domains with signaling activity as elements of the claimed products in the absence of providing sufficient structural and functional characteristics of the genera of each of these elements broadly encompassed by the instant claim language, coupled with a known or disclosed correlation between function and structure.
However, the instant application has not provided a sufficient description showing possession of the necessary functional characteristics coupled with a known or disclosed correlation between function and structure, or by a combination of such identifying characteristics, sufficient to show the applicant was in possession of the genera of  CD79 intracellular signaling domains with signaling activity, encompassing structurally related or structurally unrelated, functionally related or functionally unrelated molecules, equivalents and variants.
It is not sufficient to define the recombinant molecule by its principal biological activity, e.g. having protein A activity, because an alleged conception having no more specificity than that is simply a wish to know the identity of any material with that biological property.  Colbert v. Lofdahl, 21 USPQ2d, 1068, 1071 (BPAI 1992).
Mere idea or function is insufficient for written description; isolation and characterization at a minimum are required.  The claims do not recite sufficient written description of the structural and functional characteristics coupled with a known or disclosed correlation between function and structure, since the disclosure fails to describe the common attributes or characteristics that identify each member of the genera of CD79 intracellular signaling domains with signaling activity as broadly encompassed by the claimed invention.  Therefore, there is insufficient written description of the claimed genera of intracellular signaling domains in the absence of defining the relevant identifying characteristics such as the structure or other physical and/or chemical characteristics of the claimed genera.
Further, the Court has interpreted 35 U.S.C. §112, first paragraph, to require the patent specification to “describe the claimed invention so that one skilled in the art can recognize what is claimed.  Enzo Biochem, Inc. v. Gen-Probe Inc, 63 USPQ2d 1609 and 1618 (Fed. Cir. 2002).  In evaluating whether a patentee has fulfilled this requirement, our standard is that the patent’s “disclosure must allow one skilled in the art ‘to visualize or recognize the identity of’ the subject matter purportedly described.” Id. (quoting Regents of Univ. of Cal. v. Eli Lilly & Co., 43 USPQ2d 1398 (Fed Cir. 1997)).
The Guidelines for the Examination of Patent Applications Under the 35 U.S.C. 112, ¶ 1 Written Description Requirement make clear that the written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species by actual reduction to practice, reduction to drawings, or by disclosure of relevant, identifying characteristics, i.e., structure or other physical and/or chemical properties, by functional characteristics coupled with a known or disclosed correlation between function and structure, or by a combination of such identifying characteristics, sufficient to show the applicant was in possession of the genus.
It is not sufficient to define a genus without sufficient description of a representative number of species by actual reduction to practice, reduction to drawings, or by disclosure of relevant, identifying characteristics, i.e., structure or other physical and/or chemical properties, by functional characteristics coupled with a known or disclosed correlation between function and structure, or by a combination of such identifying characteristics.  In the absence of sufficient description of a representative number of species by actual reduction to practice, reduction to drawings, or by disclosure of relevant, identifying characteristics, the claimed invention is not described in such a way as to reasonably convey to one skilled in the relevant art that the inventors, at the time the application was filed, had possession of the claimed invention.
Vas-Cath Inc. v. Mahurkar, 19 USPQ2d 1111, makes clear that “applicant must convey with reasonable clarity to those skilled in the art that, as of the filing date sought, he or she was in possession of the invention.  The invention is, for purposes of the written description inquiry, whatever is now claimed.”  (See page 1117.)  The specification does not “clearly allow persons of ordinary skill in the art to recognize that [he or she] invented what is claimed.”  (See Vas-Cath at page 1116.).  Consequently, Applicant was not in possession of the instant claimed invention.  See University of California v. Eli Lilly and Co. 43 USPQ2d 1398.  With respect to reliance upon screening for structural and/or functional equivalents,  the Court has held that the disclosure of screening assays and general classes of compounds was not adequate to describe compounds having the desired activity: without disclosure of which peptides, polynucleotides, or small organic molecules have the desired characteristic, the claims failed to meet the description requirement of § 112.  See University of Rochester v. G.D. Searle & Co., lnc., 69 USPQ2d 1886,1895 (Fed. Cir. 2004).
Applicant is relying upon certain biological activities and the disclosure of a limited number of species (i.e. unmodified CD79A and CD79B signaling domains, 100% identical to SEQ ID NOs 210 and 211) to support entire genera.
The problem here is that the instant specification fails to provide a sufficient disclosure of
each of the modified or mutated CD79 intracellular signaling domains with signaling activity in order for the elements to retain the appropriate structural and functional requirements for intracellular signaling and T cell activation, as broadly encompassed by the claimed invention.   A skilled artisan cannot, as one can do with a fully described genus, visualize or recognize the identity of the members of the genera that exhibit the appropriate structural and functional elements for intracellular signaling and/or T cell activation.  It has been well known that minor structural differences even among structurally related compounds or compositions can result in substantially different biology, expression and activities.
Therefore, there is insufficient written description for genera of CD79 intracellular signaling domains with signaling activity, as broadly encompassed by the claimed invention at the time the invention was made and as disclosed in the specification as filed under the written description provision of 35 USC 112, first paragraph.
Applicant has been reminded that Vas-Cath makes clear that the written description provision of 35 USC 112 is severable from its enablement provision.  (See page 1115).
Conclusion
No claim is allowed.

APPLICANT REBUTTAL: Rem
arks
/Argum
ents
I.
STATUS OF THE CLAIMS
Claims 1-6, 11, 13-27 and 29-30 were pending before this amendment. After entry of this amendment, claims 1-6, 11, 13-27, 29-30, and 32-33 will be pending. Claims 7-10, 12, 28, and 31 were previously canceled. Claims 1 and 19-20 have been amended herein without prejudice or disclaimer to pursuing the subject matter of the previously pending claims in an application claiming priority from the present application. Claims 32 and 33 have been added. Support for the claims can be found throughout the application-as-filed, for example: for claim 1, paragraph [0168]; for claim 19, paragraphs [0347]- [0348]; for claim 20, paragraphs [0347]-[0348]; for claim 32, paragraphs [0179] and [0310]; and for claim 33, paragraphs [0282] and [0310]. No new matter has been added.
II.
INFORMATION DISCLOSURE STATEMENT
Applicant is submitting herewith an information disclosure statement.
III.
PRIORITY
The Office Action states "the instant claims are afforded a priority dated of 3/3/2018." However, there are no rejections in the Office Action based on prior art, and thus the priority dates are not relevant for the instant Office Action or this Response. Thus, Applicant neither confirms nor denies the priority dates asserted in the Office Action.
IV.
35 U.S.C.
112(a)
Claims 1-6, 11, 13-27, and 29-30 stand rejected under 35 U.S.C. 112(a) as failing to comply with the written description requirement for allegedly containing subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor at the time the application was filed had possession of the claimed invention. The Office Action asserts:
The claims encompass vectors and retroviral particles comprising CD79 intracellular signaling domains "with 90% sequence identity" to SEQ ID NOs 210
or 211, e.g. as recited in claim 1. According to the definitions in the specification, this element can be any domain with the recited functions of intracellular signaling and activating a T cell. Thus, the claims encompass a very broad genus of
substitutions, mutations, deletions, and insertions of the CD79 protein domains.
However, applicant was not in possession of the claimed genera of CD79 intracellular signaling domains with signaling activity as elements of the claimed products in the absence of providing sufficient structural and functional characteristics of the genera of each of these elements broadly encompassed by the instant claim language, coupled with a known or disclosed correlation between
function and structure.
Without acquiescing to the rejection, and solely in an effort to expedite prosecution, claim 1 has been amended to recite "wherein the first ITAM motif comprises the sequence YX1X2L/I, wherein X1 is any amino acid and X2 is any amino acid". The sequence of the ITAM motif provides additional structure to the first intracellular signaling domain, and the ITAM motif has a known correlation between function and structure (see, e.g., Isakov. Role of immunoreceptor tyrosine-based activation motif in signal transduction from antigen and Fc receptors. Adv Immunol. 1998:69:183-247). Thus, claim 1 and claims 2- 6, 11, 13-27, 29-30, and 32-33 dependent therefrom are described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor at the time the application was filed had possession of the claimed invention. Applicant respectfully requests withdrawal of the rejections of claims 1-6, 11, 13-27, and 29-30 under 35 U.S.C. 112(a).
Conclusion

NEXT OFFICE ACTION: EXAMINER'S AMENDMENT
An examiner’s amendment to the record appears below. Should the changes and/or additions be unacceptable to applicant, an amendment may be filed as provided by 37 CFR 1.312. To ensure consideration of such an amendment, it MUST be submitted no later than the payment of the issue fee.
The application has been amended as follows:
In the claims;
33. (Currently Amended) The vector of claim 1, wherein the first intracellular signaling domain is CD79B having at least 90% sequence identity to the sequence of SEQ ID NO:211, wherein the first intracellular signaling domain comprises a domain with at least 90% sequence identity to amino acids 16-30 of SEQ ID NO:211.
Claim Rejections - 35 USC § 112
The rejection of the claims as lacking written description is withdrawn in light of amendment of the claims and applicant’s remarks.
Reasons for Allowance
The following is an examiner’s statement of reasons for allowance: the relevant prior art does not teach or reasonably suggest the claimed vectors comprising two signaling polypeptides wherein the leucine sipper-based polypeptide comprises a signaling domain comprising an ITAM of SEQ ID NO: 210 or SEQ ID NO: 211.  Although the proteins these ITAMs were derived from (CD79A, CD79B) were known in the art, the use of these ITAM motifs in the claimed signaling polypeptide is considered to be unpredictable.
Any comments considered necessary by applicant must be submitted no later than the payment of the issue fee and, to avoid processing delays, should preferably accompany the issue fee.  Such submissions should be clearly labeled “Comments on Statement of Reasons for Allowance.”
Conclusion

CITED REFERENCE SUMMARIES: Ref US2018020818: Claims:
1. Use of a replication incompetent recombinant retroviral particle in the manufacture of a kit for genetically modifying a resting T cell or resting NK cell of a subject, wherein the use of the kit comprises: contacting the T cell or NK cell ex vivo, with the replication incompetent recombinant retroviral particle, wherein the replication incompetent recombinant retroviral particle comprises a pseudotyping element on a surface and a membrane-bound T cell activation element on the surface, wherein said contacting facilitates transduction of the T cell or NK cell by the replication incompetent recombinant retroviral particle, thereby producing a genetically modified T cell or NK cell, and wherein the contacting is performed for between 1 and 12 hours.
2. The use of claim 1 , wherein the contacting is performed for between 1 and 6 hours.
3. The use of claim 1 , wherein the membrane -bound T cell activation element is anti-CD3 scFvFc.
4. The use of claim 1 , wherein the replication incompetent recombinant retroviral particle comprises a polynucleotide comprising one or more transcriptional units operatively linked to a promoter active in T cells and/or NK cells, wherein the one or more transcriptional units encode a first polypeptide comprising a chimeric antigen receptor (CAR) and a second polypeptide comprising a T cell lymphoproliferative element, and wherein the genetically modified T cells and/or NK cells are capable of survival in ex vivo culture for at least 7 days in the absence of a target for an ASTR of the CAR and in the absence of exogenous cytokines .
5. The use of claim 4, wherein the T cell lymphoproliferative element comprises a chimeric cytokine receptor.
6. The use of claim 5, wherein the chimeric cytokine receptor comprises IL-7 tethered to the IL-7 receptor alpha, or a fragment thereof that retains the ability to promote proliferation of T cells and/or NK cells, and wherein the chimeric cytokine receptor is constitutively active.
7. The use of claim 6, wherein the chimeric cytokine receptor comprises IL-7 covalently attached to a functional extracellular fragment of the IL-7 receptor capable of binding to IL-7, an IL-7 receptor transmembrane domain, and an IL-7 receptor signaling domain.
8. A method according to claim 4, wherein the CAR is an MRB-CAR.
9. A use according to any one of claims 1 to 8, wherein the replication incompetent recombinant retroviral particle is a lentiviral particles, and wherein the genetically modified cell is a genetically modified T cell.
10. A genetically modified T cell or NK cell made by transducing resting T cells and/or resting NK cells according to a method comprising contacting the resting T cells and/or resting NK cells ex vivo, with replication incompetent recombinant retroviral particles, wherein the replication incompetent recombinant retroviral particles comprise a pseudotyping element on their surface and a membrane -bound T cell activation element on their surface, wherein said contacting facilitates transduction of the resting T cells and/or NK cells by the replication incompetent recombinant retroviral particles, thereby producing genetically modified T cells and/or NK cells, and wherein the contacting is performed for between 1 and 12 hours.
11. The genetically modified T cell or NK cell of claim 10, wherein the contacting is performed for between 1 and 6 hours.
12. The genetically modified T cell or NK cell of claim 10, wherein the membrane-bound T cell activation element is anti-CD3 scFvFc.
13. The genetically modified T cell or NK cell of claim 10, wherein the replication incompetent recombinant retroviral particle comprises a polynucleotide comprising one or more transcriptional units operatively linked to a promoter active in T cells and/or NK cells, wherein the one or more transcriptional units encode a first polypeptide comprising a chimeric antigen receptor (CAR) and a second polypeptide comprising a T cell lymphoproliferative element, and wherein the genetically modified T cells and/or NK cells are capable of survival in ex vivo culture for at least 7 days in the absence of a target for an ASTR of the CAR and in the absence of exogenous cytokines.
14. The genetically modified T cell or NK cell of claim 13, wherein the CAR is an MRB-CAR.
15. The genetically modified T cell or NK cell of claim 13, wherein the polynucleotide further comprise one or more of a Kozak-related sequence, a WPRE element, and a multiple stop sequence.
16. The genetically modified T cell or NK cell of claim 13, wherein the T cell lymphoproliferative element comprises a chimeric cytokine receptor.
17. The genetically modified T cell or NK cell of any one of claim 10 to 16, wherein the replication incompetent recombinant retroviral particle is a lenti viral particle, and wherein the genetically modified cell is a T cell.
18. A method of transducing a lymphocyte with a replication incompetent recombinant retroviral particle comprising: A. culturing a packaging cell in suspension in serum-free media, wherein the packaging cell comprises nucleic acid sequences encoding a packagable RNA genome of the replication incompetent retroviral particle, a REV protein, a gag polypeptide, a pol polypeptide, and a pseudotyping element; B. harvesting the replication incompetent recombinant retroviral particle from the serum-free media; and C. contacting the lymphocyte with the replication incompetent recombinant retroviral particle, wherein the contacting is performed for less than 12 hours, thereby transducing the lymphocyte.
19. The method of claim 18, wherein the packaging cell further comprises nucleic acids encoding an activation element.
20. The method of claim 19, wherein the activation element is an anti-CD3 antibody.
21. The method of claim 20, wherein the anti-CD3 antibody is an anti-CD3 scFvFc.
22. The method of claim 18, wherein the packaging cell is an immortalized cell comprising nucleic acids stably integrated therein that encodes the packagable RNA genome.
23. The method of claim 18, wherein the gag polypeptide and the pol polypeptide are expressed from one or more inducible promoters and wherein the method further comprises during the culturing, adding a transactivator to induce expression of the gag polypeptide and the pol polypeptide from the one or more inducible promoters.
24. The method of claim 18, wherein the replication incompetent recombinant retroviral particle comprises a polynucleotide comprising one or more transcriptional units operatively linked to a promoter active in T cells and/or NK cells, wherein the one or more transcriptional units encode a first polypeptide comprising a chimeric antigen receptor (CAR).
25. The method of claim 24, wherein the CAR is an MRB-CAR.
26. The method of claim 18, wherein the replication incompetent recombinant retroviral particle comprises a polynucleotide comprising one or more transcriptional units operatively linked to a promoter active in T cells and/or NK cells, wherein the one or more transcriptional units encode a lymphoproliferative element.
27. The method of claim 18, wherein the contacting is performed for between 1 and 12 hours.
28. The method of claim 18, wherein the lymphocyte is capable of expanding in vitro in the absence of any exogenous cytokine.
29. The method of any one of claims 18 to 28, wherein the lymphocyte is a resting T cell and wherein the replication incompetent recombinant retroviral particle is a lentiviral particle.
30. A replication incompetent recombinant retroviral particle, comprising: A. a polynucleotide comprising one or more transcriptional units operatively linked to a promoter active in T cells and/or NK cells, wherein the one or more transcriptional units encode a chimeric antigen receptor (CAR); and B. a pseudotyping element and a T cell activation element on its surface, wherein the T cell activation element is not encoded by a polynucleotide in the replication incompetent recombinant retroviral particle, and wherein the T cell activation element is an anti-CD3 scFvFc antibody.
31. The replication incompetent recombinant retroviral particle of claim 30, wherein the replication incompetent retroviral particles further comprise a membrane-bound polypeptide capable of binding to CD28.
32. The replication incompetent recombinant retroviral particle of claim 30, wherein the anti-CD3 scFvFc antibody is fused to a heterologous GPI anchor attachment sequence.
33. The replication incompetent recombinant retroviral particle of claim 30, wherein the one or more transcriptional units further encode a lymphoproliferative element.
34. The replication incompetent recombinant retroviral particle of claim 33, wherein the CAR and the lymphoproliferative element are expressed as separate polypeptides.
35. The replication incompetent recombinant retroviral particle of claim 34, wherein the lymphoproliferative element is a chimeric lymphoproliferative element.
36. The replication incompetent recombinant retroviral particle of claim 35, wherein the chimeric lymphoproliferative element is a constitutively active chimeric cytokine receptor, and wherein expression of the constitutively active chimeric cytokine receptor is under the control of a control element.
37. The replication incompetent recombinant retroviral particle of claim 36, wherein the chimeric cytokine receptor comprises an intracellular signaling domain of an IL-7 receptor, an intracellular signaling domain of an IL-12 receptor, an intracellular signaling domain of an IL-15 receptor, an intracellular signaling domain of an IL-21 receptor, an intracellular signaling domain of an !L-23 receptor, an intracellular signaling domain of an IL-27 receptor, an intracellular signaling domain of a transforming growth factor β (TGF[3) decoy receptor, or CD28.
38. The replication incompetent recombinant retroviral particle of claim 35, wherein the chimeric lymphoproliferative element comprises a cytokine receptor and the cytokine receptor comprises IL-7 tethered to the IL-7 receptor or a fragment thereof that retains the ability to promote proliferation of T cells and/or NK cells, and wherein the chimeric cytokine receptor is constitutively active.
39. The replication incompetent recombinant retroviral particle of claim 38, wherein the chimeric cytokine receptor comprises IL-7 covalently attached to a functional extracellular fragment of the IL-7 receptor capable of binding to IL-7, an IL-7 receptor transmembrane domain, and an IL-7 receptor signaling domain.
40. The replication incompetent recombinant retroviral particle of claim 30, wherein the polynucleotide further comprise one or more of a Kozak-related sequence, a WPRE element, and a multiple stop sequence.
41. The replication incompetent recombinant retroviral particle of any of claims 30-39, wherein the replication incompetent recombinant retroviral particle is a lentiviral particle and the promoter is active in T cells.
42. A replication incompetent recombinant retroviral particle, comprising a polynucleotide comprising one or more transcriptional units operatively linked to a promoter active in T cells and/or NK cells, wherein the one or more transcriptional units encode a first polypeptide comprising a chimeric antigen receptor (CAR) and a second polypeptide comprising a constitutively active chimeric lymphoproliferative element, and wherein the chimeric lymphoproliferative element does not comprise a cytokine tethered to its cognate receptor or tethered to a fragment of its cognate receptor.
43. The replication incompetent recombinant retroviral particle of claim 42, wherein the chimeric lymphoproliferative element comprises a chimeric cytokine receptor.
44. The replication incompetent recombinant retroviral particle of claim 42, wherein expression of the constitutively active chimeric lymphoproliferative element is under of a control element.
45. The replication incompetent recombinant retroviral particle of claim 42, wherein the replication incompetent recombinant retroviral particle further comprises a pseudotyping element on its surface.
46. The replication incompetent recombinant retroviral particle of claim 42, wherein the replication incompetent recombinant retroviral particle further comprises a T cell activation element on its surface, wherein the T cell activation element is not encoded by a polynucleotide in the replication incompetent recombinant retroviral particle.
47. The replication incompetent recombinant retroviral particle of claim 46, wherein the T cell activation element is an anti-CD3 antibody.
48. The replication incompetent recombinant retroviral particle of claim 47, wherein the T cell activation element is an anti-CD3 scFvFc.
49. The replication incompetent recombinant retroviral particle of any one of claims 42 to 47, wherein the replication incompetent recombinant retroviral particle is a lentiviral particle, and wherein the promoter is active in T cells.
50. A genetically modified T cell or NK cell, wherein the T cell or NK cell expresses a first engineered signaling polypeptide comprising a constitutively active chimeric lymphoproliferative element that does not comprise a cytokine tethered to its cognate receptor or tethered to a fragment of its cognate receptor, and a second engineered signaling polypeptide comprising a chimeric antigen receptor (CAR) that includes an antigen-specific targeting region (ASTR), a transmembrane domain, and an intracellular activating domain.
51. The genetically modified T cell or NK cell of claim 50, wherein expression of the first engineered signaling polypeptide is under the control of a control element.
52. The genetically modified T cell or NK cell of claim 50, wherein the cell is a T cell.
53. The genetically modified T cell or NK cell of claim 50, wherein the CAR is an MRB-CAR.
54. A method for transducing resting T cells and/or resting NK cells, comprising contacting the resting T cells and/or resting NK cells ex vivo, with replication incompetent recombinant retroviral particles, wherein the replication incompetent recombinant retroviral particles comprise a pseudotyping element on their surface and a membrane-bound T cell activation element on their surface, wherein said contacting facilitates transduction of the resting T cells and/or NK cells by the replication incompetent recombinant retroviral particles, thereby producing genetically modified T cells and/or NK cells, and wherein the contacting is performed for between 1 and 12 hours.
55. The method of claim 54, wherein the contacting is performed for between 1 and 6 hours.
56. The method of claim 54, wherein the membrane -bound T cell activation element is anti-CD3 scFvFc.
57. The method of claim 54, wherein the replication incompetent recombinant retroviral particle comprises a polynucleotide comprising one or more transcriptional units operatively linked to a promoter active in T cells and/or NK cells, wherein the one or more transcriptional units encode a first polypeptide comprising a chimeric antigen receptor (CAR) and a second polypeptide comprising a T cell lymphoproliferative element, and wherein the genetically modified T cells and/or NK cells are capable of survival in ex vivo culture for at least 7 days in the absence of the target for an ASTR of the CAR and in the absence of exogenous cytokines.
58. The method of claim 57, wherein the T cell lymphoproliferative element comprises a chimeric cytokine receptor.
59. The method of claim 58, wherein the chimeric cytokine receptor comprises IL-7 tethered to the IL-7 receptor alpha, or a fragment thereof that retains the ability to promote proliferation of T cells and/or NK cells, and wherein the chimeric cytokine receptor is constitutively active.
60. The method of claim 59, wherein the chimeric cytokine receptor comprises IL-7 covalently attached to a functional extracellular fragment of the IL-7 receptor capable of binding to IL-7, an IL-7 receptor transmembrane domain, and an IL-7 receptor signaling domain.
61. The method of claim 57, wherein the CAR is an MRB-CAR.
62. A method according to any one of claims 54 to 61, wherein the replication incompetent recombinant retroviral particle is a lenti viral particles, and wherein the genetically modified cell is a genetically modified T cell.
63. The method of claim 57, wherein between 10% and 50% of the resting T cells and/or resting NK cells are transduced.
64. A method for transducing resting T cells and/or resting NK cells, comprising contacting the resting T cells and/or resting NK cells ex vivo, with replication incompetent recombinant retroviral particles in a transduction reaction mixture, wherein the replication incompetent recombinant retroviral particles comprise a pseudotyping element on their surface and a membrane -bound anti-CD3 scFvFc antibody on their surface, wherein said contacting facilitates transduction of the resting T cells and/or NK cells by the replication incompetent recombinant retroviral particles, thereby producing genetically modified T cells and/or NK cells.
65. The method of claim 64, wherein the transduction is carried out for between 1 hour and 20 hours.
66. The method of claim 64, wherein the replication incompetent recombinant retroviral particle comprises a polynucleotide comprising one or more transcriptional units operatively linked to a promoter active in T cells and/or NK cells, wherein the one or more transcriptional units encode a first polypeptide comprising a chimeric antigen receptor (CAR) and a second polypeptide comprising a T cell lymphoproliferative element, and wherein the genetically modified T cells and/or NK cells are capable of survival in ex vivo culture for at least 7 days in the absence of the target for an ASTR of the CAR and in the absence of exogenous cytokines.
67. The method of claim 66, wherein the T cell lymphoproliferative element comprises a chimeric cytokine receptor.
68. The method of claim 67, wherein the chimeric cytokine receptor comprises IL-7 tethered to the IL-7 receptor alpha, or a fragment thereof that retains the ability to promote proliferation of T cells and/or NK cells, and wherein the chimeric cytokine receptor is constitutively active.
69. The method of claim 68, wherein the chimeric cytokine receptor comprises IL-7 covalently attached to a functional extracellular fragment of the IL-7 receptor capable of binding to IL-7, an IL-7 receptor transmembrane domain, and an IL-7 receptor signaling domain.
70. A method according to any one of claims 64 to 69, wherein the replication incompetent recombinant retroviral particle is a lenti viral particles, and wherein the genetically modified cell is a genetically modified T cell.
71. The method of claim 66, wherein between 10% and 50% of the resting T cells and/or resting NK cells are transduced.