Engineered cells

Abstract

The present disclosure relates generally to nucleic acid constructs containing a transgene under the control of a promoter, e.g., a truncated PGK promoter, and engineered cells, e.g., engineered immune cells, including CAR T cells comprising the constructs, and uses thereof in therapeutic applications.

Description

ENGINEERED CELLSFIELD

[0001] The present application claims the benefit of priority to U.S. Provisional Application No. 63 / 594,291 filed on October 30, 2023, the contents of which is hereby incorporated by reference in its entirety.REFERENCE TO SEQUENCE LISTING

[0002] This application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on October 16, 2024, is named AT-059_02WO_SL.xml and is 105,757 bytes in size.

[0003] The present disclosure relates generally to engineered cells, e.g., engineered immune cells, such as chimeric antigen receptor (CAR) T cells, methods of manufacturing such engineered cells, as well as uses in therapeutic applications.BACKGROUND

[0004] Adoptive transfer of immune cells (e.g., T cells) such as chimeric antigen receptor (CAR) modified T cells recognizing a tumor associated antigen have great potentials for treating cancer. The production of autologous CAR T cells, however, is expensive and requires long lead time, with products of varying potency due to the source of patientspecific T cells. Allogeneic CAR T cells generated from healthy donor T cells can be produced and used as off-the shelf products with better product consistency and reduced manufacturing time and costs.

[0005] Most of the CAR T therapies currently approved or in research and development use lentivirus mediated CAR random integration in engineering the CAR T cells. The drawbacks of such random integrations include unknown integration sites, uneven copy number of integrated CAR, and therefore potential increased risk for genome toxicity. Lentivirus preparation and production often requires long lead time, great resources and the outputs are sometimes variable, all of which may increase overall manufacturing time, costs and complexity. Thus, improved methods for engineering immune cell for cell-based therapies are needed.SUMMARY OF THE INVENTION

[0006] The present disclosure provides engineered cells, e.g., engineered immune cells, including CAR T cells comprising and / or expressing a receptor polypeptide and / or a nucleic acid inhibitory agent under the control of a PGK promoter, e.g., a truncated PGK promoter. In one embodiment, the receptor polypeptide is an antigen-specific chimeric antigen receptor (CAR), e.g., a CD19-specific CAR, or a CD70 binding protein, or a chimeric cytokine receptor (CCR). Further provided herein are engineered cells, e.g., engineered immune cells, comprising one or more polynucleotides. In some embodiments, the one or more polynucleotides comprise one or more coding sequences, at least two coding sequences, or two or more coding sequences encoding the receptor polypeptide and / or nucleic acid inhibitory agent.

[0007] In one aspect, the engineered cell, e.g., engineered immune cell, is a CAR T cell. In one embodiment, the CAR T cell is an autologous or allogeneic CAR T cell. In some embodiments, the CAR T cell comprises a recombinant nucleic acid sequence that has been integrated at the human T cell receptor (TCR) alpha chain constant region gene. In one embodiment, the recombinant nucleic acid sequence comprises in a 5’ to 3’ direction: (a) a 5’ region of the TCR alpha chain constant region, (b) a truncated PGK promoter that controls expression of the CAR nucleic acid sequence, (c) a CAR nucleic acid sequence, and (d) a 3' region of the human TCR alpha constant region gene. In a further embodiment, the recombinant nucleic acid sequence further comprises an additional nucleic acid sequence encoding a receptor polypeptide or a nucleic acid inhibitory agent. In other embodiments, the receptor polypeptide is a chimeric cytokine receptor (CCR) or a CD70 binding protein. In an additional embodiment, the nucleic acid inhibitory agent is an RNA interference agent. In one embodiment, the CAR nucleic acid sequence and the additional nucleic acid sequence are linked by a P2A peptide.

[0008] In another embodiment, the truncated PGK promoter comprises at least about a 50, a 60, a 70, a 80, or a 90 nucleotides (or nucleotide) deletion from the 5’ end of the long PGK promoter having the nucleotide sequence of SEQ ID NO: 34. In a further embodiment, the truncated PGK promoter is operably linked to the recombinant nucleic acid sequence, wherein recombinant nucleic acid sequence encodes a receptor polypeptide and / or a nucleic acid inhibitory agent.

[0009] In a further embodiment, the truncated PGK promoter comprises one or more deletions in the sequence shown as SEQ ID NO: 34. In one embodiment, the truncated PGK promoter comprises one or more 5’ region deletions and / or 3’ region deletions.

[0010] In some embodiments, the truncated PGK promoter comprises a nucleotide sequence comprising about a 300, about a 310, about a 320, about a 330, about a340, about a 350, about a 360, about a 370, about a 380, or about a 390 nucleotides (or nucleotide) deletion from the 5’ end of SEQ ID NO: 34 and having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 35. In other embodiments, the truncated PGK promoter comprises a nucleotide sequence comprising about a 210, about a 220, about a 230 about a 240, about a 250, about a 260, about a 270, about a 280, or about a 290 nucleotides (or nucleotide) deletion from the 5’ end of SEQ ID NO: 34 and having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 36. In a further embodiment, the truncated PGK promoter comprises a nucleotide sequence comprising about a 100, about a 110, about a 120, about a 130, about a 140, about a 150, about a 160, about a 170, about a 180, or about a 190 nucleotides (or nucleotide) deletion from the 5’ end of SEQ ID NO: 34 and having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 37. In one other embodiment, the truncated PGK promoter comprises a nucleotide sequence comprising about a 50, about a 60, about a 70, about a 80, about a 90 or about a 100 nucleotides (or nucleotide) deletion from the 5’ end of SEQ ID NO: 34 and having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 38. In one embodiment, the truncated PGK promoter comprises SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38.

[0011] In another embodiment, the CAR T cell exhibits greater expansion and / or cytotoxicity when compared to a CAR T cell that does not comprise a truncated PGK promoter. In another embodiment, the CAR T cell exhibits less exhaustion when compared to a CAR T cell that does not comprise a truncated PGK promoter.

[0012] In a further embodiment, the integrated recombinant nucleic acid sequence prevents or reduces or impairs expression of the TCR alpha constant region gene in the CAR T cell.

[0013] In other embodiments, the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain. In another embodiment, the extracellular domain comprises an antigen binding domain and / or wherein the intracellular domain comprises at least one co-stimulatory domain, as described herein. In a further embodiment, the intracellular domain comprises at least one activating domain, e.g., CD3. In another embodiment, the activating domain is CD3 comprising CD3 zeta or a variant thereof. In other embodiments, the CAR nucleic acid sequence expresses a CAR that binds to BCMA, EGFRvIII, WT-1, CD20, CD23, CD30, CD38, CD33, CD 133, MHC-WT1, TSPAN10, MHC-PRAME, Livl, ADAM10, CHRNA2, LeY, NKGD2D, CS1, CD44v6, ROR1, Claudin-18.2, Mucl7, FAP alpha, Ly6G6D, c6orf23, G6D, MEGT1, NG25, CD19, FLT3, CD70, DLL3, CD52 or CD34. In a further embodiment, the CAR T cell is an autologous or allogeneic CAR T cell.

[0014] In an additional embodiment, the present disclosure provides an isolated population of engineered cells, e.g., engineered immune cells, that comprise a CAR T cell, as described herein.

[0015] In another aspect, the present disclosure provides methods for manufacturing a CAR T cell. In one embodiment, the CAR T cell is an autologous or allogeneic CAR T cell. In one other embodiment, the method comprises introducing into a cell, e.g., an immune cell, a recombinant nucleic acid sequence that comprises in a 5’ to 3’ direction: (a) a 5’ region of the T cell receptor (TCR) alpha chain constant region, (b) a truncated PGK promoter that controls expression of the CAR nucleic acid sequence, (c) a CAR nucleic acid sequence, and (d) a 3' region of the human TCR alpha constant region gene. In another embodiment, the introducing step is performed under conditions sufficient to allow integration of the recombinant nucleic acid sequence at the human T cell receptor (TCR) alpha chain constant region gene, thereby providing a CAR T cell. In another embodiment, the integration is such that the expression of the TCR alpha constant region gene is prevented in the CAR T cell. In a further embodiment, the integrated recombinant nucleic acid sequence prevents expression of the TCR alpha constant region gene in the CAR T cell.

[0016] In other embodiments, the method further comprises performing an expansion assay using the CAR T cell and / or performing a cytotoxicity assay using the CAR T cell. Inone embodiment, the expansion assay is an in vitro or an in vivo expansion assay and / or wherein the cytotoxicity assay is an in vitro or an in vivo cytotoxicity assay.

[0017] In a further embodiment, the method comprises use of a truncated PGK promoter, wherein the truncated PGK promoter comprises at least a 50 nucleotides (or nucleotide) deletion from the 5’ end of the long PGK promoter having the nucleotide sequence of SEQ ID NO: 34. In a further embodiment, the truncated PGK promoter is operably linked to the recombinant nucleic acid sequence, wherein recombinant nucleic acid sequence encodes a receptor polypeptide and / or a nucleic acid inhibitory agent. In another embodiment, the truncated PGK promoter comprises one or more deletions in the sequence shown as SEQ ID NO: 34. In one embodiment, the truncated PGK promoter comprises one or more 5’ region deletions and / or one or more 3’ region deletions. In some embodiments, the truncated PGK promoter comprises a nucleotide sequence comprising about a 300, about a 310, about a 320, about a 330, about a 340, about a 350, about a 360, about a 370, about a 380, or about a 390 nucleotides (or nucleotide) deletion from the 5’ end of SEQ ID NO: 34 and having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 35. In other embodiments, the truncated PGK promoter comprises a nucleotide sequence comprising about a 210, about a 220, about a 230 about a 240, about a 250, about a 260, about a 270, about a 280, or about a 290 nucleotides (nucleotide) deletion from the 5’ end of SEQ ID NO: 34 and having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 36. In a further embodiment, the truncated PGK promoter comprises a nucleotide sequence comprising about a 100, about a 110, about a 120, about a 130, about a 140, about a 150, about a 160, about a 170, about a 180, or about a 190 nucleotides (or nucleotide) deletion from the 5’ end of SEQ ID NO: 34 and having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 37. In one other embodiment, the truncated PGK promoter comprises a nucleotide sequence comprising about a 50, about a 60, about a 70, about a 80, about a 90 or about a 100 nucleotides (nucleotide) deletion from the 5’ end of SEQ ID NO: 34 and having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 38. In one embodiment, the truncated PGK promoter comprises SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38.

[0018] In other embodiments, the method comprises the use of a nucleic acid sequence encoding a CAR, wherein the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain. In another embodiment, the extracellular domain comprises an antigen binding domain and / or wherein the intracellular domain comprises at least one co-stimulatory domain, as described herein. In a further embodiment, the intracellular domain comprises at least one activating domain, e.g., CD3. In another embodiment, the activating domain is CD3 comprising CD3 zeta or a variant thereof.

[0019] In a further aspect, the present disclosure provides nucleic acid constructs, nucleic acid molecules, or polynucleotides comprising a recombinant nucleic acid sequence encoding a CAR. In one embodiment, the recombinant nucleic acid sequence comprises in a 5’ to 3’ direction: (a) a 5’ region of the T cell receptor (TCR) alpha chain constant region,(b) a truncated PGK promoter that controls expression of the CAR nucleic acid sequence,(c) a CAR nucleic acid sequence, and (d) a 3' region of the human TCR alpha constant region gene. In a further embodiment, the recombinant nucleic acid sequence further comprises an additional nucleic acid sequence encoding a receptor polypeptide or a nucleic acid inhibitory agent. In one embodiment, the receptor polypeptide is a CCR or a CD70 binding protein. In another embodiment, the nucleic acid inhibitory agent is an RNA interference agent. In a further embodiment, the CAR nucleic acid sequence and the additional nucleic acid sequence are linked by a P2A peptide.

[0020] In a further embodiment, the nucleic acid constructs, nucleic acid molecules, or polynucleotides described herein comprise a truncated PGK promoter, wherein the truncated PGK promoter comprises at least a 50 nucleotides (or nucleotide) deletion from the 5’ end of the long PGK promoter having the nucleotide sequence of SEQ ID NO: 34. In a further embodiment, the truncated PGK promoter is operably linked to the recombinant nucleic acid sequence, wherein recombinant nucleic acid sequence encodes a receptor polypeptide and / or a nucleic acid inhibitory agent. In another embodiment, the truncated PGK promoter comprises one or more deletions in the sequence shown as SEQ ID NO: 34. In one embodiment, the truncated PGK promoter comprises one or more 5’ region deletions and / or 3’ region deletions. In some embodiments, the truncated PGK promoter comprises a nucleotide sequence comprising about a 300, about a 310, about a 320, about a 330, about a 340, about a 350, about a 360, about a 370, about a 380, or about a 390 nucleotides (or nucleotide) deletion from the 5’ end of SEQ ID NO: 34 and having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to the nucleotidesequence of SEQ ID NO: 35. In other embodiments, the truncated PGK promoter comprises a nucleotide sequence comprising about a 210, about a 220, about a 230 about a 240, about a 250, about a 260, about a 270, about a 280, or about a 290 nucleotides (or nucleotide) deletion from the 5’ end of SEQ ID NO: 34 and having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 36. In a further embodiment, the truncated PGK promoter comprises a nucleotide sequence comprising about a 100, about a 110, about a 120, about a 130, about a 140, about a 150, about a 160, about a 170, about a 180, or about a 190 nucleotides (or nucleotide) deletion from the 5’ end of SEQ ID NO: 34 and having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 37. In one other embodiment, the truncated PGK promoter comprises a nucleotide sequence comprising about a 50, about a 60, about a 70, about a 80, about a 90 or about 100 nucleotides (or nucleotide) deletion from the 5’ end of SEQ ID NO: 34 and having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 38. In one embodiment, the truncated PGK promoter comprises SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38.

[0021] In other embodiments, the nucleic acid constructs, nucleic acid molecules, or polynucleotides described herein comprise a nucleic acid sequence encoding a CAR, wherein the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain. In another embodiment, the extracellular domain comprises an antigen binding domain and / or wherein the intracellular domain comprises at least one costimulatory domain, as described herein. In a further embodiment, the intracellular domain comprises at least one activating domain, e.g., CD3. In another embodiment, the activating domain is CD3 comprising CD3 zeta or a variant thereof.

[0022] In related aspects, provided herein are vectors comprising the polynucleotides and engineered cells, e.g., engineered immune cells, such as engineered T cells or CAR T cells, comprising the vectors or the polynucleotides. In some embodiments, the vector is an adeno-associated virus (AAV) vector or a lentiviral vector.

[0023] In one other aspect, the present disclosure provides CAR T cells, methods of manufacturing engineered cells, e.g., engineered immune cells, nucleic acid constructs, molecules or polynucleotides that make use of a CD70 binding protein. In someembodiments, the CD70-binding protein is a recombinant CD70-binding protein. In some embodiment, the CD70-binding protein comprises an anti-CD70 antibody or an antigenbinding fragment thereof, and a transmembrane domain. In some embodiments, the anti- CD70 antibody comprises the amino acid sequence of SEQ ID NOs: 16 and / or 17. In some embodiments, the anti-CD70 antibody comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the transmembrane domain comprises a CD8 transmembrane domain comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the CD70-binding protein further comprises a CD3 signaling domain. In some embodiments, the CD70-binding protein does not comprise a costimulatory domain. In some embodiments, the CD70-binding protein comprises a CD3 signaling domain comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the CD70-binding protein comprises the amino acid sequence of SEQ ID NO: 19 or 21, or comprises an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 19 or 21, with or without a signal peptide. In some embodiments, the coding sequence that encodes the CD70-binding protein comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 20 or 22.

[0024] In one other aspect, the present disclosure provides CAR T cells, methods of manufacturing engineered cells, e.g., engineered immune cells, nucleic acid constructs, molecules or polynucleotides that make use of a CCR. In some embodiments, the CCR comprises a thrombopoietin receptor / myeloproliferative leukemia protein receptor (TPOR / MPLR) transmembrane and JAK binding domain and an intracellular recruiting domain. In some embodiments, the TPOR / MPLR transmembrane and JAK binding domain comprises the amino acid sequence of SEQ ID NO: 25 or 51. In some embodiments, the intracellular recruiting domain comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, the CCR comprises the amino acid sequence of SEQ ID NO: 27 or 29 or an amino acid sequence having at least about 85%, at least about 90%, at least about 95%, at least about 99% sequence identity to SEQ ID NO: 27 or 29. In some embodiments, the coding sequence that encodes a CCR comprises the nucleotide sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28 or 30.

[0025] In yet related aspects, populations of engineered cells, e.g., engineered immune cells, and pharmaceutical compositions of engineered cells or populations of engineeredcells are provided. In a further aspect, methods of treating a disease condition in a subject are provided comprising administering to the subject an effective amount of the engineered cells, an effective amount of the population of engineered cells, or an effective amount of the pharmaceutical compositions, described throughout. In some embodiments, the subject is a human. In some embodiments, the disease condition is cancer, including without limitation, non-Hodgkin’s lymphoma, large B cell lymphoma (LBCL), follicular lymphoma (FL), T cell lymphoma (TCL), B cell acute lymphoblastic leukemia (BALL), T cell acute lymphoblastic leukemia (TALL), primary central nervous system lymphoma (PCNSL), Mantle cell lymphoma (MCL), chronic lymphocytic leukemia (CLL), peripheral T cell lymphoma (PTCL).

[0026] In some embodiments, the disease condition is an autoimmune disease or disorder, including without limitation, lupus, systemic lupus erythematosus (SLE), lupus nephritis, rheumatoid arthritis, systemic sclerosis, scleroderma, multiple sclerosis (MS), relapse remitting multiple sclerosis (RRMS), secondary progressive multiple sclerosis (SPMS), primary progressive multiple sclerosis (PPMS), neuromyelitis optica spectrum disorder (NMOSD), myasthenia gravis, Sjogren’s syndrome, and myositis. In some embodiments, the immune cell is capable of reducing or depleting B cells, pathogenic B cells, autoreactive B cells, pathogenic T cells, autoreactive T cells, CD70+ T cells, or alloreactive T cells.

[0027] In some embodiments, the engineered cell, e.g., engineered immune cell, is autologous with respect to the subject. In some embodiments, the engineered cell, e.g., engineered immune cell, is allogeneic with respect to the subject.BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIGs. 1A-1C show results of in vitro long-term killing assays of the anti-CD19 CAR T cells with or without co-expressing a chimeric cytokine receptor (CCR).

[0029] FIGs. 2A-2B show results of in vivo cytotoxicity assay of the anti-CD19 CAR T cells with or without co-expressing the CCR (CD19CAR / CCR).

[0030] FIG. 3A depicts data showing reduced in vivo anti-tumor activity of the antiCD 19 CAR T cells co-expressing a CCR (CD 19 CAR / CCR T cells) generated by sitespecific integration as compared to CD 19 CAR / CCR T cells expressing the same CCR and CAR but generated by LVV transduction. FIG. 3B shows higher in vivo numbers of, i.e.,higher persistence of, CAR+ T cells generated by LVV transduction as compared to CAR T cells generated by site-specific integration.

[0031] FIGs. 4A-4D show results of experiments investigating the effects of different promoters driving the expression of the CCR and the CAR in CAR T cells generated by site-specific integration. FIG. 4A shows the median fluorescence intensity (MFI) of the CD 19 CAR / CCR T cells at the end of CAR T cell production. FIG. 4B shows the results of CAR T cells killing of target wild type Raji cells in a flow-based serial restimulation assay. FIG. 4C shows the CAR MFI, and FIG. 4D shows the CAR+ T cell counts, during the flow-based serial restimulation assay.

[0032] FIGs. 5A-5C compare the efficacy of CD 19 CAR / CCR T cells generated by sitespecific integration driven by either the EFS promoter or the PGK SSI-1 promoter, with the CD 19 CAR / CCR T cells generated by LVV transduction, in the standard long-term killing assay (FIG. 5A), or in a single-stimulation flow-based potency assay (FIGs. 5B-5C) against the target Raji cells

[0033] FIGs. 6A-6B compare the in vivo efficacy of CD19 CAR / CCR T cells generated by site-specific integration driven by the EFS promoter, the CypA300 promoter, or the PGK SSI-1 promoter, with the CD19 CAR / CCR T cells generated by LVV using two different CAR T cell doses 2xl06cells (FIG. 6A) or 4xl06cells (FIG. 6B).

[0034] FIG. 7A-7B depict the insertion of a transgene, such as a CAR, into a locus by site-specific integration and homologous recombination.

[0035] FIG. 8A-8D depict examples of different configurations of polynucleotides, nucleic acid constructs or molecules that contain transgenes and promoters.DETAILED DESCRIPTION OF THE INVENTION

[0036] Immune cell-based therapies starting with healthy donor cells can be produced in larger scale with better product consistency and reduced manufacturing time and costs as compared to autologous cell-based therapies. To reduce potential risk of Graft v. Host Diseases (GVHD) resulting from using non-HLA matching donor cells, genetic modifications to reduce or impair T cell receptor (TCR)aP function or activity in the allogeneic immune cells, such as allogeneic CAR T cells is important. Thus, genetic modifications of allogeneic CAR T cells to reduce or impair the expression of genesinvolved in T cell receptor (TCR)aP function or activity while still retaining CAR function is desired. Transgene expression cassettes for genetic modification of cells are often designed to both target one or more genes for disruption and insert a transgene encoding a polypeptide of interest, e.g., a receptor polypeptide such as a chimeric antigen receptor. An important component in such cassettes is the promoter that will control expression of the encoded polypeptide. The present disclosure relates to polynucleotides, nucleic acid constructs or molecules encoding one or more receptor polypeptides and / or nucleic acid inhibitory agents, e.g., RNA interference agents, wherein expression of the one or more receptor polypeptides or agents is under control of a promoter derived from or obtained from the phosphoglycerate kinase (PGK1) gene (PGK). The promoter may be a truncated PGK promoter.

[0037] Compositions and methods for expressing a receptor polypeptide and / or nucleic acid inhibitory agents, e.g., RNA interference agent are provided herein. Also provided are uses of such compositions and methods for improving the functional activities of cells, e.g., immune cells, such as T cells. For instance, the methods can be used to improve the functional activities of CAR-T cells. The methods and compositions provided herein are useful for improving the therapeutic efficacy of therapeutic cell populations, e.g., immune cell populations, including CAR T cells.General Techniques

[0038] The practice of the invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubelet al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995).Definitions

[0039] The term “extracellular ligand-binding domain” as used herein refers to a polypeptide that is capable of binding a ligand or capable of interacting with a cell surface molecule. For example, the extracellular ligand-binding domain may be chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state.

[0040] The term “stalk domain” or “hinge domain” are used interchangeably herein to refer to any polypeptide that functions to link the transmembrane domain to the extracellular ligand-binding domain. In particular, stalk domains are used to provide more flexibility and accessibility for the extracellular ligand-binding domain.

[0041] The term “intracellular signaling domain” refers to the portion of a protein which transduces the effector signal function signal and directs the cell to perform a specialized function.

[0042] A “co-stimulatory ligand” refers to a molecule on an antigen presenting cell that specifically binds a cognate co-stimulatory signal molecule on a T cell, thereby providing a signal which, in addition to the primary signal provided by, for instance, binding of a TCR / CD3 complex with an MHC molecule loaded with peptide, mediates a T cell response, including, but not limited to, proliferation activation, differentiation and the like. A co- stimulatory ligand can include but is not limited to CD7, B7-1 (CD80), B7-2 (CD86), PD- Ll, PD-L2, 4-1BBL, OX40L, inducible costimulatory igand (ICOS-L), intercellular adhesion molecule (ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin P receptor, 3 / TR6, ILT3, ILT4, an agonist or antibody that binds Toll ligand receptor and a ligand that specifically binds with B7-H3. A co-stimulatory ligand alsoencompasses, inter alia, an antibody that specifically binds with a co-stimulatory molecule present on a T cell, such as but not limited to, CD27, CD28, 4-1BB, 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LTGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83.

[0043] An “antibody” is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab’, F(ab’)2, Fv), single chain (scFv) and domain antibodies (including, for example, shark and camelid antibodies), and fusion proteins comprising an antibody, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site. An antibody includes an antibody of any class, such as IgG, IgA, IgE, IgD, or IgM (or sub-class thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant region of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2. The heavy-chain constant regions that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0044] The term “antigen binding fragment” or “antigen binding portion” of an antibody, as used herein, refers to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen. Antigen binding functions of an antibody can be performed by fragments of an intact antibody. Examples of binding fragments encompassed within the term “antigen binding fragment” of an antibody include Fab; Fab’; F(ab’)2; an Fd fragment consisting of the VH and CHI domains; an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a single domain antibody (dAb) fragment (Ward et al., Nature 341 :544-546, 1989), and an isolated complementarity determining region (CDR).

[0045] An antibody, an antigen binding fragment, an antibody conjugate, or a polypeptide that “specifically binds” to a target (e.g., a protein) is a term well understood in the art, andmethods to determine such specific binding are also well known in the art. A molecule is said to exhibit “specific binding” if it reacts or associates more frequently, more rapidly, with greater duration and / or with greater affinity with a particular cell or substance than it does with alternative cells or substances. An antibody “specifically binds” to a target if it binds with greater affinity, avidity, more readily, and / or with greater duration than it binds to other substances. For example, an antibody that specifically binds to a CD 19 epitope is an antibody that binds this epitope with greater affinity, avidity, more readily, and / or with greater duration than it binds to other CD 19 epitopes or non-CD19 epitopes. It is also understood that by reading this definition, for example, an antibody (or moiety or epitope) that specifically binds to a first target may or may not specifically bind to a second target. As such, “specific binding” does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to binding means specific binding.

[0046] A “variable region” of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. As known in the art, the variable regions of the heavy and light chain each consist of four framework regions (FR) connected by three complementarity determining regions (CDRs) also known as hypervariable regions. The CDRs in each chain are held together in close proximity by the FRs and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies. There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda MD)); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al., 1997, J. Molec. Biol. 273:927-948). As used herein, a CDR may refer to CDRs defined by either approach or by a combination of both approaches.

[0047] A “CDR” of a variable domain are amino acid residues within the variable region that are identified in accordance with the definitions of the Kabat, Chothia, the accumulation of both Kabat and Chothia, AbM, contact, and / or conformational definitions or any method of CDR determination well known in the art. Antibody CDRs may be identified as the hypervariable regions originally defined by Kabat et al. See, e.g., Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington D.C. The positions of the CDRs may also be identified as the structural loop structures originally described by Chothia and others. See, e.g., Chothia et al., Nature342:877-883, 1989. Other approaches to CDR identification include the “AbM definition,” which is a compromise between Kabat and Chothia and is derived using Oxford Molecular’s AbM antibody modeling software (now Accelrys®), or the “contact definition” of CDRs based on observed antigen contacts, set forth in MacCallum et al., J. Mol. Biol., 262:732-745, 1996. In another approach, referred to herein as the “conformational definition” of CDRs, the positions of the CDRs may be identified as the residues that make enthalpic contributions to antigen binding. See, e.g., Makabe et al., Journal of Biological Chemistry, 283: 1156-1166, 2008. Still other CDR boundary definitions may not strictly follow one of the above approaches, but will nonetheless overlap with at least a portion of the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. As used herein, a CDR may refer to CDRs defined by any approach known in the art, including combinations of approaches. The methods used herein may utilize CDRs defined according to any of these approaches. For any given embodiment containing more than one CDR, the CDRs may be defined in accordance with any of Kabat, Chothia, extended, AbM, contact, and / or conformational definitions.

[0048] As used herein, "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the invention may be made by the hybridoma method first described by Kohler and Milstein, Nature 256:495, 1975, or may be made by recombinant DNA methods such as described in U.S. Pat. No. 4,816,567. The monoclonal antibodies may also be isolated from phage libraries generated using the techniques described in McCafferty et al., Nature 348:552-554, 1990, for example.

[0049] As used herein, "humanized" antibody refers to forms of non-human (e.g. murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof(such as Fv, Fab, Fab’, F(ab')2 or other antigen binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. In one aspect, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Preferred are antibodies having Fc regions modified as described in WO 99 / 58572. Other forms of humanized antibodies have one or more CDRs (CDR LI, CDR L2, CDR L3, CDR Hl, CDR H2, or CDR H3) which are altered with respect to the original antibody, which are also termed one or more CDRs “derived from” one or more CDRs from the original antibody.

[0050] As used herein, “operably linked” means a linkage between two or more nucleic acid sequence or amino acid sequence elements. For instance, a promoter nucleic acid sequence that is operably linked to a nucleic acid sequence encoding a polypeptide allows the promoter sequence to control expression of the nucleic acid sequence encoding the polypeptide. A first sequence element may be operably linked to a second sequence element in a contiguous or non-contiguous manner. Where two nucleic acid elements are operably linked, the coding region remain in the same reading frame.

[0051] As used herein, “expression control sequence” means a nucleic acid sequence that directs transcription of a nucleic acid. An expression control sequence can be a promoter, such as a constitutive or an inducible promoter, or an enhancer. The expression control sequence is operably linked to the nucleic acid sequence to be transcribed.

[0052] “Promoter” and “promoter sequence” are used interchangeably and refer to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. A functional RNA can be an mRNA transcript that encodes a protein or a non-mRNA, e.g., miRNA, shRNA, or other types of interfering RNAs. In general, a coding sequence is located 3' to a promoter sequence. It is understood by those skilled in the art that different promoters can direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions.

[0053] As used herein, the terms “DNA sequences,” “nucleic acid sequences,” “nucleotide sequences,” and “polynucleotide sequences” are used interchangeably. As used herein, the terms “polynucleotides,” “DNA molecules,” and “nucleic acid molecules” are used interchangeably.

[0054] As used herein, “human antibody” means an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and / or which has been made using any of the techniques for making human antibodies known to those skilled in the art or disclosed herein. This definition of a human antibody includes antibodies comprising at least one human heavy chain polypeptide or at least one human light chain polypeptide. One such example is an antibody comprising murine light chain and human heavy chain polypeptides. Human antibodies can be produced using various techniques known in the art. In one embodiment, the human antibody is selected from a phage library, where that phage library expresses human antibodies (Vaughan et al., Nature Biotechnology, 14:309-314, 1996; Sheets et al., Proc. Natl. Acad. Sci. (USA) 95:6157- 6162, 1998; Hoogenboom and Winter, J. Mol. Biol., 227:381, 1991; Marks et al., J. Mol. Biol., 222:581, 1991). Human antibodies can also be made by immunization of animals into which human immunoglobulin loci have been transgenically introduced in place of the endogenous loci, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. This approach is described in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016. Alternatively, the human antibody may be prepared by immortalizing human B lymphocytes that produce an antibody directed against a target antigen (such B lymphocytes may be recovered from an individual or from single cell cloning of the cDNA, or may have been immunized in vitro). See, e.g., Cole et al. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77, 1985; Boerner et al., J. Immunol., 147 (l):86-95, 1991; and U.S. Pat. No. 5,750,373.

[0055] The term “chimeric antibody” is intended to refer to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.

[0056] A “monovalent antibody” comprises one antigen binding site per molecule (e.g., IgG or Fab). In some instances, a monovalent antibody can have more than one antigen binding sites, but the binding sites are from different antigens.

[0057] A “bivalent antibody” comprises two antigen binding sites per molecule (e.g., IgG). In some instances, the two binding sites have the same antigen specificities. However, bivalent antibodies may be bispecific.

[0058] Antibodies of the invention can be produced using techniques well known in the art, e.g., recombinant technologies, phage display technologies, synthetic technologies or combinations of such technologies or other technologies readily known in the art (see, for example, Jayasena, S.D., Clin. Chem., 45: 1628-50, 1999 and Fellouse, F.A., et al, J. Mol. Biol., 373 (4): 924-40, 2007).

[0059] As known in the art, “polynucleotide,” or “nucleic acid,” as used interchangeably herein, refer to chains of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a chain by DNA or RNA polymerase. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the chain. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, “caps”, substitution of one or more of the naturally occurring nucleotides with an analog, intemucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals,radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide(s). Further, any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid supports. The 5’ and 3’ terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups. Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2’-O-methyl-, 2’-O-allyl, 2’-fluoro- or 2’ -azido-ribose, carbocyclic sugar analogs, alpha- or beta-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(O)S(“thioate”), P(S)S (“dithioate”), (0)NR2(“amidate”), P(O)R, P(O)OR’, CO or CH2(“formacetal”), in which each R or R’ is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

[0060] As used herein, “transfection” refers to the uptake of exogenous or heterologous RNA or DNA by a cell. A cell has been “transfected” by exogenous or heterologous RNA or DNA when such RNA or DNA has been introduced inside the cell. A cell has been “transformed” by exogenous or heterologous RNA or DNA when the transfected RNA or DNA effects a phenotypic change. The transforming RNA or DNA can be integrated (covalently linked) into chromosomal DNA making up the genome of the cell.

[0061] As used herein, the term “homology arms” refers to nucleic acid sequences that are disposed at the 5’ end and the 3’ end of a polynucleotide that has a sequence encoding a transgene of interest. In general, the homology arms have homology to a region of the genome that comprises a nuclease cleavage site that is located within a target gene of interest, e.g., a gene that is involved in T cell receptor (TCR)aP function or activity. For instance, the homology arms are designed to have homology to regions of the genome thatflank the nuclease cleavage site. The homology arms may be designed to have a suitable length such that they promote the insertion of the polynucleotide at the nuclease cleavage site within the target gene of interest. For instance, the homology arms can comprise at least or about 50 base pairs, at least or about 75 base pairs, at least or about 100 base pairs, at least or about 125 base pairs, at least or about 150 base pairs, at least or about 175 base pairs, at least or about 200 base pairs, at least or about 300 base pairs, at least or about 400 base pairs, at least or about 500 base pairs, at least or about 600 base pairs, at least or about 700 base pairs, at least or about 800 base pairs, at least or about 900 base pairs, or at least or about 1000 base pairs.

[0062] As used herein, “transformation” refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” or “recombinant” or “transformed” organisms.

[0063] As known in the art, a “constant region” of an antibody refers to the constant region of the antibody light chain or the constant region of the antibody heavy chain, either alone or in combination.

[0064] As used herein, “substantially pure” refers to material which is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or even at least 99% pure (i.e., free from contaminants).

[0065] A “host cell” includes an individual cell or cell culture that can be or has been a recipient for vector(s) for incorporation of polynucleotide inserts. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide(s) of this invention.

[0066] As known in the art, the term “Fc region” is used to define a C-terminal region of an immunoglobulin heavy chain. The “Fc region” may be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. The numbering of the residues in the Fc region is that of the EU index as in Kabat. Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md., 1991. The Fc region of an immunoglobulin generally comprises two constant regions, CH2 and CH3.

[0067] As used in the art, “Fc receptor” and “FcR” describe a receptor that binds to the Fc region of an antibody. The preferred FcR is a native sequence human FcR. Moreover, a preferred FcR is one which binds an IgG antibody (a gamma receptor) and includes receptors of the FcyRI, FcyRII, and FcyRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcyRII receptors include FcyRIIA (an "activating receptor") and FcyRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in the cytoplasmic domains thereof. FcRs are reviewed in Ravetch and Kinet, Ann. Rev. Immunol., 9:457-92, 1991; Capel et al., Immunomethods, 4:25-34, 1994; and de Haas et al., J. Lab. Clin. Med., 126:330-41, 1995. “FcR” also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol., 117:587, 1976; and Kim et al., J. Immunol., 24:249, 1994).

[0068] The term “compete”, as used herein with regard to an antibody, means that a first antibody, or an antigen binding fragment (or portion) thereof, binds to an epitope in a manner sufficiently similar to the binding of a second antibody, or an antigen binding portion thereof, such that the result of binding of the first antibody with its cognate epitope is detectably decreased in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody. The alternative, where the binding of the second antibody to its epitope is also detectably decreased in the presence of the first antibody, can, but need not be the case. That is, a first antibody can inhibit the binding of a second antibody to its epitope without that second antibody inhibiting the binding of the first antibody to its respective epitope. However, where each antibody detectably inhibits the binding of the other antibody with its cognate epitope or ligand, whether to the same, greater, or lesser extent, the antibodies are said to “cross-compete” with each other for binding of their respective epitope(s). Both competing and cross-competing antibodies are encompassed by the invention. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope, or portion thereof), the skilled artisan would appreciate, based upon the teachings provided herein, that such competing and / or cross-competing antibodies are encompassed and can be useful for the methods disclosed herein.

[0069] As used herein “autologous” means that cells, a cell line, or population of cells used for treating patients are originating from said patient.

[0070] As used herein “allogeneic” means that cells or population of cells used for treating patients are not originating from said patient but from a donor.

[0071] As used herein, the term “endogenous” refers to any material from or produced inside an organism, cell, tissue or system.

[0072] As used herein, the term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue or system. In some embodiments, an exogenous or recombinant sequence or exogenous or recombinant protein is not a naturally occurring sequence or protein and not endogenous or natural to the cell, tissue or organism.

[0073] As used herein, “immune cell” refers to a cell of hematopoietic origin functionally involved in the initiation and / or execution of innate and / or adaptative immune response. Examples of immune cells include T cells, e.g., alpha / beta T cells and gamma / delta T cells, regulatory T (Treg) cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes.

[0074] As used herein, “treatment” is an approach for obtaining a beneficial or desired clinical result. For purposes of the instant disclosure, beneficial or desired clinical results include, but are not limited to, one or more of the following: reducing the proliferation of (or destroying) neoplastic or cancerous cells, inhibiting metastasis of neoplastic cells, shrinking or decreasing the size of tumor, remission of a disease (e.g., cancer), decreasing symptoms resulting from a disease (e.g., cancer), increasing the quality of life of those suffering from a disease (e.g., cancer), decreasing the dose of other medications required to treat a disease (e.g., cancer), delaying the progression of a disease (e.g., cancer), curing a disease (e.g., cancer), and / or prolong survival of subjects having a disease (e.g., cancer).

[0075] “Ameliorating” means a lessening or improvement of one or more symptoms as compared with not administering a treatment. “Ameliorating” also includes shortening or reduction in duration of a symptom. As used herein, an “effective dosage” or “effective amount” of drug, compound, or pharmaceutical composition is an amount sufficient to effect any one or more beneficial or desired results. For prophylactic use, beneficial or desired results include eliminating or reducing the risk, lessening the severity, or delaying the outset of the disease, including biochemical, histological and / or behavioral symptoms ofthe disease, its complications and intermediate pathological phenotypes presenting during development of the disease. For therapeutic use, beneficial or desired results include clinical results such as reducing incidence or amelioration of one or more symptoms of various diseases or conditions (such as for example cancer), decreasing the dose of other medications required to treat the disease, enhancing the effect of another medication, and / or delaying the progression of the disease. An effective dosage can be administered in one or more administrations. For purposes of the instant disclosure, an effective dosage of drug, compound, or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly. As is understood in the clinical context, an effective dosage of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an “effective dosage” can be considered in the context of administering one or more therapeutic agents, and a single agent can be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result can be or is achieved.

[0076] As used herein, a “subject” is any mammal, e.g., a human, or a monkey. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats. In an exemplary embodiment, the subject is a human. In an exemplary embodiment, the subject is a monkey, e.g., a cynomolgus monkey.

[0077] As used herein, the term “nucleic acid construct”, “recombinant construct”, or “expression cassette” are used interchangeably and refers to single or double- stranded nucleic acid molecules or polynucleotides. Such constructs are recombinant and include a combination of different nucleic acid fragments including, without limitation, promoter and coding sequences. In general, this combination of fragments is not found in nature. For example, the promoter sequence of one gene may be combined with the coding sequence of another gene. Constructs may be used alone or in combination with a vector. In one embodiment, an expression cassette is an expression unit(s) of one or more coding sequences operably linked to a promoter. In some embodiments, the expression of multiple coding sequences can be driven by one promoter and the more than one coding sequences are linked by the sequence encoding a 2 A peptide, e.g., P2A or T2A. In some embodiments, the expression of multiple coding sequences occurs by ribosome skipping. As an example, a bicistronic expression cassette allows expression of two proteins from the same RNA transcript driven by one promoter. In some embodiments, the expression of the multiplecoding sequences can be driven by multiple promoters, each promoter operably linked to a coding sequence.

[0078] As used herein, to “functionally express” a gene means that a gene is expressed and that expression yields a functioning gene end product. For example, if a gene encodes a protein, then a cell functionally expresses the gene if expression of the gene ultimately produces a properly functioning protein. Thus, if a gene is not transcribed, or expression of the gene ultimately produces an RNA that is not translated or translation yields only a nonfunctioning protein, e.g., the protein does not fold correctly or is not transported to its site of action (e.g. membrane, for membrane-bound proteins), for example, then the gene is not functionally expressed. Functional expression can be measured directly (e.g. by assaying for the gene product itself) or indirectly (e.g. by assaying for the effects of the gene product).

[0079] As used herein, “vector” or “recombinant vector” means a construct, which is capable of delivering, and, preferably, expressing, one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells. Vectors may include, without limitation, viral vectors, recombinant AAV vectors and other known vectors suitable for delivering a transgene to a cell, e.g., an immune cell. Vectors can be based on different viruses including, without limitation, retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses (AAVs).

[0080] An “antigen binding protein” comprises one or more antigen binding domains. An “antigen binding domain” as used herein means any polypeptide that binds a specified target antigen. In some embodiments, the antigen binding domain binds to an antigen on a tumor cell. In some embodiments, the antigen binding domain binds to an antigen on a cell involved in a hyperproliferative disease or to a viral or bacterial antigen.

[0081] Antigen binding domains include, but are not limited to, antibody binding regions that are immunologically functional fragments. The term “immunologically functional fragment” (or “fragment”) of an antigen binding domain is a species of antigen binding domain comprising a portion (regardless of how that portion is obtained or synthesized) of an antibody that lacks at least some of the amino acids present in a full-length chain, butwhich is still capable of specifically binding to a target antigen. Such fragments are biologically active in that they bind to the target antigen and can compete with other antigen binding domains, including intact antibodies, for binding to a given epitope.

[0082] Immunologically functional immunoglobulin fragments include, but are not limited to, scFv fragments, Fab fragments (Fab', F(ab')2, and the like), one or more complementarity determining regions (“CDRs”), a diabody (heavy chain variable domain on the same polypeptide as a light chain variable domain, connected via a short peptide linker that is too short to permit pairing between the two domains on the same chain), domain antibodies, bivalent antigen binding domains (comprises two antigen binding sites), multispecific antigen binding domains, and single-chain antibodies. These fragments can be derived from any mammalian source, including but not limited to human, mouse, rat, camelid or rabbit. As will be appreciated by one of skill in the art, an antigen binding domain can include non-protein components.

[0083] The variable regions typically exhibit the same general structure of relatively conserved framework regions (FR) joined by the 3 hypervariable regions (CDRs). The CDRs from the two chains of each pair typically are aligned by the framework regions, which can enable binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chain variable regions typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. By convention, CDR regions in the heavy chain are typically referred to as HC CDR1, CDR2, and CDR3. The CDR regions in the light chain are typically referred to as LC CDR1, CDR2, and CDR3.

[0084] In some embodiments, antigen binding domains comprise one or more complementarity binding regions (CDRs) present in the full-length light or heavy chain of an antibody, and in some embodiments comprise a single heavy chain and / or light chain or portion thereof. These fragments can be produced by recombinant DNA techniques or can be produced by enzymatic or chemical cleavage of antigen binding domains, including intact antibodies.

[0085] In some embodiments, the antigen binding domain is an antibody or fragment thereof, including one or more of the complementarity determining regions (CDRs) thereof. In some embodiments, the antigen binding domain is a single chain variable fragment (scFv), comprising light chain CDRs: CDR1, CDR2 and CDR3, and heavy chain CDRs: CDR1, CDR2 and CDR3.

[0086] The assignment of amino acids to each of the framework, CDR, and variable domains is typically in accordance with numbering schemes of Kabat numbering (see, e.g., Kabat et al. in Sequences of Proteins of Immunological Interest, 5th Ed., NIH Publication 91-3242, Bethesda Md. 1991), Chothia numbering (see, e.g., Chothia & Lesk, (1987), J Mol Biol 196: 901-917; Al-Lazikani et al., (1997) J Mol Biol 273: 927-948; Chothia et al., (1992) J Mol Biol 227: 799-817; Tramontane et al., (1990) J Mol Biol 215(1): 175-82; and U.S. Pat. No. 7,709,226), contact numbering, the AbM scheme (Antibody Modeling program, Oxford Molecular) or the AHo system (Honneger and Pluckthun, J Mol Biol (2001) 309(3):657-70).

[0087] In some embodiments, the antigen binding domain is a recombinant antigen receptor. The term “recombinant antigen receptor” as used herein refers broadly to a non- naturally occurring surface receptor that comprises an extracellular antigen-binding domain or an extracellular ligand-binding domain, a transmembrane domain and an intracellular domain. In some embodiments, the recombinant antigen receptor is a chimeric antigen receptor (CAR). Chimeric antigen receptors (CARs) are well-known in the art. A CAR is a fusion protein that comprises an antigen recognition moiety, a transmembrane domain and T cell activation domains (see, e.g., Eshhar et al., Proc. Natl. Acad. Sci. USA, 90(2): 720-724 (1993)).

[0088] In some embodiments, the intracellular domain of a recombinant antigen receptor comprises a co-stimulatory domain and an ITAM-containing domain. In some embodiments, the intracellular domain of a recombinant antigen receptor comprises an intracellular protein or a functional variant thereof (e.g., truncation(s), insertion(s), deletion(s) or substitution(s)).

[0089] The term “extracellular ligand-binding domain” or “extracellular antigen-binding domain” as used herein refers to a polypeptide that is capable of binding a ligand or an antigen. Preferably, the domain will be capable of interacting with a cell surface molecule, such as a ligand or a surface antigen. For example, the extracellular ligand-binding or antigen-binding domain can be chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state, e.g., a tumor-specific antigen. In some embodiments, the antigen-binding domain comprises an antibody, or an antigen binding fragment or an antigen binding portion of an antibody. In some embodiments, the antigen binding domain comprises an Fv or scFv, an Fab or scFab, anF(ab’)2 or a scF(ab’)2, an Fd, a monobody, a affibody, a camelid antibody, a VHH antibody, a single domain antibody, or a darpin. In some embodiments, the ligand-binding domain comprises a partner of a binding pair, such as a ligand that binds to a surface receptor, or an ectodomain of a surface receptor that binds to a ligand.

[0090] The term “stalk domain” or “hinge domain” are used interchangeably herein to refer to any oligo- or polypeptide that functions to link the transmembrane domain to the extracellular ligand-binding domain. In particular, stalk domains or hinge domains are often used to provide more flexibility and accessibility for the extracellular ligand-binding domain.

[0091] The term “intracellular signaling domain” refers to the portion of a protein which transduces the effector signal function signal and directs the cell to perform a specialized function.

[0092] As used herein, “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” includes any material which, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject's immune system. Examples include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil / water emulsion, and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline (PBS) or normal (0.9%) saline. Compositions of the instant disclosure comprising such carriers are formulated by well- known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy 21 st Ed. Mack Publishing, 2005).

[0093] Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to plus or minus 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10% that value or parameter per se. For example, description referring to “about X” includes description of “X .” Numeric ranges are inclusive of the numbers defining the range.

[0094] It is understood that wherever embodiments are described herein with the language “comprising,” otherwise analogous embodiments described in terms of “consisting of and / or “consisting essentially of’ are also provided.

[0095] Where aspects or embodiments of the instant disclosure are described in terms of a Markush group or other grouping of alternatives, the instant disclosure encompasses not only the entire group listed as a whole, but also each member of the group individually and all possible subgroups of the main group, and also the main group absent one or more of the group members. The instant disclosure also envisages the explicit exclusion of one or more of any of the group members in the disclosed and / or claimed embodiments.

[0096] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. In case of conflict, the present specification, including definitions, will control. Throughout this specification and claims, the word “comprise,” or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0097] Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the instant disclosure. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0098] Provided herein are engineered cells, e.g., engineered immune cells, such as engineered T cells, comprising and / or expressing an antigen-specific chimeric antigen receptor (CAR), e.g., a CD19-specific CAR, and a CD70-binding protein, and optionally comprising and / or expressing a chimeric cytokine receptor (CCR) and / or a nucleic acid inhibitory agent, e.g., an RNA interference agent. Also provided herein are engineered cells, e.g., engineered immune cells, such as engineered T cells, comprising and / or expressing an antigen-specific CAR, e.g., a CD19-specific CAR, and a chimeric cytokine receptor (CCR), and optionally comprising and / or expressing a CD70-binding protein and / or a nucleic acid inhibitory agent, e.g., an RNA interference agent. In certain embodiments, the engineered cells, e.g., engineered immune cells, are human. In other embodiments, the engineered cells, e.g., engineered immune cells, are derived or obtained from human cells, e.g., human immune cells.

[0099] Further provided herein are polynucleotides comprising one or more coding sequences encoding the antigen-specific CAR, e.g., a CD19-specific CAR, and the CD70-binding protein or the CCR and / or nucleic acid inhibitory agent, e.g., an RNA interference agent. In certain embodiments, the antigen-specific CAR, e.g., CD19-specific CAR, and the CD70-binding protein or CCR and / or nucleic acid inhibitory agent, e.g., an RNA interference agent, are expressed from a bicistronic expression cassette and are linked by a P2A or T2A self-cleaving peptide. In certain aspects, the antigen-specific cells, e.g., CD 19- specific immune cells, such as CD19-specific CAR T cells, exhibit enhanced cytotoxicity and potency against antigen-positive and / or antigen-negative / CD70 positive cells, e.g., CD 19-positive and / or CD 19 negative / CD70 positive hematologic tumor cells, reduced rejection by the host or patient alloreactive immune cells, and increased persistence and cell expansion as compared to the same antigen-specific cells, e.g., CD19-specific immune cells, such as CD19-specific CAR T cells, without expressing the CD70-binding protein and / or the CCR. In certain embodiments, the expression of the one or more coding sequences or the bicistronic expression cassette is driven by a PGK promoter, especially a truncated PGK promoter.

[0100] In certain aspects, the polynucleotides provided herein are cloned into a lentiviral vector (LVV) and introduced into the engineered cells, e.g., engineered immune cells, such as peripheral blood mononuclear cells (PBMCs), by lentiviral transduction. Transduction of LVV constructs into PBMCs results in engineered cells with randomly integrated transgene(s) in the host cell genome. Alternatively, the transgene(s) can also be introduced into cells by site-specific integration (SSI) into a predetermined genetic locus. Site-specific integration has the added benefit of ensuring the uniformity of the insertion site of the transgene(s) in the genome and reduced number of integration events as compared to LVV random integration. Thus, in certain aspects, the polynucleotides provided herein are integrated into a predetermined locus in the genome of the engineered cells, e.g., engineered immune cells, such as CD19-specific CAR T cells. In certain embodiments, the predetermined genetic locus is the T cell receptor alpha chain constant region (TRAC) locus. In certain embodiments, the predetermined genetic locus is the CD52 gene locus.

[0101] It was surprisingly demonstrated that a PGK promoter, or a truncated PGK promoter described herein (e.g., the PGK SSI promoters) can convey benefits to engineered cells (e.g., engineered immune cells), for example, including CAR T cells, e.g., providing enhanced CAR T cell expansion and / or enhanced in vitro or in vivo cytotoxicity against target tumor cells, when the transgene(s) is introduced by site-specific integration. Thus, in certain embodiments, the expression of the transgene(s), e.g., the one or more codingsequences, e.g., in a bi-cistronic or multi-cistronic expression cassette, is driven by a PGK promoter, especially a truncated PGK promoter described herein. In some embodiments, the one or more coding sequences are present in a bi-cistronic or multi-cistronic expression cassette. In some embodiments, the transgene comprises a receptor polypeptide, e.g., a CAR, a CCR, a CD70-binding protein, a dominant negative receptor or other sequences or sequences encoding other proteins or recombinant proteins and / or a nucleic acid inhibitory agent, e.g., an RNA interference agent.

[0102] In some embodiments, engineered immune cells generated by site-specific integration demonstrated surprisingly improved in vivo anti-tumor activity when the expression of the transgene(s), e.g., CD19-specific CAR, is driven by the PGK promoters, especially truncated PGK promoters, e.g., the PGK SSI promoters described herein, as compared to the EFla short (EFS) promoter. In some embodiments, engineered immune cells generated by site-specific integration demonstrated surprisingly improved in vivo antitumor activity when the expression of the transgene(s), e.g., CD19-specific CAR and the CCR, is driven by the PGK promoters, e.g., the PGK SSI promoters described herein, as compared to the EFS promoter. In some embodiments, engineered immune cells generated by site-specific integration demonstrated surprisingly improved in vivo anti-tumor activity when the expression of the transgene(s), e.g., CD19-specific CAR and the CD70-binding protein, is driven by the PGK promoters, e.g., the PGK SSI promoters described herein, as compared to the EFS promoter. In certain embodiments, the polynucleotides were integrated into the, e.g., TRAC locus. In some embodiments, the polynucleotides were integrated into the CD52 locus.1. Nucleic acid constructs and vectors

[0103] The present disclosure relates to engineered cells, e.g., engineered immune cells, that comprise one or more transgenes that have been integrated into the genome of the engineered cell, as well as methods of making and using engineered cells. In one embodiment, a transgene is integrated into the genome such that a target gene is disrupted. The target gene may be a gene that is involved in T cell receptor (TCR)aP function or activity. In other embodiments, the expression of the transgene is controlled by a promoter that is not the target gene promoter. In another embodiment, the promoter is a non- endogenous or exogenous promoter relative to the target gene. In one embodiment, the promoter is a truncated PGK promoter. In other embodiments, the expression of thetransgene is under the control of a truncated PGK promoter. In certain embodiments, the engineered cell, target gene and / or the promoter, e.g., truncated PGK promoter, are human. In other embodiments, the engineered cells, e.g., engineered immune cells, are human.

[0104] In one aspect, the present disclosure provides polynucleotides or nucleic acid constructs or molecules that comprise one or more coding sequences or transgenes. In some embodiments, a polynucleotide, nucleic acid construct or molecule according to the present invention comprises a donor template. In another embodiment, the donor template comprises one or more transgenes that include sequences encoding receptor polypeptides and / or nucleic acid inhibitory agents, e.g., RNA interference agents, that are under the control of a promoter. In one embodiment, the promoter may originate from another gene that is not directly involved in T cell receptor (TCR)aP function or activity. In one embodiment, the promoter comprises a nucleic acid sequence that is derived from the gene, including a full or partial sequence of the original promoter sequence. In one embodiment, the gene is the phosphoglycerate kinase (PGK1) gene (PGK). In another embodiment, instant disclosure provides truncated PGK promoters that comprises a partial nucleic acid sequence derived from, obtained from, or originating from the original PGK promoter sequence of the PGK gene. In certain embodiments, the PGK gene is the human PGK gene.

[0105] In one embodiment, the one or more transgenes comprise a truncated PGK promoter to control expression of the one or more sequences encoding one or more polypeptides or agents. In other embodiments, the truncated PGK promoter comprises one or more deletions in a promoter sequence from a corresponding promoter sequence of a genomic region that comprises a PGK gene. Table 1 provides exemplary PGK promoter sequences and / or regions that comprise PGK promoters.Table 1 - Promoters (Bolded, underlined text = transcription start site)0106] In one embodiment, the PGK promoter is derived from the genomic region comprising the PGK gene. For instance, the PGK promoter can be derived from the genomic region comprising the PGK gene in homo sapiens chromosome X, GRCh38.pl4 Primary Assembly (NCBI Reference Sequence: NC_000023.11). In another embodiment, the PGK promoter is derived from a polynucleotide sequence corresponding to nucleotide positions of about 78103669 to 78104334 of the genomic region comprising the PGK gene in homo sapiens chromosome X, GRCh38.pl4 Primary Assembly (NCBI Reference Sequence: NC_000023.11).

[0107] In an additional aspect, the truncated PGK promoters described herein comprise one or more nucleic acid sequences that bind to or are predicted to bind to one or more transcription factors. Alternatively, the truncated PGK promoters described herein may a) lack one or more nucleic acid sequences that bind to or are predicted to bind to one or more transcription factors or b) be characterized by the absence of one or more nucleic acid sequences that bind to or are predicted to bind to one or more transcription factors. Thepromoter sequences described herein can be analyzed using a transcription factor binding site prediction database known as JASPAR. JASPAR is an open-access database storing manually curated transcription factor (TF) binding profiles as position frequency matrices (PFMs), which summarize occurrences of each nucleotide at each position in a set of observed TF-DNA interactions. PFMs can be used to scan any DNA sequence to predict TF binding sites. In other embodiments, the transcription factor is a human transcription factor.

[0108] In one embodiment, the truncated PGK promoter comprises a nucleic acid sequence that binds to or is predicted to bind to a transcription factor selected from the group consisting of ZNF692, ZNF257, YY1, TEAD4, TEAD1, TCF4, TCF3, TBX3, SP1, SOX18, RFX7, MYOG, MYF5, KLF9, KLF6, KLF4, KLF10, HIF1, FIGLA, DUXA, DUX4, CTCFL, BHLHE22, BHLHA15, ASCL1, and ARNT::HIF1A. In one other embodiment, the transcription factor is one or more of YY1, RFX7, and HIF1. In an additional embodiment, the truncated PGK promoter comprises the nucleotide sequence of SEQ ID NO: 35 or having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 35.

[0109] In another embodiment, the truncated PGK promoter is characterized by the absence of a nucleic acid sequence that binds to or is predicted to bind to a transcription factor selected from the group consisting of ZNF281, ZNF148, VEZF1, TLX2, TFAP2E, TFAP2C, TFAP2B, TFAP2A, SP8, SOX15, RELB, RBPJ, PRDM1, PITX2, NKX3-2, NKX2-8, NKX2-3, NFKB1, NFATC2, MSX2, MEIS1, KLF16, KLF15, KLF11, ISL2, H0XD4, H0XD12, H0XD11, HOXD10, HOXC9, HOXC4, HOXC12, HOXC11, HOXC10, HOXB9, HOXB7, HOXB4, H0XA9, H0XA4, H0XA1, GSX2, FOXD2, FOXCI, EN2, EGR1, EBF1, E2F8, E2F6, E2F4, E2F1, DRGX, CEBPE, CEBPD, CEBPB, BARHL2, and BARHL1. In an additional embodiment, the truncated PGK promoter comprises the nucleotide sequence of SEQ ID NO: 35 or having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 35.

[0110] In one embodiment, the truncated PGK promoter comprises a nucleic acid sequence that binds to or is predicted to bind to a transcription factor selected from the group consisting of ZNF692, ZNF257, YY1, TEAD4, TEAD1, TCF4, TCF3, TBX3, SPI1, SP1, SOX18, SOX10, RUNX2, RFX7, PLAGL2, OSR2, OSR1, MYOG, MYF5, KLF9,KLF6, KLF4, KLF10, IKZF1, HSF4, HIF1A, FLU, FIGLA, FEV, ETV5, ETV4, ETS2, ERG, ELK4, ELK1, ELF5, ELF1, DUXA, DUX4, CTCFL, BHLHE22, BHLHA15, ASCL1, and ARNT::HIF1A. In one other embodiment, the transcription factor is one or more of YY1, RFX7, HIF1A, and ETS2. In an additional embodiment, the truncated PGK promoter comprises the nucleotide sequence of SEQ ID NO: 36 or having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 36.

[0111] In another embodiment, the truncated PGK promoter is characterized by the absence of a nucleic acid sequence that binds to or is predicted to bind to a transcription factor selected from the group consisting of ZNF281, ZNF148, VEZF1, TLX2, TFAP2C, TFAP2B, TFAP2A, SP8, SOX15, RELB, RBPJ, PRDM1, PITX2, NKX3-2, NKX2-8, NKX2-3, NFKB1, NFATC2, MSX2, KLF16, KLF15, KLF11, ISL2, H0XD4, H0XD12, H0XD11, HOXD10, HOXC9, HOXC4, HOXC12, HOXC11, HOXC10, HOXB9, HOXB7, HOXB4, HOXA9, HOXA4, HOXA1, GSX2, FOXD2, FOXCI, EN2, EGR1, EBF1, E2F8, E2F6, E2F4, E2F1, DRGX, CEBPE, CEBPD, CEBPB, BARHL2, and BARHL1. In one other embodiment, the transcription factor is one or more of NFKB1, NFATC2, EGR1, and CEBPB. In an additional embodiment, the truncated PGK promoter comprises the nucleotide sequence of SEQ ID NO: 36 or having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 36.

[0112] In one embodiment, the truncated PGK promoter comprises a nucleic acid sequence that binds to or is predicted to bind to a transcription factor selected from the group consisting of ZNF692, ZNF257, YY1, TEAD4, TEAD1, TCF4, TCF3, TBX3, STAT3, STAT1, SPI1, SPDEF, SP1, SOX18, SOX10, RUNX2, RHOXF1, RFX7, PLAGL2, OSR2, OSR1, MYOG, MYF5, KLF9, KLF6, KLF4, KLF3, KLF10, IKZF1, HSF4, HIF1A, FLU, FIGLA, FEV, ETV5, ETV4, ETS2, ERG, ELK4, ELK1, ELF5, ELF1, DUXA, DUX4, CTCFL, CREB1, BHLHE22, BHLHA15, ASCL1, and ARNT::HIF1A. In one other embodiment, the transcription factor is one or more of YY1, STAT3, STAT1, RFX7, and ETS2. In an additional embodiment, the truncated PGK promoter comprises the nucleotide sequence of SEQ ID NO: 37 or having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 37.

[0113] In another embodiment, the truncated PGK promoter is characterized by the absence of a nucleic acid sequence that binds to or is predicted to bind to a transcriptionfactor selected from the group consisting of ZNF281, ZNF148, VEZF1, TLX2, TFAP2C, TFAP2B, TFAP2A, SOX15, RELB, RBPJ, PRDM1, PITX2, NKX3-2, NKX2-8, NKX2-3, NFKB1, NFATC2, MSX2, KLF16, KLF15, ISL2, H0XD4, H0XD12, H0XD11, HOXDIO, H0XC9, H0XC4, H0XC12, H0XC11, HOXCIO, H0XB9, H0XB7, H0XB4, H0XA9, H0XA4, H0XA1, GSX2, FOXD2, FOXCI, EN2, EGR1, EBF1, E2F8, E2F6, E2F4, E2F1, DRGX, CEBPE, CEBPD, CEBPB, BARHL2, and BARHL1. In one other embodiment, the transcription factor is one or more of NFKB1, NFATC2, EGR1, and CEBPB. In an additional embodiment, the truncated PGK promoter comprises the nucleotide sequence of SEQ ID NO: 37 or having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 37.

[0114] In one embodiment, the truncated PGK promoter comprises one or more deletions, insertions or substitutions in a PGK promoter shown as SEQ ID NO: 34. In some embodiments, the one or more deletions in the truncated PGK promoter comprise one or more 5’ region deletions and / or one or more 3’ region deletions. In other embodiments, the 5’ region deletion comprises a deletion of about 300, about 301, about 302, about 303, about 304, about 305, about 306, about 307, about 308, about 309, about 310, about 311, about312, about 313, about 314, about 315, about 316, about 317, about 318, about 319, about320, about 321, about 322, about 323, about 324, about 325, about 326, about 327, about328, about 329, about 330, about 331, about 332, about 333, about 334, about 335, about336, about 337, about 338, about 339, about 340, about 341, about 342, about 343, about344, about 345, about 346, about 347, about 348, about 349, about 350, about 351, about352, about 353, about 354, about 355, about 356, about 357, about 358, about 359, about360, about 361, about 362, about 363, about 364, about 365, about 366, about 367, about368, about 369, about 370, about 371, about 372, about 373, about 374, about 375, about376, about 377, about 378, about 379, about 380, about 381, about 382, about 383, about384, about 385, about 386, about 387, about 388, about 389, or about 390 nucleotides from the 5’ end of SEQ ID NO: 34, and having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 35. In some embodiments, the truncated PGK promoter comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 35.

[0115] In other embodiments, the 5’ region deletion comprises a deletion of about 210, about 211, about 212, about 213, about 214, about 215, about 216, about 217, about 218,about 219, about 220, about 221, about 222, about 223, about 224, about 225, about 226, about 227, about 228, about 229, about 230, about 231, about 232, about 233, about 234, about 235, about 236, about 237, about 238, about 239, about 240, about 241, about 242, about 243, about 244, about 245, about 246, about 247, about 248, about 249, about 250, about 251, about 252, about 253, about 254, about 255, about 256, about 257, about 258, about 259, about 260, about 261, about 262, about 263, about 264, about 265, about 266, about 267, about 268, about 269, about 270, about 271, about 272, about 273, about 274, about 275, about 276, about 277, about 278, about 279, about 280, about 281, about 282, about 283, about 284, about 285, about 286, about 287, about 288, about 289, or about 290 nucleotides from the 5’ end of SEQ ID NO: 34, and having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 36. In some embodiments, the truncated PGK promoter comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 36.

[0116] In other embodiments, the 5’ region deletion comprises a deletion of about 110, about 111, about 112, about 113, about 114, about 115, about 116, about 117, about 118, about 119, about 120, about 121, about 122, about 123, about 124, about 125, about 126, about 127, about 128, about 129, about 130, about 131, about 132, about 133, about 134, about 135, about 136, about 137, about 138, about 139, about 140, about 141, about 142, about 143, about 144, about 145, about 146, about 147, about 148, about 149, about 150, about 151, about 152, about 153, about 154, about 155, about 156, about 157, about 158, about 159, about 160, about 161, about 162, about 163, about 164, about 165, about 166, about 167, about 168, about 169, about 170, about 171, about 172, about 173, about 174, about 175, about 176, about 177, about 178, about 179, about 180, about 181, about 182, about 183, about 184, about 185, about 186, about 187, about 188, about 189, about 190, about 191, about 192, about 193, about 194, about 195, about 196, about 197, about 198, about 199, or about 200 nucleotides from the 5’ end of SEQ ID NO: 34, and having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 37. In some embodiments, the truncated PGK promoter comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 37.

[0117] In other embodiments, the 5’ region deletion comprises a deletion of about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59,about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, or about 100 nucleotides from the 5’ end of SEQ ID NO: 34, and having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 38. In some embodiments, the truncated PGK promoter comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 38.

[0118] In some embodiments, the truncated PGK promoter consists essentially of or consists of a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 35, 36, 37 or 38.

[0119] In one other aspect, the polynucleotides, nucleic acid constructs or molecules described herein comprise a donor template. The donor template can comprise one or more transgenes or coding sequences, which encode one or more receptor polypeptides and / or one or more nucleic acid inhibitory agents, e.g., RNA interference agents, as described herein. In other embodiments, the donor template comprises a promoter, such as a truncated PGK promoter, to control expression of the one or more transgenes or coding sequences.

[0120] In other embodiments, the polynucleotide, nucleic acid construct, or molecule comprises a donor template that can integrate a transgene via homologous recombination. In one embodiment, the donor template comprises a transgene with a sequence that encodes a targeting molecule, e.g., a receptor polypeptide. The targeting molecule, e.g., receptor polypeptide, may be an antigen binding molecule. In another embodiment, the antigen binding molecule may be part of a chimeric antigen receptor (CAR) or the antigen binding molecule is a CAR. In one embodiment, the receptor polypeptide is a CAR, which comprises an antigen binding domain. In other embodiments, the template comprises a sequence encoding a chimeric cytokine receptor (CCR), or a CD70 binding protein. In one embodiment, the sequence that encodes a targeting molecule is under the control of a PGK promoter, e.g., a truncated PGK promoter.

[0121] In other embodiments, the polynucleotides, nucleic acid constructs or molecules further comprise one or more homology sequences that are homologous to a portion of a target nucleic acid sequence in a target gene. In one embodiment, the target gene is a gene that is involved in T cell receptor (TCR)aP function or activity. In an additional embodiment, the gene is the TCR alpha gene constant domain (TRAC gene). In another embodiment, the target is one or more of a component of the TCR, an HLA, TCRa, and TCRp. In other embodiments, the target gene is a human gene.

[0122] In one aspect, the target gene is a gene that is expressed by a host immune cell, wherein the host immune cell is involved in a potential immune response against the engineered cell, e.g., an engineered immune cell such as a CAR T cell, following administration to a subject. In other embodiments, the host immune cell is human. Clinical data suggests that temporary depletion of host immune cells is advantageous at the beginning of a CAR T cell treatment. A temporary depletion can be achieved by administering agents that target and deplete host immune cells that express the target gene. In one embodiment, genetic modification of cells, e.g., immune cells to reduce or impair the expression of the target gene is desired. In one embodiment, the target gene is CD52.Before, during, or after the administration of an engineered cell, e.g., an engineered immune cell, to a subject in need, a binding agent that targets CD52, e.g., an anti-CD52 antibody, is administered to the subject. In another embodiment, the binding agent depletes host immune cells expressing CD52 but does not bind engineered cells in which CD52 expression has been reduced or impaired. In one other embodiment, the binding agent does not deplete engineered cells in which CD52 expression has been reduced or impaired. In one embodiment, the anti-CD52 antibody is alemtuzumab (SEQ ID NOs: 67-74).

[0123] In some embodiments, the polynucleotides, nucleic acid constructs or molecules are used to generate engineered cells, e.g., immune cells, that are resistant to one or more chemotherapy drugs. The chemotherapy drug can be, for example, a purine nucleotide analogue (PNA), thus making the immune cell suitable for cancer treatment combining adoptive immunotherapy and chemotherapy. Exemplary PNAs include, for example, clofarabine, fludarabine, cyclophosphamide, and cytarabine, alone or in combination. PNAs are metabolized by deoxycytidine kinase (dCK) into mono-, di-, and tri-phosphate PNA. Their tri-phosphate forms compete with ATP for DNA synthesis, act as pro-apoptotic agents, and are potent inhibitors of ribonucleotide reductase (RNR), which is involved in trinucleotide production.

[0124] In another embodiment, the target gene is deoxycytidine kinase (dCK). Certain PNAs can lymphodeplete host immune cells. DCK-deficient cells are known to be resistant to lymphodepletion such regimens. In one embodiment, the target gene is dCK. Before, during, or after the administration of an engineered cell, e.g., an engineered immune cell, to a subject in need, a lymphodepletion compound, such as a PNA, is administered to the subject. In another embodiment, the lymphodepletion compound depletes host immune cells expressing dCK but does not deplete engineered cells in which dCK expression has been reduced or impaired.

[0125] In another embodiment, the target gene is the glucocorticoid receptor (GR), e.g., human GR. Certain glucocorticoids, e.g., dexamethasone, are known to have an immunosuppressive effect on host immune cells. In one embodiment, the target gene is GR. Before, during, or after the administration of an engineered cell, e.g., an engineered immune cell, to a subject in need, a glucocorticoid, e.g., dexamethasone, is administered to the subject. In another embodiment, the glucocorticoid suppresses the immune response by host immune cells that express GR but does not suppress engineered cells in which GR expression has been reduced or impaired.

[0126] In another embodiment, the target gene is a checkpoint inhibitor. Certain immune receptors, e.g., PD-1, PDL1 and CTLA-4, are involved in inhibitory effects on host immune cells. In one embodiment, the target gene is a checkpoint inhibitor. Before, during, or after the administration of an engineered cell, e.g., an engineered immune cell, to a subject in need, a binding agent that targets one or more checkpoint inhibitors, e.g., an anti-PD-1, anti- PDL1, or anti-CTLA4 antibody, is administered to the subject. In another embodiment, the binding agent depletes host immune cells expressing the checkpoint inhibitor but does not bind engineered cells in which checkpoint inhibitor expression has been reduced or impaired. In one other embodiment, the binding agent does not deplete engineered cells in which checkpoint inhibitor expression has been reduced or impaired. In another embodiment, the target gene is one or more of PD-1, PDL1, and CTLA-4. In other embodiments, the target gene is a human gene.

[0127] In one other aspect, the target gene is a molecule involved in rejection and / or recognition of an engineered cell, e.g., an engineered immune cell as described herein, by a host immune cell. In one embodiment, the target gene is a gene involved in diverse peptide presentation, such as the TAP2 component of the transporter associated with antigenprocessing (TAP). The dominant pathway by which MHC class I molecules are loaded with peptide is TAP-dependent: peptides generated by the proteasome (or the IFN-y-inducible immunoproteasome) are imported to the endoplasmic reticulum (ER) via TAP and then loaded on MHC class I. Knocking out TAP2 reduces surface [32m modestly (2-fold decrease after selection for KO cells) compared to the profound (10-100-fold) reduction in surface P2 microglobulin (P2m) in P2m KO cells (see FIG. 4A of PCT / US2022 / 14393, which is incorporated herein by reference in its entirety). In another embodiment, the target gene is TAP2 and / or P2m. In other embodiments, the target gene is a human gene.

[0128] In another embodiment, the target gene is a gene involved in the regulation of the transcription of HLA-I and HLA-II molecules. HLA-I and HLA-II molecules are tightly regulated at the transcriptional level by similar critical cis-regulatory elements: W / S, XI, X2 and Y box motifs. The Regulatory Factor, X (RFX) heterocomplex is comprised of RFX5, RFXAP and RFXANK and binds to the XI box. The X2 box is occupied by CREB / ATF1 family transcription factors and the Y box is bound by the NF-Y protein. In addition, two members of the nucleotide-binding domain and leucine-rich repeat containing receptor (NLR) family, NLRC5 and CIITA are required for formation of the HLA enhanceosome complex to promote transcription of HLA-I and HLA-II, respectively. In one embodiment, the target gene is one or more of TAP2, NLRC5, P2m, CIITA, RFX5, RFXAP and RFXANK. In other embodiments, the target gene is a human gene.

[0129] In an additional embodiment, the target gene is a cell surface receptor involved in immune cell adhesion and activation at the immune synapse. Certain cell surface molecules including, without limitation, CD58 and CD2, CD48 and CD2, and / or ICAM-1 and LFA-1, interact to provide proper cell adhesion and activation of immune cells (Dustin, M.L. The immunological synapse, Cancer Immunol Res. 2014 Nov;2(l 1): 1023-33). Furthermore, the loss of CD58 has been associated with resistance of tumor cells to T-cell mediated killing and immune evasion (Frangieh, C.J. et al. Multimodal pooled Perturb-CITE-seq screens in patient models define mechanisms of cancer immune evasion. Nat Genet. 2021 Mar;53(3):332-341, Epub 2021 Mar 1; Challa-Malladi, M. et al., Combined genetic inactivation of P2-Microglobulin and CD58 reveals frequent escape from immune recognition in diffuse large B cell lymphoma. Cancer Cell. 2011 Dec 13 ;20(6): 728-40, Epub 2011 Dec 1). In one embodiment, the target gene is one or more of CD48, CD58, and ICAM-1. In other embodiments, the target gene is a human gene.

[0130] In one other aspect of the present disclosure, the target genes of an engineered cell may be one or more genes encoding one or molecules that have a role in one or more cellular pathways the relate to rejection by host or recipient immune cells that are reactive with T cell and NK cell epitope determinants on the surface of an allogeneic cell product that is distinct from the host. In one embodiment, the target gene is one or more genes encoding i) a cell surface receptor that is known to play a role in immune cell adhesion and activation at the immune synapse (e.g., one or more of CD48, CD58, and ICAM-1) and / or ii) a transcription factor or regulator of HLA-I and HLA-II molecules (e.g., RFX5, NLRC5, CIITA, RFXAP and RFXANK). The approaches described herein to generate engineered cells having reduced or eliminated expression of one or more genes may focus only on genes that encode molecules having a role at the immune synapse but may also be supplemented with reduced or eliminated expression of genes that encode molecules having a primary role as transcription factors for HLA-I and / or HLA-II molecules.

[0131] In one other embodiment, the target gene is CD70, e.g., human CD70. CD70 is expressed on the surface of immune cells, e.g., T cells, especially activated T cells, and a CD70 binding protein can cause lysis of such CD70-expressing immune cells (see FIG. 1 A of PCT / US2022 / 33598, which is incorporated herein by reference in its entirety). In certain embodiments, the donor template comprises homology arms that target CD70 and a transgene encoding a CD70 binding protein. In another embodiment, the engineered cell, e.g., an engineered immune cell, has reduced or impaired CD70 expression and expresses a CD70 binding protein. Following the administration of an engineered cell, e.g., an engineered immune cell, to a subject in need, the CD70 binding protein causes lysis of host immune cells that express CD70 but does not cause lysis of other engineered cells which have reduced or impaired CD70 expression.

[0132] In other embodiments, the polynucleotides, nucleic acid constructs or molecules comprise a donor template that can integrate into the host cell genome via homologous recombination at the site of a double strand break of the genome. In one embodiment, the donor template comprises one or more, at least two, or two or more coding sequences or transgenes. In one embodiment, the expression of the one or more, at least two, or two or more coding sequences or transgenes is under the control of a PGK promoter, e.g., a truncated PGK promoter.

[0133] In other embodiments, the polynucleotides, nucleic acid constructs or molecules further comprise one or more homology sequences that are homologous to a portion of an integration site in the genome of the engineered immune cell (for site-specific integration). In one embodiment, the integration site is a gene (target site or target gene) that is involved in T cell receptor (TCR) a|3 function or activity. In an additional embodiment, the gene is the TCR alpha gene constant region (TRAC) gene. In another embodiment, the integration she is in TRAC, a component of the TCR, an HLA, TCRa, TCRP, p2-microglobulin (“P2m”), CD52, GR, deoxy cytidine kinase (DCK), PD-1, and CTLA-4 gene.

[0134] In other embodiments, the polynucleotide, nucleic acid construct or molecule includes a 5’ homology arm having a sequence homologous to a sequence that is positioned 5’ to a double strand break of the target nucleic acid sequence in the target gene. In one other embodiment, the polynucleotide, nucleic acid construct or molecule further includes a 3’ homology arm having a sequence homologous to a sequence that is positioned 3’ to a double strand break of the target nucleic acid sequence in the target gene. Upon delivery of a polynucleotide, nucleic acid construct, or molecule to a cell and subsequent to cleavage of the target nucleic acid sequence, a homologous recombination event occurs between the target nucleic acid sequence and the one or more homology sequences of the polynucleotide, nucleic acid construct or molecule.

[0135] Nucleotide sequence homologies are present in regions flanking upstream and downstream of the site of the double-strand break and the site of genome integration, and the nucleic acid sequence to be introduced, e.g., one or more transgenes, is located between the two homology arms. In some embodiments, in the context of site-specific integration, the terms - 5’ homology arm and 3’ homology arm, and the terms - regions flanking upstream and downstream, are used to refer to the orientation of the target site or target gene for genome integration.

[0136] Nucleotide sequence homologies are present in regions flanking upstream and downstream the site of the double-strand break and the site of genome integration, and the nucleic acid sequence to be introduced, e.g., one or more transgenes, is located between the two homology arms. In some embodiments, in the context of site-specific integration, the terms 5’ homology arm and 3’ homology arm and the terms regions flanking upstream and downstream are used to refer to the orientation of the target site or target gene for genome integration.

[0137] In some embodiments, the double strand break can be made by a rare-cutting endonuclease, for example without limitation, a zinc finger endonuclease, TALE endonuclease, or CRISPR. The polynucleotide, nucleic acid construct or molecule can be integrated into the desired integration site of the genome by homologous recombination between the 5’ homology and the 3’ homology arms and the homologous sequences at the integration site. In some embodiments, 5’ and 3’ homologous sequences are provided in Table 3, for example, SEQ ID NOs: 42-45.

[0138] In one embodiment, the polynucleotide, nucleic acid construct or molecule comprises a donor template comprising a 5’ homology arm, a 3’ homology arm, a transgene, and a promoter, such as a truncated PGK promoter. In other embodiments, the polynucleotide, nucleic acid construct or molecule comprises a 5’ homology arm, a first sequence encoding a first receptor polypeptide, a second sequence encoding a second receptor polypeptide, a 3 ’ homology arm, and a promoter, such as a truncated PGK promoter.

[0139] In some embodiments, the orientation of the one or more coding sequences or transgenes is the same orientation as the target site for genome integration or the opposite orientation to the target site for genome integration. In some embodiments, the polynucleotide, nucleic acid construct or molecule comprises a donor template comprising a 5’ homology arm, a promoter, one or more coding sequences or transgenes, and a 3’ homology arm. In some embodiments, the promoter comprises a truncated PGK promoter, e.g., the PGK SSI-1, PGK SSI-2 or PGK SSI-3 promoter as described herein.

[0140] FIG. 8A-8D depict examples of different configurations of polynucleotides, nucleic acid constructs or molecules that contain transgenes and promoters. FIG. 8A (left panel) depicts a polynucleotide, nucleic acid construct or molecule that comprises a donor template having a first homology arm (HA1), a PGK promoter (such as a truncated PGK promoter), a receptor polypeptide sequence, and a second homology arm (HA2). FIG. 8A (right panel) depicts a polynucleotide, nucleic acid construct or molecule having a donor template that comprises a first homology arm (HA1), a PGK promoter (such as a truncated PGK promoter), a first receptor polypeptide sequence, a linker sequence, a second receptor polypeptide sequence, and a second homology arm (HA2). FIG. 8B depicts a polynucleotide, nucleic acid construct or molecule having a donor template that comprises a first homology arm (HA1), a PGK promoter (such as a truncated PGK promoter), a CARsequence that includes sequences encoding an scFv, a CD8 / hinge region, a 4- IBB costimulatory region, and a CD3zeta region, and a second homology arm (HA2). FIG. 8C depicts a polynucleotide, nucleic acid construct or molecule having a donor template that comprises a first homology arm (HA1), a PGK promoter (such as a truncated PGK promoter), a chimeric cytokine receptor sequence, a linker sequence, a CAR sequence that includes sequences encoding an scFv, a CD8 / hinge region, a 4- IBB costimulatory region, and a CD3zeta region, and a second homology arm (HA2).

[0141] In one aspect, the present disclosure provides multiple polynucleotides, nucleic acid constructs, or molecules to target different genes of interest in the same cell. In one embodiment, a first nucleic acid construct (or polynucleotide or molecule) comprises a donor template with homology arms for a first target gene, a PGK promoter (such as a truncated PGK promoter), and a first receptor polypeptide sequence, while a second nucleic acid construct (or polynucleotide or molecule) comprises homology arms for a second target gene, a promoter that is not a truncated PGK promoter, and a second receptor polypeptide sequence. Both the first and the second nucleic acid construct (or polynucleotide or molecule) may be used to modify the first and second target genes in the same cell. FIG. 8D (left panel) depicts a first nucleic acid construct (or polynucleotide or molecule) with a donor template that includes a first transgene with homology arms that target a first gene for modification. The first nucleic acid construct (or polynucleotide or molecule) has a first donor template that comprises a first homology arm (HA1), a PGK promoter (such as a truncated PGK promoter), a first receptor polypeptide sequence, and a second homology arm (HA2). FIG. 8D (right panel) depicts a second nucleic acid construct (or polynucleotide or molecule) with a second donor template that includes a second transgene with homology arms that target a second gene for modification. The second nucleic acid construct (or polynucleotide or molecule) comprises a third homology arm (HA3), a promoter that is not a truncated PGK promoter, a second receptor polypeptide sequence, and a fourth homology arm (HA4). In one embodiment, the promoter that is not a truncated PGK promoter comprises an EFS promoter or an EFL promoter.

[0142] In one aspect, the nucleic acid construct (or polynucleotide or molecule) comprises a donor template with a promoter, e.g., a truncated PGK promoter, that is positioned such the promoter can initiate transcription in the same or opposite direction as that of the target gene of interest, which is under the control of an endogenous promoter. In one embodiment, the promoter is positioned between the transgene and the 3’ homology armsuch that transcription can initiate from the 3’ homology arm end towards the 5’ homology arm end. In other embodiments, the promoter initiates transcription in a direction that is opposite of transcription that would normally occur by endogenous promoter of the target gene of interest. This promoter-transgene configuration is advantageous because it allows for initiation of transcription of the transgene by the promoter, e.g., a PGK promoter, without any interfering transcription of the transgene by the endogenous promoter.

[0143] FIG. 7A-7B depict the insertion of a transgene, such as a CAR, into a gene of interest. FIG. 7A depicts an insertion of a CAR into the TRAC locus by site-specific integration and homologous recombination (TRAC knock-in or TRAC KI). The P2A peptide is from Thosea asigna virus. As shown by the large, dotted line arrow in the top diagram, transcription by the endogenous promoter for TRAC would normally proceed in one direction. Following insertion of the CAR into the TRAC locus, transcription by the CAR-associate promoter would proceed in the opposite direction, as shown by the smaller, solid line arrow in the bottom panel. FIG. 7B depicts an insertion of a CAR into the CD52 locus by site-specific integration and homologous recombination (CD52 knock-in or CD52 KI). As shown by the large, dotted line arrow in the top diagram, transcription by the endogenous promoter for CD52 would normally proceed in one direction. Following insertion of the CAR into the CD52 locus, transcription by the CAR-associate promoter would proceed in the opposite direction, as shown by the smaller, solid line arrow in the bottom panel. The diagrams in both FIGS. 7A and 7B are exemplified as using an adeno- associated virus 6 (AAV6) for the site-specific integration of CAR, under a non-endogenous or exogenous promoter, relative to the endogenous TRAC or CD52 promoter. LHA and RHA: left and right homology arms, respectively. In one embodiment, the inserted sequence that encodes the CAR is under the control of a PGK promoter, e.g., a truncated PGK promoter.

[0144] FIG. 7A depicts the insertion of a transgene with a promoter within the TRAC gene such that transcription of the transgene can be initiated in the opposite direction of the endogenous TRAC promoter. Alternatively, the promoter is positioned between the transgene and the 5’ homology arm such that transcription can initiate from the 5’ homology arm end to the 3’ homology arm end. In other embodiments, the promoter initiates transcription in the same direction as transcription of the target gene of interest by the endogenous promoter. In one embodiment, the non-endogenous promoter is a PGK promoter, e.g., a truncated PGK promoter.

[0145] In one embodiment, the polynucleotide, nucleic acid construct or molecule includes a donor template that comprises in a 5’ to 3’ direction: (a) a 5’ homology arm, a transgene, a truncated PGK promoter, and a 3’ homology arm, or (b) a 5’ homology arm, a truncated PGK promoter, a transgene, and a 3’ homology arm. In other embodiments, the polynucleotide, nucleic acid construct or molecule has a donor template that comprises in a 5’ to 3’ direction: (a) a 5’ homology arm, a first sequence encoding a first receptor polypeptide, a second sequence encoding a second receptor polypeptide, a truncated PGK promoter, and a 3’ homology arm, or (b) a 5’ homology arm, a truncated PGK promoter, a first sequence encoding a first receptor polypeptide, a second sequence encoding a second receptor polypeptide, and a 3’ homology arm.

[0146] In one other embodiment, the polynucleotide, nucleic acid construct or molecule comprises a donor template with a transgene having a first and a second sequence that are in the same orientation (relative to one another). For instance, both the first and second sequence may be disposed in the nucleic acid construct (or polynucleotide or molecule) in a 5’ to 3’ orientation or in a 3’ to 5’ orientation relative to the homology arms. In addition, both the first and second sequence may be disposed in the nucleic acid construct (or polynucleotide or molecule) in a 5’ to 3’ orientation or in a 3’ to 5’ orientation relative to the promoter such that the promoter, e.g., a truncated PGK promoter, can initiate transcription in a 5’ to 3’ direction. The first sequence may be in a 5’ position or a 3’ position relative to the second sequence. In one embodiment, the first sequence has a 5’ to 3’ orientation, while the second sequence has a 3’ to 5’ orientation.

[0147] In some embodiments, the stoichiometric co-expression of the first and second receptor polypeptides is achieved using a linker sequence between the two sequences. The linker sequence may encode a P2A peptide from the Thosea asigna virus, as further described herein.

[0148] In one aspect, the polynucleotide, nucleic acid construct or molecule has a donor template that includes a nucleic acid inhibitory sequence, such as a nucleic acid inhibitory agent, such as an RNA interference agent that includes one or more RNA interference sequences. The donor template may have a nucleic acid inhibitory sequence alone or in combination with a sequence encoding a receptor polypeptide. Upon introduction into a cell, the RNA interference sequence reduces the level of expression of a target gene relative to a comparable cell that does not have the sequence. The reduced level of expression maybe a knockout or a knockdown of expression. Different knockdown methods may be suitable, such as those that employ any of various RNA-based techniques (e.g., short hairpin RNA (shRNA), antisense RNA, microRNA (miRNA), small (or short) interfering RNA (siRNA); see, e.g., Van Hoeck et al., Biomaterials, Vol. 286, July 2022, 121510, ISSN 0142-9612; Lam et al., Mol. Ther.-Nucleic Acids 4:e252 (2015), doi: 10.1038 / mtna.2015.23; Sridharan and Gogtay, Brit. J. Clin. Pharmacol. 82: 659-72 (2016), as well as Krause et al. U.S. Patent No. 9556433, which is incorporated herein by reference in its entirety). In another embodiment, the RNA interference agent is a microRNA-adapted shRNA, in which a miRNA scaffold comprises one or more shRNA sequences. An RNA-based reagent may be delivered to a cell, e.g., an immune cell, such that one or more genes are knocked down. In one embodiment, the RNA-based reagent may be configured to target or is targeted to the one or more genes. In one embodiment, the RNA interference sequence may be between about 15 and about 30 nucleotides in length. In another embodiment, the nucleic acid inhibitory agent, e.g., an RNA interference agent comprising one or more RNA interference sequences, reduces expression of a gene that is involved in T cell receptor (TCR)aP function or activity. In other embodiments, the gene is selected from TRAC, a component of the TCR, an HLA, TCRa, TCRP, P2-microglobulin (“P2m”), CD52, GR, deoxycytidine kinase (DCK), PD-1, and CTLA-4.

[0149] In one aspect, the polynucleotide, nucleic acid construct or molecule comprises a donor template with homology arms, an RNA interference sequence, and a promoter, such as a truncated PGK promoter. In one embodiment, the donor template further comprises a receptor polypeptide sequence. In one other embodiment, the polynucleotide, nucleic acid construct or molecule has a donor template that comprises in a 5’ to 3’ direction: (a) a 5’ homology arm, a truncated PGK promoter, an RNA interference sequence, and a 3’ homology arm, or (b) a 5’ homology arm, an RNA interference sequence, a truncated PGK promoter, and a 3’ homology arm. In one other embodiment, the donor template comprises a first and a second RNA interference sequence that are in the same orientation (relative to one another). For instance, both the first and second RNA interference sequence may be disposed in the nucleic acid construct (or polynucleotide or molecule) in a 5’ to 3’ orientation or in a 3’ to 5’ orientation relative to the homology arms. In addition, both the first and second RNA interference sequence may be disposed in the nucleic acid construct (or polynucleotide or molecule) in a 5’ to 3’ orientation or in a 3’ to 5’ orientation relative to the promoter such that the promoter, e.g., a truncated PGK promoter, can initiatetranscription in a 5’ to 3’ direction. The first RNA interference sequence may be in a 5’ position or a 3’ position relative to the second RNA interference sequence. In one embodiment, the first RNA interference sequence has a 5’ to 3’ orientation, while the second RNA interference sequence has a 3’ to 5’ orientation.

[0150] In another aspect, the polynucleotides, nucleic acid constructs or molecules are provided as part of one or more recombinant vectors, such as a viral-based vector, suitable for introduction into one or more cells, e.g., immune cells, in order to modify a target gene of interest. In some embodiments, the vector comprises a donor template that comprises homology arm sequences that have homology to sequences of a target gene. In one embodiment, the target gene is a gene that is involved in T cell receptor (TCR)aP function or activity. In an additional embodiment, the gene is the TCR alpha gene constant domain (TRAC gene). In one other embodiment, the recombinant viral-based vector is an adenovirus associated virus (AAV). In one embodiment, the AAV vector contains AAV serotype sequences, such as viral inverted terminal repeat (ITR) sequence. The polynucleotides, nucleic acid constructs, or molecules may comprise one or more AAV serotype sequences including, without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-DJ, or AAV-DJ / 8 serotype sequences. A recombinant AAV vector incorporating a nucleic acid sequence that comprises a transgene can be generated and recombinant AAV particles can be produced for infection of a cell, e.g., an immune cell, in order to modify a target gene of interest.

[0151] In some embodiments, the vector comprises a donor template that further comprises a transgene. In another embodiment, the transgene comprises a polynucleotide encoding a receptor polypeptide and / or an RNA interference sequence. In some embodiments, the polynucleotide encoding the receptor polypeptide and / or the RNA interference sequence is present in an expression vector for stable expression in the cells. In some embodiments, the polynucleotide is present in a viral vector for stable expression in the cells. In some embodiments, the viral vector may be, for example, an AAV vector.

[0152] In one aspect, the present disclosure provides expression vectors containing a polynucleotide, nucleic acid construct or molecule, as described herein. In one embodiment, the expression vector comprises a polynucleotide, nucleic acid construct or molecule having a donor template that includes a 5’ homology arm, a transgene, a promoter (e.g., a truncated PGK promoter), and a 3’ homology arm. The donor template in the vectormay comprise in a 5’ to 3’ direction: (a) a 5’ homology arm, a transgene, a truncated PGK promoter, and a 3’ homology arm, or (b) a 5’ homology arm, a truncated PGK promoter, a transgene, and a 3’ homology arm. In other embodiments, the donor template in the vector comprises in a 5’ to 3’ direction: (a) a 5’ homology arm, a first sequence encoding a first receptor polypeptide (or first or second nucleic acid inhibitory agent), a second sequence encoding a second receptor polypeptide (or first or second nucleic acid inhibitory agent), a truncated PGK promoter, and a 3’ homology arm, or (b) a 5’ homology arm, a truncated PGK promoter, a first sequence encoding a first receptor polypeptide (or first or second nucleic acid inhibitory agent), a second sequence encoding a second receptor polypeptide (or first or second nucleic acid inhibitory agent), and a 3’ homology arm.

[0153] In another aspect, the disclosure provides a method of making any of the nucleic acid constructs or molecules, expression vectors, or polynucleotides that comprise the nucleic acid sequences described herein. Polynucleotides complementary to any such sequences are also encompassed by the disclosure. Polynucleotides may be single-stranded (coding or antisense) or double- stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules include HnRNA molecules, which contain introns and correspond to a DNA molecule in a one-to-one manner, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide of the disclosure, and a polynucleotide may, but need not, be linked to other molecules and / or support materials.

[0154] In another aspect, the present disclosure provides variants of the nucleic acid sequences described herein. Nucleic acid variants of a given polynucleotide may contain a deletion and / or an insertion of at least one nucleotide position in the given polynucleotide. In one embodiment, variants of the given polynucleotide are configured such that the open reading frame of the variant is preserved. Variants preferably exhibit at least about 70% identity, more preferably, at least about 80% identity, yet more preferably, at least about 90% identity, and most preferably, at least about 95% identity to a polynucleotide sequence described herein.

[0155] Two polynucleotide or polypeptide sequences are said to be "identical" if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify andcompare local regions of sequence similarity. A "comparison window" as used herein, refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, or 40 to about 50, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.

[0156] Optimal alignment of sequences for comparison may be conducted using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wl), using default parameters. This program embodies several alignment schemes described in the following references: Dayhoff, M.O., 1978, A model of evolutionary change in proteins - Matrices for detecting distant relationships. In Dayhoff, M.O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J., 1990, Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, CA; Higgins, D.G. and Sharp, P.M., 1989, CABIOS 5: 151 -153; Myers, E.W. and Muller W., 1988, CABIOS 4: 1 1 -17; Robinson, E.D., 1971 , Comb. Theor. 1 1 : 105; Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4:406-425; Sneath, P.H.A. and Sokal, R.R., 1973, Numerical Taxonomy the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, W.J. and Lipman, D.J., 1983, Proc. Natl. Acad. Sci. USA 80:726-730.

[0157] Preferably, the "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a window of comparison of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12 percent, as compared to the reference sequences (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid bases or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size) and multiplying the results by 100 to yield the percentage of sequence identity.

[0158] Variants may also, or alternatively, be substantially homologous to a native gene, or a portion or complement thereof. Such polynucleotide variants are capable of hybridizingunder moderately stringent conditions to a naturally occurring DNA sequence encoding a native antibody (or a complementary sequence).

[0159] Suitable "moderately stringent conditions" include prewashing in a solution of 5 X SSC, 0.5% SDS, 1 .0 mM EDTA (pH 8.0); hybridizing at 50°C-65°C, 5 X SSC, overnight; followed by washing twice at 65°C for 20 minutes with each of 2X, 0.5X and 0.2X SSC containing 0.1 % SDS.

[0160] As used herein, "highly stringent conditions" or "high stringency conditions" are those that: (1 ) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1 % sodium dodecyl sulfate at 50°C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v / v) formamide with 0.1 % bovine serum albumin / 0.1 % Ficoll / 0.1 % polyvinylpyrrolidone / 50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCI, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1 % sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 pg / m\), 0.1 % SDS, and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2 x SSC (sodium chloride / sodium citrate) and 50% formamide at 55°C, followed by a high- stringency wash consisting of 0.1 x SSC containing EDTA at 55°C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.

[0161] It will be appreciated by those of ordinary skill in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide as described herein. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. Nonetheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the disclosure. Further, alleles of the genes comprising the polynucleotide sequences provided herein are within the scope of the disclosure. Alleles are endogenous genes that are altered as a result of one or more mutations, such as deletions, additions and / or substitutions of nucleotides. The resulting mRNA and protein may, but need not, have an altered structure or function. Alleles may be identified using standard techniques (such as hybridization, amplification and / or database sequence comparison).

[0162] The polynucleotides of this disclosure can be obtained using chemical synthesis, recombinant methods, or PCR. Methods of chemical polynucleotide synthesis are well known in the art and need not be described in detail herein. One of skill in the art can use the sequences provided herein and a commercial DNA synthesizer to produce a desired DNA sequence.

[0163] For preparing polynucleotides using recombinant methods, a polynucleotide comprising a desired sequence can be inserted into a suitable vector, and the vector in turn can be introduced into a suitable host cell for replication and amplification, as further discussed herein. Polynucleotides may be inserted into host cells by any means known in the art. Cells are transformed by introducing an exogenous polynucleotide by direct uptake, endocytosis, transfection, F-mating or electroporation. Once introduced, the exogenous polynucleotide can be maintained within the cell as a non-integrated vector (such as a plasmid) or integrated into the host cell genome. The polynucleotide so amplified can be isolated from the host cell by methods well known within the art. See, e.g., Sambrook et al., 1989.

[0164] Alternatively, PCR allows reproduction of DNA sequences. PCR technology is well known in the art and is described in U.S. Patent Nos. 4,683,195, 4,800,159, 4,754,065 and 4,683,202, as well as PCR: The Polymerase Chain Reaction, Mullis et al. eds., Birkauswer Press, Boston, 1994.

[0165] Accordingly, provided herein are polynucleotides comprising a promoter and a first and a second coding sequences, wherein either the first or the second coding sequences encodes an antigen-specific CAR, e.g., a CD19-specific CAR.

[0166] RNA can be obtained by using the isolated DNA in an appropriate vector and inserting it into a suitable host cell. When the cell replicates and the DNA is transcribed into RNA, the RNA can then be isolated using methods well known to those of skill in the art, as set forth in Sambrook et al., 1989, supra, for example.

[0167] Suitable cloning vectors may be constructed according to standard techniques, or may be selected from a large number of cloning vectors available in the art. While the cloning vector selected may vary according to the host cell intended to be used, useful cloning vectors will generally have the ability to self-replicate, may possess a single target for a particular restriction endonuclease, and / or may carry genes for a marker that can be used in selecting clones containing the vector. Suitable examples include plasmids andbacterial viruses, e.g., pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mpl8, mpl9, pBR322, pMB9, ColEl, pCRl, RP4, phage DNAs, and shuttle vectors such as pSA3 and pAT28. These and many other cloning vectors are available from commercial vendors such as BioRad, Strategene, and Invitrogen.

[0168] Expression vectors generally are replicable polynucleotide constructs that contain a polynucleotide according to the disclosure. It is implied that an expression vector must be replicable in the host cells either as episomes or as an integral part of the chromosomal DNA. Suitable expression vectors include but are not limited to plasmids, viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, cosmids, and expression vector(s) disclosed in PCT Publication No. WO 87 / 04462. Vector components may generally include, but are not limited to, one or more of the following: a signal sequence; an origin of replication; one or more marker genes; suitable transcriptional controlling elements (such as promoters, enhancers and terminator). For expression (i.e., translation), one or more translational controlling elements are also usually required, such as ribosome binding sites, translation initiation sites, and stop codons.

[0169] The vectors containing the polynucleotides of interest can be introduced into the host cell by any of a number of appropriate means, including electroporation, transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; and infection (e.g., where the vector is an infectious agent such as vaccinia virus). The choice of introducing vectors or polynucleotides will often depend on features of the host cell.

[0170] A polynucleotide encoding a receptor polypeptide (e.g., one or more of a CAR, a CCR, or CD70 binding protein) and / or a nucleic acid inhibitory agent, e.g., an RNA interference agent, as disclosed herein may exist in an expression cassette or expression vector (e.g., a plasmid for introduction into a bacterial host cell, or a viral vector such as a baculovirus vector for transfection of an insect host cell, or a plasmid or viral vector such as a lentivirus for transfection of a mammalian host cell). In some embodiments, a polynucleotide or vector can include a nucleic acid sequence encoding ribosomal skip sequences such as, for example without limitation, a sequence encoding a 2A peptide. 2A peptides, which were identified in the Aphthovirus subgroup of picomaviruses, cause a ribosomal "skip" from one codon to the next without the formation of a peptide bond between the two amino acids encoded by the codons (see (Donnelly and Elliott 2001;Atkins, Wills et al. 2007; Doronina, Wu et al. 2008)). By "codon" is meant three nucleotides on an mRNA (or on the sense strand of a DNA molecule) that are translated by a ribosome into one amino acid residue. Thus, two polypeptides can be synthesized from a single, contiguous open reading frame within an mRNA when the polypeptides are separated by a 2A oligopeptide sequence that is in frame. Such ribosomal skip mechanisms are well known in the art and are known to be used by several vectors for the expression of several proteins encoded by a single messenger RNA.

[0171] Further provided herein are polynucleotides comprising one or more coding sequences encoding a CAR and the CD70-binding protein or the CCR. In certain embodiments, the CAR and the CD70-binding protein or CCR are expressed from a bicistronic expression cassette and are linked by a P2A or T2A peptide. In certain embodiments, the expression of the one or more coding sequences or the bicistronic expression cassette is driven by a PGK promoter, especially a truncated PGK promoter.

[0172] To direct transmembrane polypeptides into the secretory pathway of a host cell, in some embodiments, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in a polynucleotide sequence or vector sequence. The secretory signal sequence is operably linked to the transmembrane nucleic acid sequence, i.e., the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell. Secretory signal sequences are commonly positioned 5' to the nucleic acid sequence encoding the polypeptide of interest, although certain secretory signal sequences may be positioned elsewhere in the nucleic acid sequence of interest (see, e.g., Welch et al., U.S. Patent No.5,037,743; Holland et al., U.S. Patent No. 5,143,830). Those skilled in the art will recognize that, in view of the degeneracy of the genetic code, considerable sequence variation is possible among these polynucleotide molecules. In some embodiments, nucleic acid sequences of the disclosure are codon-optimized for expression in mammalian cells, preferably for expression in human cells. Codon-optimization refers to the exchange in a sequence of interest of codons that are generally rare in highly expressed genes of a given species for codons that are generally frequent in highly expressed genes of such species, such codons encoding the same amino acids as the codons that are being exchanged.

[0173] Polynucleotides can comprise a native sequence (e.g., an endogenous sequence that encodes an antibody or a portion thereof or an endogenous promoter sequence) or cancomprise a variant of such a sequence. Polynucleotide variants contain one or more substitutions, additions, deletions and / or insertions such that the activity of the encoded polypeptide is not diminished, relative to a native molecule. The effect on the immunoreactivity of the encoded polypeptide can generally be assessed as described herein. Variants preferably exhibit at least about 70% identity, more preferably, at least about 80% identity, yet more preferably, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94%, and most preferably, at least about 95%, at least about 96% identity, at least about 97% identity, at least about 98% identity or about 99% identity to a polynucleotide sequence.2. Receptor polypeptides

[0174] In one aspect, the present disclosure provides polynucleotides, nucleic acid constructs, or molecules with sequences that encode receptor polypeptides, and engineered cells, e.g., engineered immune cells, that express the receptor polypeptides, as well as methods of making and using the nucleic acid constructs (or polynucleotides or molecules) and engineered cells. In one embodiment, the engineered cells are human.

[0175] In one embodiment, the one or more receptor polypeptides include, without limitation, an antigen binding fragment or portion, a chimeric antigen receptor (CAR), a chimeric cytokine receptor (CCR), and a CD70 binding protein. Different CCRs include, without limitation, inducible CCRs and constitutively active CCRs, as described in WO 19 / 169290, WO20 / 180694, WO20 / 180664, and WO21 / 041806, each of which is incorporated herein by reference in its entirety. CD70 binding proteins are described in PCT / US2022 / 033598, which is incorporated herein by reference in its entirety.

[0176] As used herein, chimeric antigen receptors (CARs) are proteins that specifically recognize target antigens (e.g., target antigens on cancer cells). When bound to the target antigen, the CAR can activate the immune cell to attack and destroy the cell bearing that antigen (e.g., the cancer cell). CARs can also incorporate costimulatory or signaling domains to increase their potency. See Krause etal., J. Exp. Med., Volume 188, No. 4, 1998 (619-626); Finney etal., Journal of Immunology, 1998, 161 : 2791-2797, Song et al., Blood 119:696-706 (2012); Kalos et al., Sci. Tr ansi. Med. 3:95 (2011); Porter et al., N. Engl. J. Med. 365:725-33 (2011), and Gross et al., Annu. Rev. Pharmacol. Toxicol. 56:59-83 (2016); U.S. Patent Nos. 7,741,465, and 6,319,494.

[0177] Chimeric antigen receptors described herein comprise an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises an antigen binding domain that specifically binds to a target. In some embodiments, CAR is a CD19-specific CAR that comprises the following elements from N- to C- terminus: a signal sequence, a CD 19 antigen binding domain (e.g., an anti-CD19 scFv), a hinge and transmembrane region, and one or more successive signaling domains. In certain embodiments, the CD19-specific CAR disclosed herein comprises the following elements from N- to C-terminus: a CD8a signal sequence, a CD19 scFv described herein, a CD8a hinge and transmembrane region, a 4- IBB cytoplasmic signaling domain, and a CD3(^ cytoplasmic signaling domain. Exemplary sequences are shown in Table 3.

[0178] In some embodiments, antigen-specific CARs further comprise a safety switch and / or one or more monoclonal antibody specific-epitope. a. Antigen Binding Domains

[0179] As discussed above, CARs described herein comprise an antigen binding domain. An “antigen binding domain” as used herein means any polypeptide that binds a specified target antigen. In some embodiments, the antigen binding domain binds to an antigen on a tumor cell. In some embodiments, the antigen binding domain binds to an antigen on a cell involved in a hyperproliferative disease.

[0180] In some embodiments, the antigen binding domain comprises a variable heavy chain, variable light chain, and / or one or more CDRs described herein. In some embodiments, the antigen binding domain is a single chain variable fragment (scFv), comprising light chain CDRs CDR1, CDR2 and CDR3, and heavy chain CDRs CDR1, CDR2 and CDR3.

[0181] An antigen binding domain is said to be “selective” when it binds to one target more tightly or with higher affinity than it binds to a second target.

[0182] In some embodiments, the antigen binding domain specifically binds BCMA, MUC16 (also known as CA125), EGFR, EGFRvIII, MUC1, Flt-3, WT-1, CD20, CD23, CD30, CD38, CD70, CD33, CD133, MHC-WT1, TSPAN10, MHC-PRAME, MHC-NY- ESO1, HER2 (ERBB2), CAIX (Carbonic anhydrase IX), LIV1, ADAM10, CHRNA2, LeY, NKG2D, CS1, CD44v6, ROR1, CD19, Claudin-18.2 (Claudin-18A2, or Claudinl8 isoform 2), PSCA, DLL3 (Delta-like protein 3, Drosophila Delta homolog 3, Delta3 ), Mud 7(Mucinl7, Muc3, Muc3), FAP alpha (Fibroblast Activation Protein alpha), Ly6G6D (Lymphocyte antigen 6 complex locus protein G6d, c6orf23, G6D, MEGT1, NG25), PSMA, MSLN, or RNF43 (E3 ubiquitin-protein ligase RNF43, RING finger protein 43). CARs and / or antibodies that target the antigens are disclosed, for example, in the following: BCMA- W0201616630, W02020150339, WO2019196713, WO2016014565, W02017025038 (each of which is incorporated herein by reference in its entirety); MUC16: US9, 169,328, WO2016149368, W02020023888 (each of which is incorporated herein by reference in its entirety); EGFRvIII: WO2017125830, W02016016341 (each of which is incorporated herein by reference in its entirety); Flt3 : WO2018222935, W02020010284, W02017173410 (each of which is incorporated herein by reference in its entirety); CD20: WO2018145649, W02020010235, WO2020123691 (each of which is incorporated herein by reference in its entirety); CD38: WO2017025323 (which is incorporated herein by reference in its entirety); CD70: WO2019152742, WO2018152181 (each of which is incorporated herein by reference in its entirety); CD33: WO2016014576 (which is incorporated herein by reference in its entirety); CD133: W02018072025 (which is incorporated herein by reference in its entirety); CS1 : W02019030240 (which is incorporated herein by reference in its entirety); R0R1 : WO2016115559 (which is incorporated herein by reference in its entirety); CD19: W02002077029, US11,077,144 (each of which is incorporated herein in its entirety); Claudin: WO2018006882, W02021008463 (each of which is incorporated herein by reference in its entirety); DLL3: W02020180591 (which is incorporated herein by reference in its entirety); WT1 : US20160152725A1, US7622119B2 (each of which is incorporated herein by reference in its entirety); CD23: US6011138A, CN1568198A (each of which is incorporated herein by reference in its entirety); CD30: US10815301B2, US10808035B2 (each of which is incorporated herein by reference in its entirety); PRAME: US20180148503A1, W02020186204Al(each of which is incorporated herein by reference in its entirety); LIVE US20200231699A1 (which is incorporated herein by reference in its entirety); NKG2D: WO2021179353A1, US20210269501 Al (each of which is incorporated herein by reference in its entirety); FAP Alpha: US20200246383A1, US20210115102A1 (each of which is incorporated herein by reference in its entirety); PSMA: US20210277141A1, W02020108646A1 (each of which is incorporated herein by reference in its entirety); and MSLN: CN109680002A, CN109628492A (each of which is incorporated herein by reference in its entirety).

[0183] In some embodiments, the cancer antigen is selected from the group consisting of carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CDS, CD7, CDIO, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD 123, CD 133, CD 138, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen), epithelial glycoprotein (EGP 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein kinases erb- B2,3,4, folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptors, Ganglioside G2 (GD2), Ganglioside G3 (GD3), human Epidermal Growth Factor Receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), Interleukin- 13 receptor subunit alpha-2 (IL-13Ra2), K-light chain, kinase insert domain receptor (KDR), Lewis A (CAI 9.9), , LI cell adhesion molecule (LICAM), melanoma antigen family A, 1 (MAGE- AI), Mucin 16 (Muc-16), Mucin 1 (Muc-1), Mesothelin (MSLN), NKG2D ligands, cancertestis antigen NY-ESO-1, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), tumor- associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF- R2), and Wilms tumor protein (WT-1).

[0184] Variants of the antigen binding domains (e.g., variants of the CDRs, VH and / or VL) are also within the scope of the disclosure, e.g., variable light and / or variable heavy chains that each have at least 70-80%, 80-85%, 85-90%, 90-95%, 95-97%, 97-99%, or above 99% identity to the amino acid sequences of antigen binding domain sequences. In some instances, such molecules include at least one heavy chain and one light chain, whereas in other instances the variant forms contain two variable light chains and two variable heavy chains (or subparts thereof). A skilled artisan will be able to determine suitable variants of the antigen binding domains as set forth herein using well-known techniques. In certain embodiments, one skilled in the art can identify suitable areas of the molecule that can be changed without destroying activity by targeting regions not believed to be important for activity.

[0185] In certain some embodiments, the polypeptide structure of the antigen binding domains is based on antibodies, including, but not limited to, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimetics”), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as “antibody conjugates”), and fragments thereof, respectively. In some embodiments, the antigen binding domain comprises or consists of avimers.

[0186] In some embodiments, an antigen binding domain is a scFv.

[0187] In some embodiments, an antigen-selective CAR comprises a leader or signal peptide.

[0188] In some embodiments, the CAR comprises a leader or signal peptide; in some embodiments the leader peptide comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the amino acid sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 1). In some embodiments, the leader (signal) peptide comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the leader (signal) peptide is encoded by a nucleic acid sequence comprising: ATGGCACTCCCCGTAACTGCTCTGCTGCTGCCGTTGGCATTGCTCCTGCACGCCG CACGCCCG (SEQ ID NO: 48).

[0189] In other embodiments, the disclosure relates to isolated polynucleotides encoding any one of the antigen binding domains described herein. In some embodiments, the disclosure relates to isolated polynucleotides encoding a CAR. Also provided herein are vectors comprising the polynucleotides, and methods of making same.

[0190] In other embodiments, the disclosure relates to isolated polynucleotides encoding any one of the antigen binding domains described herein. In some embodiments, the disclosure relates to isolated polynucleotides encoding a CAR. Also provided herein are vectors comprising the polynucleotides, and methods of making same.

[0191] In some embodiments, a CAR-immune cell (e.g., CAR-T cell) which can form a component of an engineered immune cell population (derived from donor cells of a donor cell population as described herein) generated by practicing the methods of the instant disclosure comprises a polynucleotide encoding a safety switch polypeptide, such as for example RQR8. See, e.g., WO2013153391 A, which is hereby incorporated by reference in its entirety. In a CAR-immune cell (e.g., a CAR-T cell) comprising the polynucleotide, the safety switch polypeptide can be expressed at the surface of a CAR-immune cell (e.g., CAR-T cell). b. Hinge Domains

[0192] The extracellular domain of the CARs of the disclosure can comprise a “hinge” domain (or hinge region). The term generally refers to any polypeptide that functions to link the transmembrane domain in a CAR to the extracellular antigen binding domain in a CAR. In particular, hinge domains can be used to provide more flexibility and accessibility for the extracellular antigen binding domain.

[0193] A hinge domain can comprise up to 300 amino acids — in some embodiments 10 to 100 amino acids or in some embodiments 25 to 50 amino acids. The hinge domain can be derived from all or part of naturally occurring molecules, such as from all or part of the extracellular region of CD8, CD4, CD28, 4- IBB, or IgG (in particular, the hinge region of an IgG; it will be appreciated that the hinge region can contain some or all of a member of the immunoglobulin family such as IgGl, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, or fragment thereof), or from all or part of an antibody heavy-chain constant region. Alternatively, the hinge domain can be a synthetic sequence that corresponds to a naturally occurring hinge sequence, or can be an entirely synthetic hinge sequence. In some embodiments said hinge domain is a part of human CD8a chain (e.g., NP 001139345.1). In other embodiments, said hinge and transmembrane domains comprise a part of human CD8a chain. In some embodiments, the hinge domain of CARs described herein comprises a subsequence of CD8a, an IgGl, IgG4, PD-1 or an FcyRIIIa, in particular the hinge region of any of an CD8a, an IgGl, IgG4, PD-1 or an FcyRIIIa. In some embodiments, the hinge domain comprises a human CD8a hinge, a human IgGl hinge, a human IgG4 hinge, a human PD-1 hinge, or a human FcyRIIIa hinge. In some embodiments, the CARs diclosed herein comprise an scFv, a CD8a human hinge and transmembrane domains, the CD3(^ signaling domain, and 4- IBB signaling domain. Table 3 provides amino acid sequences for exemplary hinge domains, as provided herein. In some embodiments, the hinge in the CAR of the disclosure is a CD8 hinge comprising the amino acid sequence of TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 9). In other embodiments, the hinge in the CAR of the disclosure is a CD28 hinge comprising the amino acid sequence of IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 93). c. Transmembrane Domains

[0194] The CARs of the disclosure are designed with a transmembrane domain that is fused to the extracellular domain of the CAR. It can similarly be fused to the intracellulardomain of the CAR. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex. In some embodiments, short linkers can form linkages between any or some of the extracellular, transmembrane, and intracellular domains of the CAR.

[0195] Suitable transmembrane domains for a CAR disclosed herein have the ability to (a) be expressed at the surface an immune cell such as, for example without limitation, a lymphocyte cell, such as a T helper (Th) cell, cytotoxic T (Tc) cell, T regulatory (Treg) cell, or Natural killer (NK) cells, and / or (b) interact with the extracellular antigen binding domain and intracellular signaling domain for directing the cellular response of an immune cell against a target cell.

[0196] The transmembrane domain can be derived either from a natural or from a synthetic source. Where the source is natural, the domain can be derived from any membrane-bound or transmembrane protein.

[0197] Transmembrane regions of particular use in this disclosure can be derived from (comprise, or correspond to) CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen- 1 (LFA-1, CDl-la / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP- 10, Fc gamma receptor, MHC class 1 molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 lb, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8),SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD 19a, a ligand that specifically binds with CD83, or any combination thereof.

[0198] As non-limiting examples, the transmembrane region can be derived from, or be a portion of a T cell receptor such as a, P, y or 5, polypeptide constituting CD3 complex, IL-2 receptor p55 (a chain), p75 (P chain) or y chain, subunit chain of Fc receptors, in particular Fey receptor III or CD proteins. Alternatively, the transmembrane domain can be synthetic and can comprise predominantly hydrophobic residues such as leucine and valine. In some embodiments said transmembrane domain is derived from the human CD8a chain (e.g., NP_001139345.1).

[0199] In some embodiments, the transmembrane domain in the CAR of the disclosure is a CD8a transmembrane domain. In some embodiments, the transmembrane domain in the CAR of the disclosure is a CD8a transmembrane domain comprising the amino acid sequence IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 97). In some embodiments, the hinge and transmembrane domain in the CAR of the disclosure is a CD8a hinge and CD8a transmembrane domain.

[0200] In some embodiments, the transmembrane domain in the CAR of the disclosure is a CD28 transmembrane domain. In some embodiments, the transmembrane domain in the CAR of the disclosure is a CD28 transmembrane domain comprising the amino acid sequence of FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 56). In some embodiments, the hinge and transmembrane domain in the CAR of the disclosure is a CD28 hinge and CD28 transmembrane domain. In other embodiments, the CD28 hinge and CD28 transmembrane domain comprises the amino acid sequence of lEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLV TVAFIIFWV (SEQ ID NO: 94). In some embodiments, the CAR of the disclosure comprises a CD8 hinge and a CD28 transmembrane domain. In other embodiments, the CD8 hinge and CD28 transmembrane domain comprises the amino acid sequence of TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVL ACYSLLVTVAFIIFWV (SEQ ID NO: 95). d. Intracellular Domains

[0201] The intracellular (cytoplasmic) domain of the CARs of the disclosure can provide activation of at least one of the normal effector functions of the immune cell comprising theCAR, e.g., Signal 1 / activation and / or Signal 2 / costimulation. Effector function of a T cell, for example, may refer to cytolytic activity or helper activity, including the secretion of cytokines.

[0202] In some embodiments, an activating intracellular signaling domain for use in a CAR can be the cytoplasmic sequences of, for example without limitation, the T cell receptor and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability.

[0203] It will be appreciated that suitable (e.g., activating) intracellular domains include, but are not limited to signaling domains derived from (or corresponding to) CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD1- la / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 lb, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, a ligand that specifically binds with CD83, or any combination thereof.

[0204] The intracellular domains of the CARs of the disclosure can incorporate, in addition to the activating domains described above, co-stimulatory signaling domains (interchangeably referred to herein as costimulatory molecules) to increase their potency. Co-stimulatory domains can provide a signal in addition to the primary signal provided by an activating molecule as described herein.

[0205] A “co-stimulatory molecule” as used herein refers to the cognate binding partner on immune cells, e.g. T cells, that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the cell, such as, but not limited to proliferation. Co- stimulatory molecules include, but are not limited to an MHC class I molecule, BTLA and Toll ligand receptor. Examples of costimulatory molecules include CD27, CD28, CD8, 4- 1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 and a ligand that specifically binds with CD83 and the like.

[0206] It will be appreciated that suitable co-stimulatory domains within the scope of the disclosure can be derived from (or correspond to) for example, CD28, 0X40, 4- 1BB / CD137, CD2, CD3 (alpha, beta, delta, epsilon, gamma, zeta), CD4, CD5, CD7, CD9, CD16, CD22, CD27, CD30, CD 33, CD37, CD40, CD 45, CD64, CD80, CD86, CD134, CD137, CD154, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1 (CD1 la / CD18), CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNFR, integrin, signaling lymphocytic activation molecule, BTLA, Toll ligand receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl-ld, ITGAE, CD 103, ITGAL, CDl-la, LFA-1, ITGAM, CDl-lb, ITGAX, CDl-lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD 19a, CD83 ligand, or fragments or combinations thereof. It will be appreciated that additional costimulatory molecules, or fragments thereof, not listed above are within the scope of the disclosure.

[0207] In some embodiments, the intracellular / cytoplasmic domain of the CAR can be designed to comprise the 4-1BB / CD137 domain by itself or combined with any other desired intracellular domain(s) useful in the context of the CAR of the disclosure. The complete native amino acid sequence of 4-1BB / CD137 is described in NCBI ReferenceSequence: NP_ 001552.2. The complete native 4-1BB / CD137 nucleic acid sequence is described in NCBI Reference Sequence: NM_ 001561.5.

[0208] In some embodiments, the intracellular / cytoplasmic domain of the CAR can be designed to comprise the CD28 domain by itself or combined with any other desired intracellular domain(s) useful in the context of the CAR of the disclosure. The complete native amino acid sequence of CD28 is described in NCBI Reference Sequence:NP 006130.1. The complete native CD28 nucleic acid sequence is described in NCBI Reference Sequence: NM_006139.1.

[0209] In some embodiments, the intracellular / cytoplasmic domain of the CAR can be designed to comprise the CD3 zeta domain by itself or combined with any other desired intracellular domain(s) useful in the context of the CAR of the disclosure.

[0210] For example, the intracellular domain of the CAR can comprise a CD3 zeta chain portion and a portion of a costimulatory signaling molecule. The intracellular signaling sequences within the intracellular signaling portion of the CAR of the disclosure can be linked to each other in a random or specified order. In some embodiments, the intracellular domain is designed to comprise the activating domain of CD3 zeta and a signaling domain of CD28. In some embodiments, the intracellular domain is designed to comprise the activating domain of CD3 zeta and a signaling domain of 4- IBB.

[0211] In some embodiments, the 4- IBB (intracellular domain) comprises the amino acid sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 12). In some embodiments, the 4-1BB (intracellular domain) is encoded by the nucleic acid sequence:AAGCGCGGCAGGAAGAAGCTCCTCTACATTTTTAAGCAGCCTTTTATGAGGCCC GTACAGACAACACAGGAGGAAGATGGCTGTAGCTGCAGATTTCCCGAGGAGGA GGAAGGTGGGTGCGAGCTG (SEQ ID NO: 57).

[0212] In some embodiments, the intracellular domain in the CAR is designed to comprise a portion of CD28 and CD3 zeta, wherein the intracellular CD28 comprises the amino acid sequence of SEQ ID NO: 13 and is encoded by the nucleic acid sequence set forth in SEQ ID NO: 58.

[0213] In some embodiments, the CD3 zeta amino acid sequence may comprise SEQ ID NO: 11 and the nucleic acid sequence that encodes the CD3 zeta amino acid sequence may comprise SEQ ID NO: 59:AGGGTGAAGTTTTCCAGATCTGCAGATGCACCAGCGTATCAGCAGGGCCAGAA CCAACTGTATAACGAGCTCAACCTGGGACGCAGGGAAGAGTATGACGTTTTGG ACAAGCGCAGAGGACGGGACCCTGAGATGGGTGGCAAACCAAGACGAAAAAA CCCCCAGGAGGGTCTCTATAATGAGCTGCAGAAGGATAAGATGGCTGAAGCCT ATTCTGAAATAGGCATGAAAGGAGAGCGGAGAAGGGGAAAAGGGCACGACGG TTTGTACCAGGGACTCAGCACTGCTACGAAGGATACTTATGACGCTCTCCACAT GCAAGCCCTGCCACCTAGG (SEQ ID NO: 59).

[0214] In some embodiments the intracellular signaling domain of the CAR of the disclosure comprises a domain of a co-stimulatory molecule. In some embodiments, the intracellular signaling domain of a CAR of the disclosure comprises a part of co-stimulatory molecule selected from the group consisting of fragment of 4-1BB (GenBank: AAA53133.) and CD28 (NP_006130.1).

[0215] In some embodiments, the intracellular signaling domain of the CAR of the present disclosure comprises an amino acid sequence which comprises at least 70%, at least 80%, at least 90%, 95%, 97%, or 99% sequence identity with an amino acid sequence shown in SEQ ID NO: 12. In some embodiments, the intracellular signaling domain of the CAR of the disclosure comprises amino acid sequence which comprises at least 70%, at least 80%, at least 90%, 95%, 97%, or 99% sequence identity with an amino acid sequence shown in SEQ ID NO: 13.

[0216] In exemplary embodiments, a CAR of the disclosure comprises, from N- terminus to C-terminus: a (cleavable) CD8a signal sequence, an anti-CD19 scFv, a CD8a hinge and transmembrane region, a 4-1BB cytoplasmic (costimulatory) signaling domain, and a CD3(^ cytoplasmic signaling domain.

[0217] In certain aspects, provided herein are engineered immune cells comprising and / or expressing a CD19-specific CAR, and a CD70-binding protein and / or a chimeric cytokine receptor (CCR). e. CD-70 binding proteins

[0218] In a related aspect, the current disclosure provides a CD70-binding proteins as described herein, wherein the CD70-binding proteins comprise an extracellular ligand or antigen binding domain that binds to CD70 (or a CD70-binding domain) and a transmembrane domain. The CD70-binding proteins can comprise no to one or more intracellular signaling domains as described herein. In some embodiments, the CD70- binding protein is a non-naturally occurring, or recombinant CD70-binding protein.

[0219] In some embodiments, the CD70-binding proteins provided herein comprise an extracellular domain that binds to CD70 (e.g., an anti-CD70 single chain variable fragment (scFv)) and a transmembrane domain. In some embodiments, the CD70-binding protein or CD70 CARs provided herein comprise an extracellular ligand-binding domain (e.g., scFv), a transmembrane domain, and an intracellular signaling domain. In some embodiments, the anti-CD70 scFv comprises the amino acid sequence of SEQ ID NO: 16, 17 or 18. In some embodiments, the anti-CD70 scFv comprises the amino acid sequence of SEQ ID NO: 18.

[0220] In some embodiments, the CD70-binding protein comprises one or more intracellular signaling domains selected from the group consisting of a CD3^ signaling domain, a CD36 signaling domain, a CD3y signaling domain, a CD3s signaling domain, a CD28 signaling domain, a CD2 signaling domain, an 0X40 signaling domain, and a 4-1BB signaling domain, or a variant thereof. In some embodiments, the intracellular signaling domain comprises the amino acid sequence of one or more of SEQ ID NOs: 11, 12, 13, 82, 83, 84, 85, 86, 87, 88, 89, 90, or 91. In some embodiments, the intracellular signaling domain comprises the amino acid sequence of one or more of SEQ ID NO: 11, 88, 89, or 90. In some embodiments, the CD70-binding protein comprises a CD3^ signaling domain, or a variant thereof, and does not comprise a costimulatory domain. In some embodiments, the CD70-binding protein comprises a 4- IBB signaling domain, or a variant thereof, and does not comprise a CD3 signaling domain. In some embodiments, the CD70-binding protein comprises a 4- IBB signaling domain and a CD3^ signaling domain. In some embodiments, the CD70-binding protein does not comprise an intracellular signaling domain. Different intracellular signaling domain or combination thereof can confer different signaling strength that can contribute to T cell proliferation, potency, survival, persistence, and / or resistant to host immune cell rejection. Described herein are CD70-binding proteins comprising no to one or more intracellular signaling domains. In some embodiments, the CD70-binding protein comprises the amino acid sequence of SEQ ID NO: 18, SEQ ID NO:10, and SEQ ID NO: 11 and having an amino acid sequence having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 19 or 21. In some embodiments, the CD70-binding protein comprises the amino acid sequence having one or more conservative amino acid substitutions of the amino acid sequence of SEQ ID NO: 19 or 21. In some embodiments, the CD70-binding protein comprises the amino acid sequence of SEQ ID NO: 19 or 21.

[0221] CD70 is expressed on T cells, especially activated T cells, including host alloreactive T cells. In some embodiments, engineered immune cells comprising the CD70- binding proteins described herein can exhibit different levels of persistence and / or resistance to rejection by host immune cells and can be suitable for use in lymphodepletion in vivo when administered to a patient. In some embodiments, engineered immune cells comprising the CD70-binding proteins described herein can inhibit proliferation and / or activities of host immune cells to different degrees that can allow for fine-tuning of the depth of lymphodepletion in vivo when administered to a patient. For example, engineered immune cells comprising a CD70-binding protein that demonstrated extended expansion and / or inhibition of host immune cells proliferation or activities, in e.g., an MLR assay, can be used for an extended lymphodepletion. In contrast, engineered immune cells comprising a CD70-binding protein that demonstrated less extended expansion and / or inhibition of host immune cells in the same or similar assay can be used when a less complete or a less thorough lymphodepletion is desired.

[0222] In certain aspects, the engineered cells, e.g., engineered immune cells, such as CAR T cells that comprise and / or express a receptor-specific CAR and a CD70-binding protein described herein can target receptor-positive as well as receptor-negative / CD70 positive tumor cells. The receptor-specific CAR T cells co-expressing the CD70-binding protein advantageously prevent receptor antigen escape of tumor cells. The receptor-specific CAR T cells co-expressing the CD70-binding protein and / or the chimeric cytokine receptor (CCR) demonstrate further enhanced activities. f. Chimeric Cytokine Receptors (CCRs)

[0223] The CCRs of the disclosure comprise transmembrane domains. The transmembrane domains of the disclosure contain sequences such that they allow forconstitutive dimerization of two monomers, thus allowing constitutive JAK activation on the intracellular portion, and constitutive recruitment and phosphorylation of, for example, STAT on the cytoplasmic region of the receptor.

[0224] The transmembrane domains are on the N-terminus and are coupled to intracellular / cytoplasmic domains on the C-terminus. In some embodiments, the coupling is achieved optionally through a linker.

[0225] As used herein, the transmembrane domains are capable of insertion into the membrane of a cell in which it is expressed. In some embodiments, the CCRs of the disclosure comprise a transmembrane domain spanning a cellular membrane, and an extracellular portion, and / or an intracellular portion. In some embodiments, the CCRs of the disclosure comprise a transmembrane domain spanning a cellular membrane, and an extracellular ligand binding domain, and / or an intracellular signaling domain. In some embodiments, the CCRs of the disclosure comprise a transmembrane domain spanning a cellular membrane, and further comprise an intracellular domain, and do not comprise an extracellular ligand binding domain. In some embodiments, the CCR comprises the amino acid sequence of SEQ ID NO: 27 or 29.

[0226] In some embodiments, the transmembrane domains of the disclosure are engineered (synthetic) and do not resemble any naturally occurring transmembrane domain, e.g., they are non-naturally occurring.

[0227] In other embodiments, the transmembrane domains of the disclosure are derived from or obtained from naturally occurring receptors.

[0228] In some embodiments, the transmembrane domains and / or JAK -binding / activating domains (or simply referred to as JAK -binding domains) of the disclosure are derived from, for example, one or more of the following receptors: erythropoietin receptor (EpoR), Interleukin 6 signal transducer (GP130 or IL6ST), prolactin receptor (PrlR), growth hormone receptor (GHR), granulocyte colony-stimulating factor receptor (GCSFR), and thrombopoietin receptor / myeloproliferative leukemia protein receptor (TPOR / MPLR). When derived from naturally occurring receptors, the entire receptor, or the entire transmembrane sequence of the receptor may not be necessary to effectuate constitutive activation and constitutive JAK binding / activation on the intracellular portion. In some embodiments, the transmembrane domain of the disclosure is derived from a truncated version of the naturally occurring TPOR / MPLR (myeloproliferative leukemia protein)receptor as shown in SEQ ID NO: 51. Accordingly fragments of naturally occurring receptors may be utilized. Furthermore, certain mutations may be introduced into the transmembrane domains derived from naturally occurring receptors, to further tune the downstream signaling. In some embodiments, the transmembrane domain of the disclosure is derived from TPOR / MPLR with the H499L / S505N / W515K substitutions as shown in SEQ ID NO: 25.

[0229] The OCRs of the disclosure comprise cytoplasmic recruiting domains. The recruiting domain can be a STAT -recruiting domain, an API -recruiting domain, a Myc / Max-recruiting domain; or an NFkB -recruiting domain. In some embodiments, the recruiting domain is a Signal Transducer and Activator of Transcription (STAT)-recruiting (STAT-activating) domains, e.g. from receptor tails (cytotails) or from cytokine receptor tails. These intracellular recruiting domains of the CCRs of the disclosure allow for the propagation of Signal 3 in an immune cell comprising a CAR and a chimeric cytokine receptor (e.g. a CAR-T-cell with a chimeric cytokine receptor of the disclosure). Cytokine signaling propagated through the Stat-recruiting domain allows for the cytokine-based immune potentiation of the cell. In some embodiments, the immune-potentiation is homeostatic, e.g. signaling gives rise to increase in immune cells bearing the CAR. In some embodiments, the immune-potentiation is inflammatory, e.g. signaling gives rise to increase in the potency of the immune cells bearing the CAR. In some embodiments, the immune- potentiation prevents exhaustion, e.g. signaling maintains the long-term functionality of immune cells bearing the CAR.

[0230] In some embodiments, the recruiting domains of the disclosure are synthetic, and do not resemble any naturally occurring receptor fragment. In some embodiments, the immune-potentiation prevents exhaustion, e.g. signaling maintains the long-term functionality of immune cells bearing the CAR. In some embodiments, the Stat-recruiting domains of the disclosure are synthetic, and do not resemble any naturally occurring receptor fragment.

[0231] In other embodiments, the Stat-recruiting domains of the disclosure are derived from cytoplasmic tails of naturally occurring receptors, e.g. derived from naturally occurring cytokine receptors. These cytoplasmic tails of naturally occurring receptors may be the regions downstream of the JAK-binding domains of the transmembrane domain of the receptor. The Stat-recruiting domains of the chimeric cytokine receptors comprise atleast one STAT -recruiting domain from at least one receptor. In some embodiments, the Stat-recruiting domain comprises at least one STAT 1 -recruiting domain. In some embodiments, the Stat-recruiting domain comprises at least one STAT2-recruiting domain. In some embodiments, the Stat-recruiting domain comprises at least one STAT3-recruiting domain. In some embodiments, the Stat-recruiting domain comprises at least one STAT4- recruiting domain. In some embodiments, the Stat-recruiting domain comprises at least one STAT5-recruiting domain. In some embodiments, the Stat-recruiting domain comprises at least one STAT6 -recruiting domain. In some embodiments, the Stat-recruiting domain comprises at least one STAT7-recruiting domain.

[0232] In some embodiments, the naturally occurring receptor from which the Statrecruiting domain is derived, is not a cytokine receptor.

[0233] In some embodiments, the naturally occurring receptor from which the Statrecruiting domain is derived, is a cytokine receptor. Exemplary cytokine receptors through which T-cell-immune potentiating cytokines signal include, but are not limited to IL-2 receptor, IL-7 receptor, IL- 15 receptor and IL-21 receptor. In alternative embodiments, the receptor from which the Stat-recruiting domain is derived, is not a cytokine receptor. By choosing the Stat-recruiting domain of the CCR, the receptor can be redirected to signaling of choice. In some embodiments, the recruiting domain comprises the amino acid sequence of SEQ ID NO: 26.

[0234] In some embodiments, the CCR of the disclosure comprises a recruiting domain connected to the C-terminus of the transmembrane / JAK2 binding domain, with or without a linker. In some embodiments, the linker comprises one or more amino acid residues. In some embodiments, the CCR comprises the amino acid sequence of SEQ ID NO: 27 or 29.

[0235] In some embodiments, the CCR of the disclosure comprises an extracellular ligand binding domain. In some embodiments, the extracellular ligand binding domain binds to an immune inhibitory molecule, such as PDL1. In some embodiments, the extracellular ligand binding domain of the CCR comprises an antibody that binds to PDL-1. In some embodiments, the extracellular ligand binding domain of the CCR comprises an ectodomain of PD-1 (PD-1 CCR). In some embodiments, binding to PDL-1 by the PD-1 ectodomain of the PD-1 CCR can turn the inhibitory signal of PDL-1 into a stimulatory signal via the signal transduction via the intracellular cytoplasmic recruiting domain. In some embodiments, the PD-1 CCR comprises the TPOR / MPLR transmembrane / JAK bindingdomain comprising the amino acid sequence of SEQ ID NO: 50 and an intracellular recruiting domain of SEQ ID NO: 26. In some embodiments, the PD-1 ectodomain comprises a wild-type PD-1 ectodomain and comprises the amino acid sequence of SEQ ID NO: 52.

[0236] In some embodiments, the PD-1 ectodomain comprises mutations to the wild-type PD-1 ectodomain sequence. In some embodiments, the PD-1 ectodomain sequence is a high affinity PD-1 ectodomain. In some embodiments, the PD-1 ectodomain sequence is a high affinity PD-1 ectodomain and comprises the amino acid sequence of SEQ ID NO: 53. In certain embodiments, the PD-1 ectodomain comprises one or more tandem repeat of the high affinity PD-1 ectodomains. g. Dominant negative receptor

[0237] In a further aspect, provided herein are PD-1 dominant receptor that comprises a PD-1 ectodomain and a transmembrane domain, without a functional intracellular signaling domain. In some embodiments, the PD-1 ectodomain comprises the wild-type PD-1 ectodomain. In some embodiments, the PD-1 ectodomain comprises a mutant PD-1 ectodomain. In some embodiments, the PD-1 ectodomain comprises a high-affinity PD-1 ectodomain. In some embodiments, the PD-1 ectodomain comprises the amino acid sequences of SEQ ID NO: 53.

[0238] In further embodiments, the transmembrane domain comprises a PD-1 transmembrane domain. In some embodiments, the transmembrane domain comprises a CD8a transmembrane domain. In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NOS: 10, 50, or 60. Conservative amino acid substitutions of the polypeptides disclosed herein are contemplated.

[0239] In some embodiments, the PD-1 dominant negative receptor comprises the amino acid sequence of SEQ ID NO: 52 or 53. In some embodiments, the PD-1 dominant negative receptor comprises the amino acid sequence of SEQ ID NO: 52 or 53 and having at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 54 or 55. In some embodiments, the PD-1 dominant negative receptor comprises the amino acid sequence having one or more conservative amino acid substitutions of the amino acid sequence ofSEQ ID NO: 54 or 55. In some embodiments, the PD-1 dominant negative receptor comprises the amino acid sequence of SEQ ID NO: 54 or 55.

[0240] The disclosure encompasses modifications to the polypeptides disclosed herein, e.g., a CAR, a CCR, a CD70-binding protein, a dominant negative receptor, etc., which do not significantly affect their properties and variants which have enhanced or decreased activity and / or affinity as desired. Modification of polypeptides is routine practice in the art and need not be described in detail herein. Examples of modified polypeptides include polypeptides with conservative substitutions of amino acid residues, one or more deletions or additions of amino acids which do not significantly deleteriously change the functional activity, or which mature (enhance) the affinity of the polypeptide for its ligand, or use of chemical analogs.

[0241] Amino acid sequence insertions include amino- and / or carboxyl -terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue or the antibody fused to an epitope tag. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody of an enzyme or a polypeptide which increases the half-life of the antibody in the blood circulation.

[0242] Substitution variants have at least one amino acid residue in the antigen binding domain removed and a different residue inserted in its place. In some embodiments, sites of interest for substitutional mutagenesis include the hypervariable regions / CDRs, but FR alterations are also contemplated. Conservative substitutions are shown in Table 2 under the heading of "conservative substitutions." If such substitutions result in a change in biological activity, then more substantial changes, denominated "exemplary substitutions" in Table 2, or as further described below in reference to amino acid classes, may be introduced and the products screened.Table 2: Amino Acid Substitutions

[0243] In certain aspects, the disclosure provides immune cells or engineered cells, e.g., engineered immune cells, that comprise the polynucleotides that comprise a first and a second coding sequences, wherein either the first or second coding sequence encodes an anti-CD19 CAR. In some embodiments, the engineered immune cells further comprise an additional polynucleotide comprising one or more coding sequences.

[0244] In some embodiments, the one or more coding sequences comprise a coding sequence that encodes a PD-1 dominant negative receptor. In some embodiments, the PD-1 dominant negative receptor comprises a wild-type PD-1 ectodomain. In some embodiments, the PD-1 dominant negative receptor comprises a high affinity PD-1 ectodomain. In some embodiments, the PD-1 dominant receptor does not comprise a functional intracellularsignaling domain. In some embodiments, the PD-1 dominant negative receptor comprises the amino acid sequence of SE ID NO: 54.

[0245] In some embodiments, the additional polynucleotide comprises one or more coding sequences. In some embodiments, the one or more coding sequences encode a CCR. In some embodiments, the CCR comprises the amino acid sequence of SEQ ID NO: 27 or 29. In some embodiments, the one or more coding sequences encode a CD70-binding protein. In some embodiments, the CD70-binding protein comprises the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 21. In some embodiments, the one or more coding sequences encode a PD-1 CCR. In some embodiments, the PD-1 CCR comprises a PD-1 ectodomain comprising the amino acid sequence of SEQ ID NO: 54 or SEQ ID NO: 55, a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 50, and an intracellular signaling domain comprising the amino acid sequence of SEQ ID NO: 26. In some embodiments, the one or more coding sequences encode a PD-1 dominant negative receptor, In some embodiments, the PD-1 dominant negative receptor comprises the amino acid sequence of SEQ ID NO: 54 or SEQ ID NO: 55.

[0246] In some embodiments, the additional polynucleotide comprises, from 5’ to 3’, a promoter operably linked to a third coding sequence and a fourth coding sequence. In some embodiments, the third coding sequence encodes a CCR or a CD70-binding protein. In some embodiments, the fourth coding sequence encodes a PD-1 CCR or a PD-1 dominant negative receptor. In some embodiments, the third coding sequence encodes a PD-1 CCR or a PD-1 dominant negative receptor. In some embodiments, the fourth coding sequence encodes a CCR or a CD70-binding protein.

[0247] In further aspects, provided herein are engineered immune cells, e.g., CAR T cells that express a CD19-specific CAR and a CD70-binding protein and / or a CCR. In some embodiments, the engineered immune cells, e.g., CD19 CAR T cells, further express a PD-1 dominant negative receptor.

[0248] In some embodiments, the engineered cells, e.g., engineered immune cells, comprise a polynucleotide or an integrated nucleic acid sequence comprising, from 5’ to 3’, a first and a second coding sequences driven by a truncated PGK promoter, wherein the first coding sequence encodes an antigen-specific CAR, e.g., a CD19-specific CAR and the second coding sequence encodes a CD70-binding protein, as described herein, or wherein the first coding sequence encodes a CD70-binding protein, and the second coding sequenceencodes an antigen-specific CAR, e.g., a CD19-specific CAR, as described herein, or wherein the first coding sequence encodes an antigen-specific CAR, e.g., a CD19-specific CAR, and the second coding sequence encodes a nucleic acid inhibitory agent, e.g., an RNA interference agent, as described herein, or wherein the first coding sequence encodes a CD70 binding protein, and the second coding sequence encodes a nucleic acid inhibitory agent, e.g., an RNA interference agent, as described herein.

[0249] In some embodiments, the engineered cells, e.g., engineered immune cells, further comprise an additional polynucleotide or an additional integrated nucleic acid sequence comprising one or more coding sequences, wherein the one or more coding sequences encode a CCR, and optionally a PD-1 dominant negative receptor or a nucleic acid inhibitory agent, e.g., an RNA interference agent, as described herein. In some embodiments, the additional polynucleotide or additional integrated nucleic acid sequence comprises, from 5’ to 3’, a third and a fourth coding sequences, wherein the third coding sequence encodes a CCR and the fourth coding sequence encodes a PD-1 dominant negative receptor or a nucleic acid inhibitory agent, e.g., an RNA interference agent, as described herein, or wherein the third coding sequence encodes a PD-1 dominant negative receptor and the fourth coding sequence encodes a CCR or a nucleic acid inhibitory agent, e.g., an RNA interference agent, as described herein.

[0250] In some embodiments, the engineered cells, e.g., engineered immune cells, comprise a polynucleotide or an integrated nucleic acid sequence comprising, from 5’ to 3’, a first and a second coding sequences driven by a truncated PGK promoter, wherein the first coding sequence encodes a CCR and the second coding sequence encodes an antigenspecific CAR, e.g., a CD19-specific CAR, as described herein, or wherein the first coding sequence encodes an antigen-specific CAR, e.g., a CD19-specific CAR, and the fourth coding sequence encodes a CCR, as described herein. In some embodiments, the engineered cells, e.g., engineered immune cells, further comprise an additional polynucleotide or additional integrated sequence comprising one or more coding sequences, wherein the one or more coding sequences encode a CD70-binding protein, and optionally a PD-1 dominant negative receptor, as described herein. In some embodiments, the additional polynucleotide comprises, from 5’ to 3’, a third and a fourth coding sequences, wherein the third coding sequence encodes a CD70-binding protein and the fourth coding sequence encodes a PD-1 dominant negative receptor, as described herein, or wherein the third coding sequenceencodes a PD-1 dominant negative receptor and the fourth coding sequence encodes a CD70-binding protein, as described herein.

[0251] Also provided are populations of engineered immune cells, e.g., CAR T cells. In some embodiments, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the population of engineered immune cells express the CD19 CAR described herein. In some embodiments, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the population of engineered immune cells express the CD 19 CAR and the CD70-binding protein described herein. In some embodiments, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the population of engineered immune cells express the CD 19 CAR and the CCR described herein. In some embodiments, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the population of engineered immune cells express the CD 19 CAR, the CD70-binding protein and the CCR described herein.3. Engineered cells

[0252] Provided herein are engineered cells, e.g., engineered immune cells, expressing a receptor polypeptide and / or a nucleic acid inhibitory agent of the present disclosure. The engineered cells can be allogeneic or autologous with respect to the subject to which the engineered cells are administered.

[0253] In some embodiments, the engineered cell is an engineered immune cell selected from a T cell (e.g., inflammatory T lymphocyte, cytotoxic T lymphocyte, regulatory T lymphocyte (Treg), helper T lymphocyte, tumor infiltrating lymphocyte (TIL)), natural killer (NK) cell, natural killer T cell (NKT), TCR-expressing cell, dendritic cell, killer dendritic cell, a mast cell, or a B-cell. In some embodiments, the engineered immune cell can be derived from the group consisting of CD4+ T-lymphocytes and CD8+ T- lymphocytes. In some exemplary embodiments, the engineered immune cell is a T cell. In some exemplary embodiments, the engineered immune cell is a gamma delta T cell. In some exemplary embodiments, the engineered immune cell is a macrophage. In some exemplary embodiments, the engineered immune cell is a natural killer (NK) cell.

[0254] In some embodiments, the engineered immune cell can be derived from, for example without limitation, a stem cell. The stem cells can be adult stem cells, non-human embryonic stem cells, more particularly non-human stem cells, cord blood stem cells,progenitor cells, bone marrow stem cells, induced pluripotent stem cells (iPSCs), totipotent stem cells or hematopoietic stem cells.

[0255] In some embodiments, the cell is obtained or prepared from peripheral blood. In some embodiments, the cell is obtained or prepared from peripheral blood mononuclear cells (PBMCs). In some embodiments, the cell is obtained or prepared from bone marrow. In some embodiments, the cell is obtained or prepared from umbilical cord blood. In some embodiments, the cell is a human cell.

[0256] In some embodiments, the cell is transfected or transduced by the polynucleotide, the nucleic acid construct, or the nucleic acid vector using a method selected from the group consisting of electroporation, sonoporation, biolistics (e.g., Gene Gun), lipid transfection, polymer transfection, nanoparticles, viral transfection or transduction (e.g., retrovirus, lentivirus, AAV) or polyplexes.

[0257] In some embodiments, the engineered cells, e.g., engineered immune cells, expressing at their cell surface membrane an antigen-specific CAR, e.g., a CD19-specific CAR, a CD70-binding protein and / or a CCR of the disclosure comprise a percentage of stem cell memory and central memory cells greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In some embodiments, the engineered cells, e.g., engineered immune cells, expressing at their cell surface membrane an antigen-specific CAR, e.g., a CD19-specific CAR, a CD70-binding protein and / or a CCR of the disclosure comprise a percentage of stem cell memory and central memory cells of about 10% to about 100%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, about 15% to about 100%, about 15% to about 90%, about 15% to about 80%, about 15% to about 70%, about 15% to about 60%, about 15% to about 50%, about 15% to about 40%, about 15% to about 30%, about 20% to about 100%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 20% to about 40%, about 20% to about 30%, about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, about 30% to about 50%, about 30% to about 40%, about 40% to about 100%, about 40% to about 90%, about 40% to about 80%, about 40% to about 70%, about 40% to about 60%, about 40% to about 50%, about 50% to about 100%, about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 50% to about 60%,about 60% to about 100%, about 60% to about 90%, about 60% to about 80%, about 60% to about 70%, about 70% to about 90%, about 70% to about 80%, about 80% to about 100%, about 80% to about 90%, about 90% to about 100%, about 25% to about 50%, about 75% to about 100%, or about 50% to about 75%.

[0258] In some embodiments, the cell is an immune cell, wherein the immune cell is an inflammatory T-lymphocyte that expresses the antigen-specific CAR, e.g., a CD19-specific CAR, described herein. In some embodiments, the immune cell is a cytotoxic T- lymphocyte that expresses any one of the antigen-specific CARs described herein. In some embodiments, the immune cell is a regulatory T-lymphocyte that expresses any one of the antigen-specific CARs described herein. In some embodiments, the immune cell is a helper T-lymphocyte that expresses any one of the CARs described herein.

[0259] Prior to expansion and genetic modification, a source of cells can be obtained from a subject through a variety of non-limiting methods. Cells can be obtained from a number of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, any number of T cell lines available and known to those skilled in the art, may be used. In some embodiments, cells can be derived from a healthy donor or from a patient e.g., a patient diagnosed with cancer or from a patient diagnosed with an infection. In some embodiments, cells can be part of a mixed population of cells which present different phenotypic characteristics.

[0260] In another aspect, the present disclosure provides engineered cells, e.g., engineered immune cells, comprising a transgene that is expressed under the control of a PGK promoter, such as a truncated PGK promoter. In one embodiment, the engineered cell comprises a recombinant nucleic acid sequence that encodes a receptor polypeptide (e.g., a CAR, CCR, CD70 binding protein) or a nucleic acid inhibitory agent, as described herein.

[0261] In some embodiments, an engineered cell, e.g., an engineered immune cell, according to the present disclosure may comprise one or more disrupted or inactivated genes. In some embodiments, an engineered cell according to the present disclosure comprises one disrupted or inactivated gene selected from the group consisting of CD52, GR, DCK, PDL1, PD-1, CTLA-4, LAG3, TIM3, BTLA, BY55, TIGIT, B7H5, LAIR1, SIGLEC10, 2B4, HLA, TCRa and TCR[3 and / or expresses a CAR, a multi-chain CAR and / or a pTa transgene. In some embodiments, an isolated cell comprises polynucleotidesencoding polypeptides comprising a multi-chain CAR. In some embodiments, the isolated cell according to the present disclosure comprises two disrupted or inactivated genes selected from the group consisting of: CD52 and GR, CD52 and TCRa, CD52 and TCRP, GR and TCRa, GR and TCRP, TCRa and TCRP, PD-1 and TCRa, PD-1 and TCRP, CTLA- 4 and TCRa, CTLA-4 and TCRP, LAG3 and TCRa, LAG3 and TCRP, TIM3and TCRa, Tim3 and TCRP, BTLA and TCRa, BTLA and TCRP, BY55 and TCRa, BY55 and TCRP, TIGIT and TCRa, TIGIT and TCRP, B7H5 and TCRa, B7H5 and TCRP, LAIR1 and TCRa, LAIR1 and TCRP, SIGLEC10 and TCRa, SIGLEC10 and TCRP, 2B4 and TCRa, 2B4 and TCRP and / or expresses a CAR, a multi-chain CAR and a pTa transgene. In some embodiments the method comprises disrupting or inactivating one or more genes by introducing into the cells an endonuclease able to selectively inactivate a gene by selective DNA cleavage. In some embodiments the endonuclease can be, for example, a zinc finger nuclease (ZFN), megaTAL nuclease, meganuclease, transcription activator-like effector nuclease (TALE-nuclease / TALEN), or CRISPR (e.g., Cas9, Casl2a or Casl2b) endonuclease.

[0262] In another embodiment, the recombinant nucleic acid sequence is integrated at a first site within the genome of the cell such that the receptor polypeptide is expressed by the cell at the surface of the cell and / or the nucleic acid inhibitory agent is expressed within the cell (e.g., an RNA interference sequence is expressed within the cell), and wherein integration of the recombinant nucleic acid at the first site reduces or prevents expression of a target gene. In one embodiment, the integration reduces or prevents expression of the target gene at the surface of the cell.

[0263] In other embodiments, the integration reduces or prevents expression of a functional T cell receptor (TCR) complex at the surface of the cell. In certain embodiments, the nucleic acid sequence encoding the receptor polypeptide and / or the nuclei acid inhibitory agent is integrated at a single site within the genome of the cell.

[0264] In certain embodiments, the nucleic acid sequence encoding the receptor polypeptide and / or nucleic acid inhibitory agent is integrated at two sites within the genome of the cell. In certain embodiments, the first site is an exon of the gene encoding a protein of the TCR complex. In other embodiments, the first site is an exon of the gene encoding a protein that is involved in T cell receptor (TCR)aP function or activity. In another embodiment, the target gene is a gene that is involved in T cell receptor (TCR)aP functionor activity. In one embodiment, the first site is within an exon of a gene selected from TRAC, a component of the TCR, an HLA, TCRa, TCRP, p2-microglobulin (“P2m”), CD52, GR, deoxy cytidine kinase (DCK), PD-1, and CTLA-4. In one other embodiment, the target gene is selected from TRAC, a component of the TCR, an HLA, TCRa, TCRP, P2- microglobulin (“P2m”), CD52, GR, deoxycytidine kinase (DCK), PD-1, and CTLA-4.

[0265] The present disclosure relates to an engineered cell, e.g., an engineered immune cell, that comprise a transgene integrated within a target gene of the cell, e.g., an immune cell, wherein the expression of the transgene is controlled by a promoter that is not the target gene promoter, as well as methods of making and using such engineered cells. In another embodiment, the promoter is a non-endogenous or exogenous promoter relative to the target gene. In one embodiment, the promoter is a truncated PGK promoter. In other embodiments, the expression of the transgene is under the control of a truncated PGK promoter.

[0266] In one embodiment, the engineered cell comprises a polynucleotide, nucleic acid construct or molecule comprising a donor template for integration into the genome and subsequent expression by the cell, wherein the donor template comprises a transgene. In another embodiment, the transgene comprises a recombinant nucleic acid sequence that encodes a receptor polypeptide (e.g., a CAR, CCR, CD70 binding protein) or a nucleic acid inhibitory agent, as described herein. In one other embodiment, the transgene comprises one or more sequences encoding one or more receptor polypeptides and / or one or more nucleic acid inhibitory agents, as described herein.

[0267] In one aspect, the present disclosure provides genetically modified immune cells for use in immunotherapies. The invention also provides genetically modified immune cells that comprise a transgene with a nucleic acid sequence that encodes a naturally occurring or non-naturally occurring polypeptide, such as a recombinant or chimeric polypeptide, inserted at a gene involved in T cell receptor (TCR)aP function or activity. In one embodiment, the encoded polypeptide is a chimeric antigen receptor (CAR), a chimeric cytokine receptor (CCR), or a CD70 binding protein. In another embodiment, the nucleic acid sequence of the transgene encodes a nucleic acid inhibitory agent.

[0268] In certain embodiments, the transgene comprises a promoter that is exogenous relative to the gene targeted for insertion of the transgene. The promoter may originate from another gene that is not directly involved in T cell receptor (TCR)a[3 function oractivity. In one embodiment, the promoter comprises a nucleic acid sequence that is derived from the gene, including a full or partial sequence of the original promoter sequence. In one embodiment, the gene is the phosphoglycerate kinase (PGK1) gene (PGK). In another embodiment, instant disclosure provides truncated PGK promoters that comprises a partial nucleic acid sequence derived from, obtained from, or originating from the original PGK promoter sequence of the PGK gene.

[0269] In other embodiments, the modified immune cells having transgenes comprising a CAR nucleic acid sequence that is under the control of a truncated PGK promoter exhibit improved expansion and / or cytotoxicity, as compared to modified immune cells having transgenes comprising a CAR nucleic acid sequence that is not under the control of a truncated PGK promoter. In one embodiment, the CAR nucleic acid sequence is inserted at a gene involved in in T cell receptor (TCR)aP function or activity. In another embodiment, the gene is selected from TRAC, a component of the TCR, an HLA, TCRa, TCRP, P2- microglobulin (“P2m”), CD52, GR, deoxycytidine kinase (DCK), PD-1, and CTLA-4. In one other embodiment, the target gene is selected from TRAC, a component of the TCR, an HLA, TCRa, TCRP, P2-microglobulin (“P2m”), CD52, GR, deoxycytidine kinase (DCK), PD-1, and CTLA-4.

[0270] In some embodiments, TCR is rendered not functional in the cells according to the disclosure by disrupting or inactivating TCRa gene and / or TCRP gene(s). Modified cells, which can proliferate independently of the TCRa signaling pathway are encompassed in the scope of the present disclosure. In some embodiments, a method to obtain modified cells derived from an individual is provided, wherein the cells can proliferate independently of the major histocompatibility complex (MHC) signaling pathway. Modified cells, which can proliferate independently of the MHC signaling pathway, susceptible to be obtained by this method are encompassed in the scope of the present disclosure. Modified cells disclosed herein can be used in for treating patients in need thereof against Host versus Graft (HvG) rejection and Graft versus Host Disease (GvHD); therefore in the scope of the present disclosure is a method of treating patients in need thereof against Host versus Graft (HvG) rejection and Graft versus Host Disease (GvHD) comprising treating said patient by administering to said patient an effective amount of modified cells comprising disrupted or inactivated TCRa and / or TCRP genes.

[0271] Provided herein are antigen-specific CAR-T cells, e.g., CD19-specific CAR-T cells, comprising a disrupted or inactivated dCK gene. In some embodiments, the dCK knockout cells are made by transfection of T cells using polynucleotides encoding specific TAL-nuclease directed against dCK genes by, for example, electroporation of mRNA encoding the specific TAL-nuclease. The dCK knockout antigen-specific CAR-T cells, e.g., CD19-specific CAR-T cells, are resistant to PNAs, including for example clorofarabine and / or fludarabine, and maintain T cell cytotoxic activity toward antigen-expressing cells, e.g., CD19-expressing cells.

[0272] In some embodiments, isolated cells or cell lines of the disclosure can comprise a pTa or a functional variant thereof. In some embodiments, an isolated cell or cell line can be further genetically modified by disrupting or inactivating the TCRa gene.

[0273] The disclosure also provides engineered immune cells comprising any of the CAR polynucleotides described herein. In some embodiments, a CAR can be introduced into an immune cell as a transgene via a plasmid vector. In some embodiments, the plasmid vector can also contain, for example, a selection marker which provides for identification and / or selection of cells which received the vector.

[0274] In one other aspect, the receptor polypeptides, e.g., CAR, CCR or CD70 binding polypeptides, may be synthesized in situ in the cell after introduction of polynucleotides encoding the receptor polypeptides into the cell. Alternatively, receptor polypeptides may be produced outside of cells, and then introduced into cells. Methods for introducing a polynucleotide or nucleic acid construct into cells are known in the art. In some embodiments, stable transformation methods (e.g., using a lentiviral vector) can be used to integrate the polynucleotide or nucleic acid construct into the genome of the cell. In other embodiments, transient transformation methods can be used to transiently express the polynucleotide or nucleic acid construct, and the polynucleotide or nucleic acid construct not integrated into the genome of the cell. In other embodiments, virus-mediated methods can be used. The polynucleotides or nucleic acid constructs may be introduced into a cell by any suitable means such as for example, recombinant viral vectors (e.g., retroviruses, adenoviruses), liposomes, and the like. Transient transformation methods include, for example without limitation, microinjection, electroporation or particle bombardment. Polynucleotides or nucleic acid constructs may be included in vectors, such as for example plasmid vectors or viral vectors.

[0275] In other embodiments, the modified immune cells are for use in immunotherapy.

[0276] An engineered immune can be genetically engineered to express one or more transgenes. The transgene can be introduced to the genome of the immune cell by a variety of methods, including retroviral or lentiviral transduction. The transgene delivered to the cell in a retroviral or lentiviral vector becomes randomly integrated into the genome of the cell after viral transduction. Random integration of the transgene may negatively undesirably affect the activities of the engineered cells. Thus, in some embodiments, the one or more transgenes are introduced into the engineered immune cell by site-specific integration.

[0277] In some embodiments, the engineered immune cells express higher level of the one or more transgenes introduced by site-specific integration as compared to, e.g., introduced by viral transduction. In some embodiments, the engineered immune cells exhibit improved activities of the one or more transgenes introduced by site-specific integration as compared to, e.g., introduced by viral transduction. In some embodiments, the engineered immune cells show less genome translocation when one or more transgenes are introduced by site-specific integration as compared to, e.g., introduced by viral transduction. In some embodiments, the one or more transgene is introduced into the CD52 locus by sitespecific integration. In some embodiments, the immune cell comprises additional genetic modifications, including without limitation, knock-out or knock-down of endogenous gene(s), enhancement of endogenous gene(s), and integration of exogenous gene(s). In some embodiments, the engineered immune comprises a transgene integrated into the CD52 locus and further comprises one or more genetic modifications at the TRAC locus. In some embodiments, the one or more genetic modifications at the TRAC locus comprises integration of a transgene at the TRAC locus.

[0278] In some embodiments, the engineered cells, e.g., engineered immune cells, comprise the polynucleotides integrated into the TRAC gene. In some embodiments, the engineered cells further comprise an additional polynucleotide that comprises a promoter operably linked to one or more coding sequences, wherein the one or more coding sequences expresses either a CCR, a CD70-binding protein, or a nucleic acid inhibitory agent, as described herein. In some embodiments, the one or more coding sequences of the additional polynucleotide further express a PD-1 dominant negative receptor. In some embodiments, the additional polynucleotide further comprises a 5’ homology arm and a 3’homology arm, wherein the 5’ and 3’ homology arms each are at least 100 nucleotides in length. In some embodiments, the 5’ and 3’ homology arms each comprises a nucleotide sequence homologous to a nucleotide sequence of a CD52 gene. In some embodiments, the promoter comprises an EFS promoter or a truncated PGK promoter described herein. In some embodiments, the additional polynucleotide is integrated into the CD52 gene. In some embodiments, the immune cell is a T cell, an NK cell, an NK-T cell, a tumor infiltrating lymphocyte (TIL), or a T cell of NK cell derived from an iPSC.

[0279] In related aspects, provided are engineered cells, e.g., engineered immune cells, comprising a recombinant polynucleotide sequence integrated at a human TRAC gene, wherein the recombinant polynucleotide sequence comprises a 5’ region of the human TRAC gene, the polynucleotide as described herein, and a 3’ region of the human TRAC gene, wherein the integrated recombinant polynucleotide sequence prevents expression of the human TRAC gene. In some embodiments, the engineered cells further comprise an additional recombinant polynucleotide sequence integrated at a human CD52 gene, wherein the additional recombinant polynucleotide sequence comprises a 5’ region of the human CD52 gene, one or more coding sequences, and a 3’ region of the human CD52 gene.

[0280] Also provided herein are cell lines obtained from a transformed immune cell (e.g., T-cell) according to any of the above-described methods. Also provided herein are modified cells resistant to an immunosuppressive treatment. In some embodiments, an isolated cell according to the disclosure comprises a polynucleotide encoding a CAR.

[0281] The immune cells of the disclosure can be activated and expanded, either prior to or after genetic modification of the immune cells, using methods as generally known. Generally, the engineered immune cells of the disclosure can be expanded, for example, by contacting with an agent that stimulates a CD3 TCR complex and a costimulatory molecule on the surface of the T-cells to create an activation signal for the T cell. For example, chemicals such as calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA), or mitogenic lectins like phytohemagglutinin (PHA) can be used to create an activation signal for the T cell.

[0282] In some embodiments, cell populations, such as T cell populations may be stimulated in vitro by contact with, for example, an anti-CD3 antibody such as an OKT3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction witha calcium ionophore. For co-stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody (e.g., an 0KT3 antibody) and an anti- CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. The anti-CD3 antibody and an anti-CD28 antibody can be disposed on a bead, such as a plastic or magnetic bead, or plate or other substrate. Conditions appropriate for T cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-y, IL-4, IL-7, GM-CSF, IL- 10, IL-2, IL- 15, TGFbeta, and TNF, or any other additives for the growth of cells known to the skilled artisan. Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl- cysteine and 2-mercaptoethanoi. Media can include RPMI 1640, A1M-V, DMEM, MEM, a- MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or an amount of cytokine(s) sufficient for the growth and expansion of T cells (e.g., IL-7 and / or IL-15). Antibiotics, e.g., penicillin and streptomycin, are included only in experimental cultures, not in cultures of cells that are to be infused into a subject. The target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37° C) and atmosphere (e.g., air plus 5% CO2). T cells that have been exposed to varied stimulation times may exhibit different characteristics. In some embodiments, the cells of the disclosure can be expanded by co-culturing with tissue or cells. The cells can also be expanded in vivo, for example in the subject's blood after administering the cell into the subject.4. Methods for generating engineered cells

[0283] A variety of known techniques can be utilized in making the polynucleotides, polypeptides, vectors, antigen binding domains, engineered cells (e.g., engineered immune cells), compositions, and the like according to the disclosure. Prior to the in vitro manipulation or genetic modification of the immune cells described herein, the cells may be obtained from a subject (e.g., a patient) or from a healthy donor.

[0284] In one aspect, the engineered cells are derived from a suitable source material, such as immune cells. In some embodiments, the immune cells comprise T cells. T cellscan be obtained from a number of sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph nodes tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cells can be obtained from a unit of blood collected from the subject using any number of techniques known to the skilled person, such as FICOLL™ separation.

[0285] Cells may be obtained from the circulating blood of an individual by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In certain embodiments, the cells collected by apheresis may be washed to remove the plasma fraction, and placed in an appropriate buffer or media for subsequent processing.

[0286] In certain embodiments, T cells are isolated from PBMCs by lysing the red blood cells and depleting the monocytes, for example, using centrifugation through a PERCOLL™ gradient. A specific subpopulation of T cells, (e.g., CD28+, CD4+, CDS+, CD45RA-, CD45RO+, CDS+, CD62-, CD95-, CD95+, IL2Rp+, TL2R0-, CCR7+, CCR7-, CDL-, CD62L+ and combinations thereof) can be further isolated by positive or negative selection techniques known in the art. In one example the subpopulation of T cells is CD45RA+, CD95-, IL-2RP-, CCR7+, CD62L+. In one example the subpopulation of T cells is CD45RA+, CD95+, IL-2RP+, CCR7+, CD62L+. In one example the subpopulation of T cells is CD45RO+, CD95+, IL-2R0+, CCR7+, CD62L+. In one example the subpopulation of T cells is CD45RO+, CD95+, IL-2RP+, CCR7-, CD62L-. In one example the subpopulation of T cells is CD45RA+, CD95+, IL-2R0+, CCR7-, CD62L-. For example, enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method for use herein is cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD1 lb, CD16, HLA-DR, and CD8. Flow cytometry and cell sorting may also be used to isolate cell populations of interest for use in the present disclosure.

[0287] PBMCs may be used directly for genetic modification with the immune cells (such as CARs or TCRs) using methods as described herein. In certain embodiments, afterisolating the PBMCs, T lymphocytes can be further isolated and both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations either before or after genetic modification and / or expansion.

[0288] In some embodiments, CD8+ cells are further sorted into naive, stem cell memory, central memory, and effector cells by identifying characteristic cell surface antigens that are associated with each of these types of CD8+ cells. In some embodiments, the expression of phenotypic markers of central memory T cells include CD45RO, CD62L, CCR7, CD28, CD3, and CD 127 and are negative for granzyme B. In some embodiments, stem cell memory T cells are CD45RO-, CD62L+, CD8+ T cells. In some embodiments, central memory T cells are CD45RO+, CD62L+, CD8+ T cells. In some embodiments, effector T cells are negative for CD62L, CCR7, CD28, and CD127, and positive for granzyme B and perforin.

[0289] In certain embodiments, CD4+ T cells are further sorted into subpopulations. For example, CD4+ T helper cells can be sorted into naive, central memory, and effector cells by identifying cell populations that have characteristic cell surface antigens.

[0290] In another aspect, the engineered cells, e.g., engineered immune cells, are stem cell-derived cells, e.g., stem cell-derived immune cells. In some embodiments, the immune cells may be derived from stem cells, such as a progenitor cell, a bone barrow stem cell, an inducible pluripotent stem cell, an iPSC, a hematopoietic stem cell, and a mesenchymal stem cell. iPS cells and other types of stem cells may be cultivated immortal cell lines or isolated directly from a patient. Various methods for isolating, developing, and / or cultivating stem cells are known in the art and may be used to practice the present invention.

[0291] In some embodiments, the cell, e.g., the immune cell, is an induced pluripotent stem cell (iPSC) derived from a reprogrammed cell, such as a T-cell. In some embodiments, the source material may be an induced pluripotent stem cell (iPSC) derived from a T cell or non-T cell. The source material may alternatively be a B cell, or any other cell from peripheral blood mononuclear cell isolates, hematopoietic progenitor, hematopoietic stem cell, mesenchymal stem cell, adipose stem cell, or any other somatic cell type.

[0292] Methods of preparing the engineered cells, e.g., engineered immune cells, for use in immunotherapy are provided herein. In some embodiments, the methods comprise obtaining cells from a donor, e.g., donor immune cells, introducing a receptor polypeptidesequence or a nucleic acid inhibitory agent, e.g., an RNA interference agent, under the control of a PGK promoter (such as a truncated PGK promoter), as described herein, into the donor cells, and expanding the cells.

[0293] The present disclosure provides methods for generating populations of engineered cells, e.g., engineered immune cells, that comprise one or more engineered cells that express one or more receptor polypeptides, e.g., a CAR, CCR, or CD70 binding protein, and / or nucleic acid inhibitory agents, e.g., RNA interference agents. In one embodiment, the transgenes include the sequences encoding such receptor polypeptides or nucleic acid inhibitory agents, e.g., RNA interference agents, may be introduced into cells using a targeted approach such that the transgene is inserted at a specific location in target gene of interest. In one embodiment, the targeted approach comprises use of a nuclease, such as a rare-cutting nuclease, that can cleave at a specific site within the target gene to allow insertion of polynucleotides, nucleic acid constructs, or molecules comprising the transgene at the specific cleavage site. In one embodiment, the expression of the receptor polypeptides and / or nucleic acid inhibitory agents is under the control of a PGK promoter, e.g., a truncated PGK promoter.

[0294] Prior to engineering of cells, a source of cells can be obtained from a subject through a variety of non-limiting methods. Cells can be obtained from a number of nonlimiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, any number of T cell lines available and known to those skilled in the art, may be used. In some embodiments, cells can be derived from a healthy donor. In some embodiments, cells can be part of a mixed population of cells which present different phenotypic characteristics.

[0295] In one aspect, the invention provides methods for making genetically modified immune cells that comprise one or more genetic modifications to one or more target genes that are involved in T cell receptor (TCR)a[3 function or activity. In one embodiment, the one or more target genes are TRAC and / or CD52. The genetic modifications are made such that the expression and / or activities of the one or more target genes are disrupted or inactivated. By disruption or inactivating a target gene, it is intended that the target gene is not expressed in a functional protein form and the activity of the gene product is impaired.

[0296] In some embodiments, the method comprises inactivating one or more target genes by introducing into the cells a rare-cutting endonuclease capable of selectively inactivating the target gene by introducing a specific double strand break at the target sequence within the target gene. In other embodiments the method comprises inactivating or reducing the expression level of one or more genes by introducing into the cells a rare-cutting endonuclease able to selectively inactivate a gene by selective DNA cleavage. In some embodiments the rare-cutting endonuclease can be, for example, a transcription activatorlike effector nuclease (TALE-nuclease or TALEN), a megaTAL endonuclease, a zinc finger endonuclease, a meganuclease, or a CRISPR endonuclease. In one embodiment, the rare- cutting endonuclease is a TALEN. In one other embodiment, the CRISPR endonuclease is a Cas9 endonuclease or a Cast 2 endonuclease, including Cast 2a and Cast 2b.

[0297] In one aspect, the inactivation of a target gene is via the use of a CRISPR endonuclease. A CRISPR endonuclease refers to a clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease, such as Cas9 or Casl2. Such endonucleases associate with a guide RNA (gRNA) that can direct cleavage by the endonuclease at a specific site through hybridization at a target region. In this case, one or more gRNA molecules can be designed to hybridize to one or more specific sites within one or more target genes to direct cleavage by at least one CRISPR endonuclease. Exemplary gRNAs targeting the TRAC gene and CD52 gene are provided in Table 3 as SEQ ID NOs: 46 and 47, respectively.

[0298] In another aspect, the rare-cutting endonuclease may be introduced to the cells before, simultaneously or after genetic modification of the cells, e.g., immune cells. In one embodiment, a polynucleotide encoding the rare-cutting endonuclease is introduced into the cells by a suitable method including, without limitation, transfection, lipofection, transduction, electroporation, sonoporation, biolistics (e.g., Gene Gun), lipid transfection, polymer transfection, nanoparticles, viral transfection or transduction (e.g., retrovirus, lentivirus, AAV) or polyplexes.

[0299] In some embodiments, the polynucleotides encoding the rare-cutting endonuclease according to the present disclosure can be mRNA which is introduced directly into the cells, for example by electroporation. In some embodiments, cytoPulse technology can be used to transiently permeabilize living cells for delivery of material into the cells. Parameters can bemodified in order to determine conditions for high transfection efficiency with minimal mortality. In some embodiments, the rare-cutting endonuclease is TALE-nuclease.

[0300] Also provided herein are methods of transfecting a cell, such as an immune cell e.g., a T cell. In some embodiments, the method comprises: contacting a cell with a nucleic acid, e.g., an RNA, and applying to the cell an agile pulse sequence consisting of (a) an electrical pulse with a voltage range from about 2250 to 3000 V per centimeter; (b) a pulse width of 0.1 ms; (c) a pulse interval of about 0.2 to 10 ms between the electrical pulses of step (a) and (b); (d) an electrical pulse with a voltage range from about 2250 to 3000 V per centimeter with a pulse width of about 100 ms and a pulse interval of about 100 ms between the electrical pulse of step (b) and the first electrical pulse of step (c); and (e) four electrical pulses with a voltage of about 325 V with a pulse width of about 0.2 ms and a pulse interval of 2 ms between each of 4 electrical pulses.

[0301] In some embodiments, a method of transfecting a cell, such as an immune cell, e.g., a T cell, comprises contacting the cell with RNA and applying to the cell an agile pulse sequence comprising: (a) an electrical pulse with a voltage of about 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2400, 2450, 2500, 2600, 2700, 2800, 2900 or 3000V per centimeter; (b) a pulse width of 0.1 ms; (c) and a pulse interval of about 0.2, 0.5, 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 ms between the electrical pulses of step (a) and (b); (d) one electrical pulse with a voltage range from about 2250 to 3000 V per centimeter, e.g. of 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2400, 2450, 2500, 2600, 2700, 2800, 2900 or 3000V per centimeter with a pulse width of 100 ms and a pulse interval of 100 ms between the electrical pulse of step (b) and the first electrical pulse of step (c); and (e) 4 electrical pulses with a voltage of about 325 V with a pulse width of about 0.2 ms and a pulse interval of about 2 ms between each of 4 electrical pulses. Any values included in the value range described above are disclosed in the present application. Electroporation medium can be any suitable medium known in the art. In some embodiments, the electroporation medium has conductivity in a range spanning about 0.01 to about 1.0 milliSiemens.

[0302] In other embodiments, the rare-cutting endonuclease has specificity for a nucleic acid recognition sequence within the target gene. In another embodiment, the endonuclease is expressed in the cell following introduction and generates a double-stranded break or cleavage site at the nucleic acid recognition sequence within the target gene. The presenceof the double-stranded break or cleavage site allows for a site-specific integration of a transgene via the recombinant vector, as further described herein.

[0303] An engineered cell, e.g., an engineered immune cell, can be genetically engineered to express one or more transgenes. The transgene can be introduced into the genome of a cell by a variety of methods, including retroviral or lentiviral transduction. The transgene delivered to the cell in a retroviral or lentiviral vector becomes randomly integrated into the genome of the cell after viral transduction. Random integration of the transgene may negatively undesirably affect the activities of the engineered cells. Thus, in some embodiments, the one or more transgenes are introduced into the engineered immune cell by site-specific integration.

[0304] In another aspect, the site-specific integration methods for making genetically modified cells, e.g., genetically modified immune cells, comprise the use of homology directed repair (HDR). In one embodiment, the HDR comprises homologous recombination (HR). In another embodiment, the method comprises use of a recombinant vector configured to use HDR, e.g., HR, to facilitate integration of a transgene into a cell, e.g., an immune cell. In another embodiment, the methods comprise the use of a polynucleotide that contains one or more sequences with homology to sequences of a target gene. In one other embodiment, the methods do not comprise the use of non-homologous end joining (NHEJ) and / or are not performed without the use of a polynucleotide that contains one or more sequences with homology to sequences of a target gene.

[0305] In one aspect, the site-specific integration method comprises the introduction of a recombinant viral vector, e.g., an AAV vector, to a population of cells, e.g., immune cells, wherein the recombinant viral vector is targeted to the double-stranded break or cleavage site in the target sequence of a target gene. The introduction of the recombinant viral vector to the cell may be before, simultaneously or after introduction of a rare-cutting endonuclease to the cell, as further described herein. In one embodiment, the method comprises providing a recombinant viral vector comprising a donor template. The donor template comprises a transgene encoding a receptor polypeptide or a nucleic acid inhibitory agent. In other embodiments, the donor template further comprises flanking homology arms. In one other embodiment, the flanking homology arms comprise a 5’ homology arm and a 3’ homology arm. In another embodiment, the 5’ homology arm comprises sequences homologous to sequences 5’ upstream of the double stranded break or cleavage site in thetarget sequence, and the 3’ homology arm comprises sequences homologous to sequences 3’ downstream of the double stranded break or cleavage site in the target sequence. In another embodiment, the donor template is integrated into the genomes of a population of cells, e.g., a population of immune cells, at the double-stranded break or cleavage site. In one additional embodiment, the method comprises culturing the cell following introduction of the recombinant viral vector under conditions sufficient to allow integration of the transgene. In one embodiment, the expression of the receptor polypeptide and / or the nucleic acid inhibitory agent is under the control of a PGK promoter, e.g., a truncated PGK promoter.

[0306] In some embodiments, the methods comprise introducing a donor template comprising a transgene into cells, e.g., immune cells, and expanding the cells. In some embodiments, the disclosure relates to a method of engineering a cell, e.g., an immune cell, the method comprising the step of providing a cell, e.g., an immune cell, and expressing i) at the surface of the cell at least one receptor polypeptide, and / or ii) within the cell a nucleic acid inhibitory agent, such as an RNA interference agent. In some embodiments, the method comprises: introducing into the cell (such as by transfecting the cell) with at least one polynucleotide encoding a receptor polypeptide and / or at least one nucleic acid inhibitory agent, such as an RNA interference agent, and expressing the at least one polynucleotide and / or the at least one nucleic acid inhibitory agent in the cell. In one embodiment, the expression of the receptor polypeptide and / or the nucleic acid inhibitory agent is under the control of a PGK promoter, e.g., a truncated PGK promoter.

[0307] In some embodiments, the cells, e.g., engineered immune cells, generated using a site-specific integration (SSI) method express a higher level of the one or more transgenes introduced as compared to cells where transgenes were introduced by a non- SSI method, e.g., a retroviral or lentiviral transduction method. In some embodiments, the engineered cells, e.g., engineered immune cells, generated by an SSI method exhibit improved activities of the one or more transgenes introduced as compared to cells where transgenes were introduced by a non-SSI method, e.g., a retroviral or lentiviral transduction method. In some embodiments, the engineered cells, e.g., engineered immune cells, generated using an SSI method show less genome translocation of the one or more transgenes as compared to cells wherein transgenes were introduced by a non-SSI method, e.g., a retroviral or lentiviral transduction method.

[0308] In some embodiments, the one or more transgenes are introduced into the target gene locus by site-specific integration. In some embodiments, the engineered cell, e.g., engineered immune cell, comprises additional genetic modifications, including without limitation, knock-out or knock-down of endogenous gene(s), enhancement of endogenous gene(s), and integration of exogenous gene(s). In some embodiments, the engineered immune comprises a transgene integrated into a first genetic locus and further comprises one or more genetic modifications at a second genetic locus. In some embodiments, the one or more genetic modifications at the first and / or second genetic locus comprises integration of a transgene at the first and / or second genetic locus. In one embodiment, the expression of the coding sequences from the transgene (e.g., coding sequences for a receptor polypeptide and / or a nucleic acid inhibitory agent) is under the control of a PGK promoter, e.g., a truncated PGK promoter.

[0309] In some embodiments, the method comprises a step of introducing into the cells a polynucleotide, nucleic acid construct or molecule, wherein the polynucleotide, nucleic construct or molecule comprises a polynucleotide template for homologous recombination. In other embodiments, the polynucleotide template comprises at least a sequence homologous to a portion of a nucleic acid sequence in a target gene, such that homologous recombination occurs between the target gene nucleic acid sequence and the polynucleotide template. In some embodiments, said polynucleotide template comprises a 5’ homology arm homologous to a sequence 5’ to the double strand break of the target sequence in the target gene, a sequence encoding a transgene, and a 3’ homology arm homologous to a sequence 3’ to the double strand break of the target sequence in the target gene. Following cleavage of the target nucleic acid sequence, a homologous recombination event occurs between the target nucleic acid sequence and the polynucleotide template.5. Methods of treatment

[0310] Engineered cells, e.g. engineered immune cells (such as CAR T cells), obtained by the methods described herein, or cell lines derived from such engineered cells, can be used as a medicament or to prepare a medicament. In some embodiments, such a medicament can be used for treating a disorder, disease, or condition. The disclosure comprises methods for treating or preventing a condition associated with an antigen or an undesired and / or elevated level of the antigen, as described herein, in a subject. In one embodiment, the methods comprise administering to a patient in need thereof an effective amount of at least oneimmune cell comprising a CAR comprising an antigen binding domain specific for the antigen, as disclosed herein.

[0311] In some embodiments, engineered cells e.g., engineered immune cells (such as CAR T cells) according to the instant disclosure, or cell lines derived from such engineered cells, can be used in the manufacture of a medicament for treatment of a disease or a disorder in a subject in need thereof.

[0312] Methods are provided for treating diseases or disorders, including cancer and autoimmune diseases. In some embodiments, the disclosure relates to creating a T cell- mediated immune response in a subject, comprising administering an effective amount of the engineered immune cells of the present application to the subject. In some embodiments, the T cell-mediated immune response is directed against a target cell or cells. In some embodiments, the engineered immune cell comprises a chimeric antigen receptor (CAR). The CAR containing immune cells of the disclosure can be used to treat a disease or disorder, e.g., a CD19-associated disease or disorder and / or a CD70-associated disease or disorder. In some embodiments, the disease or disorder can be an autoimmune disease, including, without limitation, lupus, systemic lupus erythematosus (SLE), lupus nephritis, rheumatoid arthritis, systemic sclerosis, scleroderma, and myositis. In some embodiments, the target cell is a tumor cell. In some aspects the disclosure comprises a method for treating or preventing a malignancy, said method comprising administering to a subject in need thereof an effective amount of at least one isolated antigen binding domain described herein. In some aspects, the disclosure comprises a method for treating or preventing a malignancy, said method comprising administering to a subject in need thereof an effective amount of an immune cell, wherein the immune cell comprises at least one chimeric antigen receptor, and a CD70-binding protein and / or CCR as described herein. In some embodiments, the method for treating or preventing a malignancy comprises administering to a subject in need thereof an effective amount of a population of immune cells, wherein the immune cells comprise at least one chimeric antigen receptor, and a CD70-binding protein and / or CCR as described herein. The CAR containing immune cells of the disclosure can be used to treat malignancies associated with an antigen, e.g., CD 19, or involving aberrant expression of the antigen, e.g., CD 19. In some embodiments, CAR containing immune cells of the disclosure can be used to treat such malignancies as nonHodgkin’s lymphoma (NHL), refractory and / or relapse NHK, large B cell lymphoma (LBCL), follicular lymphoma (FL), T cell lymphoma (TCL), B cell acute lymphoblasticleukemia (BALL), T cell acute lymphoblastic leukemia (TALL), primary central nervous system lymphoma (PCNSL), Mantle cell lymphoma (MCL), chronic lymphocytic leukemia (CLL), peripheral T cell lymphoma (PTCL). In exemplary embodiments, the CAR- containing immune cells of the disclosure, e.g., anti-CD19 CAR-T cells, are used to treat LBCL.

[0313] Also provided are methods for reducing the size of a tumor in a subject, comprising administering to the subject an engineered cell of the present disclosure to the subject, wherein the cell comprises a chimeric antigen receptor comprising an antigen binding domain, e.g., a CD 19 antigen binding domain, and binds to an antigen, e.g., a CD 19 antigen, on the tumor.

[0314] In some embodiments, the subject has a solid tumor, or a blood malignancy such as lymphoma or leukemia. In some embodiments, the engineered cell is delivered to a tumor bed, such as a tumor bed found in small cell lung cancer. In some embodiments, the cancer is present in the bone marrow of the subject. In some embodiments, the engineered cells are autologous immune cells, e.g., autologous T cells. In some embodiments, the engineered cells are allogeneic immune cells, e.g., allogeneic T cells. In some embodiments, the engineered cells are heterologous immune cells, e.g., heterologous T cells. In some embodiments, the engineered cells are transfected or transduced ex vivo. As used herein, the term “in vitro cell” refers to any cell that is cultured ex vivo.

[0315] In certain embodiments, the cells comprising a chimeric antigen receptor comprising an antigen binding domain, e.g., a CD 19 antigen binding domain, exhibit enhanced cytotoxicity and potency against pathological cells associated with an autoimmune indication, e.g., CD19 positive pathological cells associated with an autoimmune indication.

[0316] An “effective amount” is any amount that, when used alone or in combination with another agent, provides desired or beneficial results. A “therapeutically effective amount,” “effective dose,” or “therapeutically effective dosage” of a therapeutic agent, e.g., engineered CAR T cells, is any amount that, when used alone or in combination with another therapeutic agent, protects a subject against the onset of a disease or promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. The ability of a therapeutic agent to promote diseaseregression can be evaluated using a variety of methods known to the skilled practitioner (e.g., a physician or clinician), such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0317] The terms “patient” and “subject” are used interchangeably and include human and non-human animal subjects as well as those with formally diagnosed disorders, those without formally recognized disorders, those receiving medical attention, those at risk of developing the disorders, etc.

[0318] The term “treat” and “treatment” includes therapeutic treatments, prophylactic treatments, and applications in which one reduces the risk that a subject will develop a disorder or other risk factor. Treatment does not require the complete curing of a disorder and encompasses embodiments in which one reduces symptoms or underlying risk factors. The term “prevent” does not require the 100% elimination of the possibility of an event. Rather, it denotes that the likelihood of the occurrence of the event has been reduced in the presence of the compound, immune cells, therapeutic agent or method.

[0319] Desired treatment total amounts of cells in the composition comprise at least 2 cells (for example, at least one CD8+ T cell and at least one CD4+ T cell, or two CD8+ T cells, or two CD4+ T cells) or is more typically greater than 102cells, and up to 106, up to and including 108or 109cells and can be 1010or 1012or more cells. The number of cells will depend upon the desired use for which the composition is intended, and the type of cells included therein. The density of the desired cells is typically greater than 106cells / ml and generally is greater than 107cells / ml, generally 108cells / ml or greater. The clinically relevant number of immune cells can be apportioned into multiple infusions that cumulatively equal or exceed 105, 106, 107, 108, 109, 1010, 1011, or 1012cells. In some aspects of the present disclosure, particularly since all the infused cells will be redirected to a particular target antigen (e.g., CD19), lower numbers of cells, in the range of 106 / kilogram ( 106-l 011per patient) may be administered. CAR treatments may be administered multiple times at dosages within these ranges. The cells may be autologous, allogeneic, or heterologous with respect to the patient undergoing therapy.

[0320] In some embodiments, the therapeutically effective amount of the CAR T cells is about 1 X 105cells / kg, about 2 X 105cells / kg, about 3 X 105cells / kg, about 4 X 105cells / kg, about 5 X 105cells / kg, about 6 X 105cells / kg, about 7 X 105cells / kg, about 8 X105cells / kg, about 9 X 105cells / kg, 2 X 106cells / kg, about 3 X 106cells / kg, about 4 X 106cells / kg, about 5 X 106cells / kg, about 6 X 106cells / kg, about 7 X 106cells / kg, about 8 X106cells / kg, about 9 X 106cells / kg, about 1 X 107cells / kg, about 2 X 107cells / kg, about 3 X 107cells / kg, about 4 X 107cells / kg, about 5 X 107cells / kg, about 6 X 107cells / kg, about 7 X 107cells / kg, about 8 X 107cells / kg, or about 9 X 107cells / kg.

[0321] In some embodiments, target doses for CAR+ / CAR-T+ cells range from about 1 * 106to about 1 * IO10cells / kg, for example about 1 x 106cells / kg, about 1 x 107cells / kg, about I x lO8cells / kg, about I x lO9cells / kg or about I x lO10cells / kg. It will be appreciated that doses above and below this range may be appropriate for certain subjects, and appropriate dose levels can be determined by the healthcare provider as needed. Additionally, multiple doses of cells can be provided in accordance with the disclosure.

[0322] In some aspects, the disclosure comprises a pharmaceutical composition comprising at least one antigen-specific CAR as described herein and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition further comprises an additional active agent.

[0323] The CAR expressing cell populations of the present disclosure may be administered either alone, or as a pharmaceutical composition in combination with diluents and / or with other components such as IL-2 or other cytokines or cell populations.Pharmaceutical compositions of the present disclosure may comprise a CAR expressing cell population, such as T cells, as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Compositions of the present disclosure may be formulated for intravenous administration.

[0324] The pharmaceutical compositions (solutions, suspensions or the like), may include one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono- or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. For therapeutic applications, an injectable pharmaceutical composition is preferably sterile.

[0325] In some embodiments, upon administration to a patient, engineered immune cells expressing at their cell surface any of the antigen-specific CARs, e.g., CD19-specific CARs, described herein may reduce, kill or lyse endogenous antigen-expressing cells, e.g., CD 19- expression cells, of the patient. In one embodiment, a percentage reduction or lysis of antigen-expressing endogenous cells, e.g., CD19-expressing endogenous cells, or cells of a cell line expressing the antigen, e.g., a cell line expressing CD 19, by engineered immune cells expressing any one of the antigen-specific CARs, e.g., a CD19-specific CAR, described herein is at least about or greater than 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In one embodiment, a percentage reduction or lysis of antigen-expressing endogenous cells, e.g., CD19-expressing endogenous cells, or cells of a cell line expressing the antigen, e.g., a cell line expressing CD19, by engineered immune cells expressing any one of the antigen-specific CARs, e.g., a CD19-specific CAR, described herein is about 5% to about 95%, about 10% to about 95%, about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, about 25% to about 75%, or about 25% to about 60%. In one embodiment, the endogenous antigen-expressing cells, e.g., CD19-expressing cells, are endogenous antigenexpressing bone marrow cells, e.g., endogenous CD19-expressing bone marrow cells.

[0326] In one embodiment, the percent reduction or lysis of target cells, e.g., a cell line expressing the antigen, such as CD 19, by engineered immune cells expressing at their cell surface membrane an antigen-specific CAR, e.g., a CD19-specific CAR, of the disclosure can be measured using the assay disclosed herein.

[0327] The methods can further comprise administering one or more chemotherapeutic agents to a patient prior to administering the engineered cells provided herein. In certain embodiments, the chemotherapeutic agent is a lymphodepleting (preconditioning) chemotherapeutic. For example, methods of conditioning a patient in need of a T celltherapy comprising administering to the patient specified beneficial doses of cyclophosphamide (between 200 mg / m2 / day and 2000 mg / m2 / day, about 100 mg / m2 / day and about 2000 mg / m2 / day; e.g., about 100 mg / m2 / day, about 200 mg / m2 / day, about 300 mg / m2 / day, about 400 mg / m2 / day, about 500 mg / m2 / day, about 600 mg / m2 / day, about 700 mg / m2 / day, about 800 mg / m2 / day, about 900 mg / m2 / day, about 1000 mg / m2 / day, about 1500 mg / m2 / day or about 2000 mg / m2 / day) and specified doses of fludarabine (between 20 mg / m2 / day and 900 mg / m2 / day, between about 10 mg / m2 / day and about 900 mg / m2 / day; e.g., about 10 mg / m2 / day, about 20 mg / m2 / day, about 30 mg / m2 / day, about 40 mg / m2 / day, about 40 mg / m2 / day, about 50 mg / m2 / day, about 60 mg / m2 / day, about 70 mg / m2 / day, about 80 mg / m2 / day, about 90 mg / m2 / day, about 100 mg / m2 / day, about 500 mg / m2 / day or about 900 mg / m2 / day). An exemplary dosing regimen involves treating a patient comprising administering daily to the patient about 300 mg / m2 / day of cyclophosphamide in combination or before or after administering about 30 mg / m2 / day of fludarabine for three days prior to administration of a therapeutically effective amount of engineered T cells to the patient.

[0328] In some embodiments, notably in the case when the engineered cells provided herein have been gene edited to eliminate or minimize surface expression of CD52, lymphodepletion further comprises administration of an anti-CD52 antibody, such as alemtuzumab. In some embodiments, the CD52 antibody is administered at a dose of about 1-20 mg / day IV, e.g., about 13 mg / day IV, e.g., about 20 mg / day IV, e.g., about 30 mg / day IV, for 1, 2, 3 or more days. The antibody can be administered in combination with, before, or after administration of other elements of a lymphodepletion regime (e.g., cyclophosphamide and / or fludarabine). Exemplary anti-CD52 antibody sequences are provided in Table 3 as SEQ ID NOs: 67-74.

[0329] In other embodiments, the antigen binding domain, transduced (or otherwise engineered) cells and the chemotherapeutic agent are administered each in an amount effective to treat the disease or condition in the subject.

[0330] In certain embodiments, compositions comprising CAR-expressing immune effector cells disclosed herein may be administered in conjunction with any number of chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine resume; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2, 2', 2"- trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel (TAXOL™, Bristol-Myers Squibb) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RF S2000; difluoromethylomithine (DMFO); retinoic acid derivatives such as Targretin™ (bexarotene), Panretin™, (alitretinoin); ONTAK™(denileukin diftitox); esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4- hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti -androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Combinations of chemotherapeutic agents are also administered where appropriate, including, but not limited to CHOP, i.e., Cyclophosphamide (Cytoxan®), Doxorubicin (hydroxydoxorubicin), Vincristine (Oncovin®), and Prednisone.

[0331] In some embodiments, the chemotherapeutic agent is administered at the same time or within one week after the administration of the engineered cell, polypeptide, or nucleic acid. In other embodiments, the chemotherapeutic agent is administered from about 1-7 days, about 1 to about 4 weeks or from about 1 week to about 1 month, about 1 week to about 2 months, about 1 week to about 3 months, about 1 week to about 6 months, about 1 week to about 9 months, or about 1 week to about 12 months after the administration of the engineered cell, polypeptide, or nucleic acid. In other embodiments, the chemotherapeutic agent is administered at least 1 month before administering the cell, polypeptide, or nucleic acid. In some embodiments, the methods further comprise administering two or more chemotherapeutic agents.

[0332] A variety of additional therapeutic agents may be used in conjunction with the compositions described herein. For example, potentially useful additional therapeutic agents include PD-1 inhibitors such as nivolumab (Opdivo®), pembrolizumab (Keytruda®), pembrolizumab, pidilizumab, and atezolizumab.

[0333] Additional therapeutic agents suitable for use in combination with the disclosure include, but are not limited to, ibrutinib (Imbruvica®), ofatumumab(Arzerra®, rituximab (Rituxan®), bevacizumab (Avastin®), trastuzumab (Herceptin®), trastuzumab emtansine (KADCYLA®, imatinib (Gleevec®), cetuximab (Erbitux®, panitumumab) (Vectibix®), catumaxomab, ibritumomab, ofatumumab, tositumomab, brentuximab, alemtuzumab, gemtuzumab, erlotinib, gefitinib, vandetanib, afatinib, lapatinib, neratinib, axitinib, masitinib, pazopanib, sunitinib, sorafenib, toceranib, lestaurtinib, axitinib, cediranib, lenvatinib, nintedanib, pazopanib, regorafenib, semaxanib, sorafenib, sunitinib, tivozanib,toceranib, vandetanib, entrectinib, cabozantinib, imatinib, dasatinib, nilotinib, ponatinib, radotinib, bosutinib, lestaurtinib, ruxolitinib, pacritinib, cobimetinib, selumetinib, trametinib, binimetinib, alectinib, ceritinib, crizotinib, aflibercept, adipotide, denileukin diftitox, mTOR inhibitors such as Everolimus and Temsirolimus, hedgehog inhibitors such as sonidegib and vismodegib, CDK inhibitors such as CDK inhibitor (palbociclib).

[0334] In some embodiments, the composition comprising CAR-containing immune cells may be administered with a therapeutic regimen to prevent or reduce cytokine release syndrome (CRS) or neurotoxicity. The therapeutic regimen to prevent cytokine release syndrome (CRS) or neurotoxicity may include lenzilumab, tocilizumab, atrial natriuretic peptide (ANP), anakinra, iNOS inhibitors (e.g., L-NIL or 1400W). In additional embodiments, the composition comprising CAR-containing immune cells can be administered with an anti-inflammatory agent. Anti-inflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), nonsteroidal antiinflammatory drugs (NSAIDS) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamide and mycophenolate. Exemplary NSAIDs include ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors, and sialylates. Exemplary analgesics include acetaminophen, oxycodone, tramadol of proporxyphene hydrochloride. Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone.Exemplary biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors, such as the TNF antagonists, (e.g., etanercept (ENBREL®), adalimumab (HUMIRA®) and infliximab (REMICADE®), chemokine inhibitors and adhesion molecule inhibitors. The biological response modifiers include monoclonal antibodies as well as recombinant forms of molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofin) and intramuscular) and minocycline.

[0335] In certain embodiments, the compositions described herein are administered in conjunction with a cytokine. Examples of cytokines are lymphokines, monokines, and traditional polypeptide hormones. Included among the cytokines are growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growthhormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor (HGF); fibroblast growth factor (FGF); prolactin; placental lactogen; mullerian -inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors (NGFs) such as NGF-beta; plateletgrowth factor; transforming growth factors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-alpha, beta, and -gamma; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte- CSF (G-CSF); interleukins (ILs) such as IL-1, IL-lalpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, IL-21 a tumor necrosis factor such as TNF -alpha or TNF-beta; and other polypeptide factors including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture, and biologically active equivalents of the native sequence cytokines.6. Kits and Articles of Manufacture

[0336] The present application provides kits comprising any one of the CD19 CAR containing immune cells described herein, and pharmaceutical compositions of the same. In an embodiment of a kit the engineered CAR cells (e.g., CAR T cells) are frozen in a suitable medium, such as CryoStor® CS10, CryoStor® CS2 or CryoStor® CS5 (BioLife Solutions).

[0337] In some exemplary embodiments, a kit of the disclosure comprises allogeneic CD19 CAR-containing T-cells for administering to the subject a lymphodepletion regiment and a CAR-T regimen.

[0338] The present application also provides articles of manufacture comprising any one of the therapeutic compositions or kits described herein. Examples of an article of manufacture include vials (e.g., sealed vials).

[0339] All references cited herein, including patents, patent applications, papers, textbooks, and the like, and the references cited therein, to the extent that they are not already, are hereby incorporated by reference in their entirety.Table 3IllEXAMPLESExperimental Methods

[0340] To make lentivirus encoding the desired constructs, HEK293T-cells were plated at 0.45 million cells per mL in 2mL of DMEM (Gibco) supplemented with 10% FBS (Hyclone) per well of a 6-well plate the day before transfection. On the day of transfection, the lentivirus was prepared by mixing together lentiviral packaging vectors 1.5 ug psPAX2, 0.5 ug pMD2G, and 0.5 ug of the appropriate transfer CAR vector in 250 uL Opti-MEM (Gibco) per well of the 6-well plate (“DNA mix”). 10 uL Lipofectamine 2000 (Invitrogen) in 250 uL Opti-MEM was incubated at room temperature for 5 minutes and then added to the DNA mix. The mixture was incubated at room temperature for 20 minutes and the total volume of 500 uL was slowly added to the sides of the wells containing HEK293T. 1 day post-transfection, the media from each well of HEK293 T-cells in the 6-well plate was replaced with 2mL per well of T-cell transduction media, i.e., X-Vivo-15 supplemented with 10% FBS. 2 days post-transfection, The lentiviral supernatants from HEK293T-cells were harvested and passed through a 0.45 micron filter (EMD Millipore) to remove cell debris, concentrated 25-folds using the Lenti-X Concentrator (Takara Bio) according to manufacturer’s instructions and flash-frozen in aliquots. Lentiviral titers were determined by thawing an aliquot of the frozen lentivirus, making 4-fold serial dilutions and performing limiting dilution titration on JurkaT-cells (Clone E6-1; ATCC). On Day 0, purified T-cells were activated in X-Vivo-15 medium (Lonza) supplemented with 100 lU / mL human IL-2 (Miltenyi Biotec), 10% FBS (Hyclone), and human T TransAct (Miltenyi Biotec, Cat# 130- 111-160, 1 : 100 dilution) in a Grex-24 plate (Wilson Wolf, cat# 80192M). On Day2, T-cells were resuspended at 0.5 million cells per mL in T-cell transduction media, transduced with the respective lentiviral stocks at MOI=5 along with 100 lU / mL human IL-2 in a Grex-24 plate. On Day 5 when transduction was complete, cells were harvested and washed to remove residual IL-2. They were then resuspended in T-cell expansion media, i.e., X-Vivo- 15 supplemented with 5% human AB serum (Gemini Bio), and each sample was divided equally into 2 parts, with one part receiving 100 lU / mL human IL-2 as per standard protocol, and the other receiving a lower concentration of 25 lU / mL human IL-2. Cells were expanded into larger G-Rex vessels (Wilson Wolf) as needed using T-cell expansion media and the respective concentrations of human IL-2. On Days 5, 9 and 14, the absolute numberof T-cells in each sample was counted, and transduction efficiency was determined by detecting the percentage of T-cells that bound a FITC-conjugated v5 tag monoclonal antibody (Thermo Fisher) using flow cytometry. On Day 14 or 15, the CAR-T-cell products were cryopreserved and thawed as needed for further assays.

[0341] For cells generated with site specific integration with AAV6, two days after T cell activation, TransAct was removed by centrifugation. The cells were then washed in PBS and electroporated using the AMAXA 4D nucleofector electroporation apparatus with 0.5ug of TALEN® mRNA per arm of the nuclease every IxlO6cells. Recombinant AAV6 comprising one or more transgenes was added at MOI 10,000-15,000 to the T cells after the gene editing step. After electroporation, cells were incubated at 30°C overnight. Cells were then returned to 37°C and T cell culturing conditions as described above. At day 14 postactivation, TCRa / p depletion was performed using EasySep™ human TCRa / p depletion kit (STEMCELL Technologies) as instructed by the manufacturer protocol. At day 15 or 16 post-activation, T cells were cryopreserved in 90% FBS / 10% DMSO using rate-controlled freezing chambers and stored in liquid nitrogen vapor phase.

[0342] AAV-mediated site-specific integration can also be done with a CRISPR endonuclease, e.g., Casl2i endonuclease, or CRISPR reagents. Pan T cell donors were thawed and activated with GMP -grade TransAct and IL-2 for 2 days. Prior to electroporation, Casl2i nuclease and TRAC targeting guide RNA (gRNA) were incubated together at room temperature to form a ribonucleoprotein complex (RNP). T cells were then mixed with the RNP and electroporated. Cells were then immediately incubated with AAV comprising one or more transgenes at a specified MOI overnight, then transferred into IL-2 supplemented complete T cell media in a GREX plate. Cells were then expanded another 12 days before freezing. Flow cytometry analysis was performed 4, 7, and 11 days after electroporation. The transgene cassette was expected to be inserted at the target sequence GACCCTGCC within TRAC exon 1.

[0343] Standard Long-term Killing Assay (LTKA). CAR T cells were co-cultured with luciferase-GFP-expressing Raji cells at an effector-to-target ratio of 8: 1. Every 2-3 days, half the cells were passaged onto fresh Raji cells. The remaining half of cells was then used to determine target cell killing using Bright-glo reagent (Promega).

[0344] Flow-based serial restimulation assay. In a 24-well G-rex® (Wilson Wolf, Cat # 80192M), 5xl05CAR T cells were co-cultured with 5xl05Raji Luciferase-GFP cells inRPMI 1640 + 10% FBS media (Hyclone). A small aliquot of the co-cultured CAR T and target cells were taken to perform an antibody stain with 123count eBeads™ Counting Beads (Thermo Fisher Scientific, Cat # 01-1234-42) to determine baseline phenotype and cell counts at the start of the assay. Every 3-4 days, 50% of supernatant was removed from the G-rex® and remaining cell suspension was mixed thoroughly, and a small aliquot was taken for phenotyping and cell counting as described above. After cell counts are determined, fresh Raji cells were added back into each well to bring the E:T ratio back to 1 : 1. Serial restimulation is repeated until CAR T no longer kills the Raji target cells.

[0345] Single-stimulation FACS assay. Similar to the flow-based serial stimulation assay, CAR T and Raji cells were co-cultured at a 1 :2 or 1 :4 E:T ratio in a 24-well G-rex® rather than a 1 : 1 E:T. Every 3-4 days, 50% of supernatant were removed from the G-rex® and remaining cell suspension were mixed thoroughly, and a small aliquot was taken for phenotyping and cell counting as described above. Instead of adding fresh Raji cells back, only fresh media was added back. Timepoints are collected every 3-4 days until Raji cells are either completely cleared by CAR T or until Raji cells outgrow the T cells.

[0346] Primed T mixed lymphocyte reaction (MLR). Human PBMCs (host) were primed against irradiated unedited T cells derived from donors used to make gene edited graft T cells above to promote expansion of alloreactive T cell clones. Briefly, graft PBMCs were irradiated at 30 Gy and co-cultured with host PBMCs at a 1 : 1 ratio in R10 + 20 lU / mL IL-2 + 10 ng / mL IL-7 + 10 ng / mL IL-15 (Miltenyi, Cat # 130-095-765) for 4 days. Media was exchanged to R10 without cytokines and the cells were continued to culture for 3 more days. Afterwards, pan T cells were isolated using MACS negative selection (Miltenyi, human pan T cell isolation kit, Cat #130-096-535) per the manufacturer’s recommendations. In a 96-well plate, 20,000 gene edited graft T cells were seeded with 20,000 primed host T cells and cultured in R10 + 20 lU / mL IL-2 for 2 days at 37°C, 5% CO2. Survival of graft T cells was determined by flow cytometry using absolute counts by gating on live TCRa[3- CAR+ T cells.Example 1: Generation of CAR T cells expressing an anti-CD19 CAR from a lentiviral vector

[0347] Constructs for the expression of second generation anti -CD 19 CARs were designed based on the anti-CD19 scFv of SEQ ID NO: 4, CD3z signaling domain and 4- 1BB costimulatory domain and cloned in a lentiviral vector (LVV). CAR T cells generatedfrom donor PBMCs either express the CD 19 CAR alone, or a chimeric cytokine receptor (CCR) and the CD 19 CAR from a bicistronic expression cassette linked via a cleavable P2A peptide (CD 19 CAR / CCR). The CCR contains a TPOR transmembrane / JAK binding and activation domain and IL2R intracellular signaling domains exemplified as in SEQ ID NO: 27. Gene expression in all LVV constructs was driven by the elongation factor 1 alpha (EFla) promoter. CAR T cells were generated by transducing activated donor PBMC cells with the LVV. The cytotoxic activities of CAR T cells harboring different constructs generated from three different donors were evaluated by an in vitro long-term killing assay (LTKA) against CD19 positive Raji cells and Daudi cells. As shown in FIGs. 1A-1C, in most cases, co-expressing a CCR improved CD 19 CAR T cell cytotoxicity.

[0348] In vivo cytotoxicity of CAR T cells engineered with the selected constructs was evaluated in an orthotopic animal tumor model established by injecting Raji cells into NSG mice. Briefly, luciferase-expressing Raji cells were injected into mice intravenously. CAR T cells were injected 4 days after tumor cell transfer. Tumor burden was measured using an IVIS Spectrum instrument. As shown in FIGs. 2A-2B, CD 19 CAR T cells co-expressing a CCR effectively reduced tumor cells in the animal model at a dose level of 3xl06CAR T cells.Example 2: CAR T cells expressing an anti-CD19 CAR by site-specific integration

[0349] Transduction of LVV constructs into PBMCs results in engineered cells with randomly integrated transgene(s) in the host cell genome. The transgene(s) can be introduced into cells by site-specific integration (SSI) into a predetermined genetic locus to ensure uniformity of the insertion site of the transgene(s) in the genome and limit the number of integration events. Site-specific integration aided by, for example, adeno- associated virus vector (AAV), can also accommodate one or more transgenes that can be larger in size than what can be incorporated into a lentiviral vector, while maintaining high transduction efficiency. The CD 19 CAR / CCR construct cloned in an adeno-associated virus vector (AAV) was inserted into the TRAC locus by homologous recombination, and the expression of the CCR and CD19 CAR was driven by a short EFla promoter (EFS). The resulting CAR T cells were evaluated for in vivo cytotoxicity in the Raji orthotopic tumor model in NSG mice, as compared with CAR T cells generated by LVV transduction expressing the same CCR and CD19 CAR (N=8 mice / group, 2.5 x 106CAR+cells / mouse).

[0350] The results in FIG. 3A show that, unexpectedly, the CAR T cells generated by site-specific integration exhibited significantly reduced tumor control in vivo than the CAR T cells generated by LVV transduction. The results were reproduced in CAR T cells generated using cells from a different donor. Consistent with the poor in vivo cytotoxicity, the CAR T cells generated by site-specific integration failed to expand and failed to persist in the treated mice for the duration of the experiment, in contrast to the CAR T cells generated by LVV transduction (FIG. 3B). Similar in vitro results were also observed in FIGs. 5A-5C, in which the CAR T cells generated by site-specific integration (SSI) driven by the EFS promoter (SSI EFS CD19 / CCR) showed reduced cytotoxicity in a standard long-term killing assay, and reduced expansion than the CAR T cells generated by LVV transduction.Example 3: Improved anti-CD19 CAR construct for site-specific integration

[0351] To improve the in vitro and in vivo anti-tumor activity of our SSI-engineered CAR T cells, we tested a panel of different promoters to drive expression of the CCR and the CAR. All SSI constructs were designed to integrate into the T cell receptor alpha constant (TRAC) locus. The surface expression of the CD 19 CAR was measured using an antiidiotype (anti-ID) antibody (ACROBiosystems) via flow cytometry - to measure the mean fluorescence intensity (MFI) of the CAR - and the anti-tumor activity of the CAR T cells were examined. Several heterologous promoters were tested, including 3 promoters derived from the human phosphoglycerate kinase 1 (PGKT) gene, designated PGK-SSI-1, PGK- SSI-2, and PGK-SSI-3. SSI-engineered CAR T cells having a construct with the endogenous promoter (Endo) of SEQ ID NO: 33 was also tested (FIG. 4A-B).

[0352] The CAR MFI was measured at the end of manufacturing. The CAR construct driven by the human ubiquitin B (UBB) promoter (UBB300) had the highest CAR expression at the end of manufacturing (FIG. 4A). The 3 PGK SSI promoters, as well as the human elongation factor 1 -alpha 1 short (EFS) promoter and the human cyclophilin A (CypA300) promoter resulted in similar CAR expression at the end of manufacturing (FIG. 4A). Next, CAR T cells were analyzed in a flow cytometry-based serial restimulation cytotoxicity assay against a CD 19+ tumor cell line, Raji, at an effector to target (E:T) ratio of 1 : 1. Surprisingly, the PGK SSI promoter constructs led to superior anti-tumor activity compared to all other promoters tested (FIG. 4B).

[0353] To understand the mechanism behind this difference in anti-tumor efficacy, we monitored the CAR expression (MFI) during the serial restimulation assay. We noticed that although the CAR MFI at the end of the process was similar between the EFS and PGK SSI promoters (FIG. 4A), the promoters modulated CAR expression differently after exposure to CD 19+ tumor cells. All promoters increased CAR expression by day 3 after target exposure, with the EFS and UBB promoters achieving the highest level of expression (FIG. 4C). By day 17, all promoters down-regulated CAR expression, however the EFS and UBB promoters maintained significantly higher expression throughout the rest of the assay, as compared to the PGK SSI promoters (FIG. 4C). Surprisingly, this sustained high-level CAR expression level after day 17 negatively correlates with CAR T cell expansion (FIG. 4D) and anti-tumor activity (FIG. 4B). In contrast, the PGK SSI promoters, which maintained low-level CAR expression from day 17 on but showed the highest peak expansion (FIG. 4D) and best anti-tumor activity (FIG. 4B) as compared to the other constructs. Without wishing to be bound by specific mechanisms, the results suggest that prolonged high expression of the CAR by the EFS and UBB promoters may have led to exhaustion and dysfunction of the CAR T cells, whereas lower-level expression of the CAR after target exposure by the PGK SSI promoters preserved CAR T cell function and led to better CAR T cells expansion and better anti-tumor activity.

[0354] With these results, we next tested the PGK SSI promoters, e.g., PGK SSI-1, in comparison with the EFS promoter in CAR T cells generated by site-specific integration, and CAR T cells generated by LVV transduction. The results in FIG. 5A show that, when tested in a standard LTKA against wild type Raji cells at an E:T ratio of 4: 1, CD19 CAR T cells co-expressing the CCR under the PGK SSI-1 promoter, but not the EFS promoter, performed comparably with that of LVV expressing the same CD 19 CAR and CCR. When tested in a single-stimulation FACS based potency assay against wild type Raji cells at an E:T ratio of 1 :2, the PGK SSI-1 driven SSI construct outperformed not only the EFS constructs but also the LVV transduced CAR T cells as evidenced by both the number of remaining target cells (FIG. 5B) and the expansion of CAR T+ cells (FIG. 5C). All results shown in FIGs. 5A-C were obtained using CAR T cells generated from the cells of the same donor. In another LTKA experiment, CAR T cells utilizing the PGK SSI-1 promoter were found to outperform CAR T cells that utilized an optimized endogenous promoter - SEQ ID NO: 96 (data not shown).

[0355] The results in the in vitro killing assay were confirmed in vivo in the Raji orthotopic tumor NSG mouse model. In the animal tumor model, CD19 CAR T cells driven by the PGK SSI-1 promoter outperformed LVV transduced CAR T cells and the other constructs driven by the Cyp300A and the EFS promoters, at the two different cell doses tested (FIG. 6A-6B).

[0356] Although the disclosed teachings have been described with reference to various applications, methods, kits, and compositions, it will be appreciated that various changes and modifications can be made without departing from the teachings herein and the claimed invention below. The foregoing examples are provided to better illustrate the disclosed teachings and are not intended to limit the scope of the teachings presented herein. While the present teachings have been described in terms of these exemplary embodiments, the skilled artisan will readily understand that numerous variations and modifications of these exemplary embodiments are possible without undue experimentation. All such variations and modifications are within the scope of the current teachings.

Claims

What is claimed is:

1. A chimeric antigen receptor (CAR) T cell comprising: a recombinant nucleic acid sequence that has been integrated at the human T cell receptor (TCR) alpha chain constant region gene, wherein the recombinant nucleic acid sequence comprises in a 5’ to 3’ direction:(a) a 5’ region of the TCR alpha chain constant region,(b) a truncated PGK promoter that controls expression of the CAR nucleic acid sequence,(c) a CAR nucleic acid sequence, and(d) a 3' region of the human TCR alpha constant region gene.

2. The CAR T cell of claim 1, which exhibits greater expansion and / or cytotoxicity when compared to a CAR T cell that does not comprise a truncated PGK promoter.

3. The CAR T cell of claim 1, which exhibits less exhaustion when compared to a CAR T cell that does not comprise a truncated PGK promoter.

4. The CAR T cell of claim 1 or 2, wherein the integrated recombinant nucleic acid sequence prevents expression of the TCR alpha constant region gene in the CAR T cell.

5. The CAR T cell of claim 1, wherein the recombinant nucleic acid sequence further comprises an additional nucleic acid sequence encoding a receptor polypeptide or a nucleic acid inhibitory agent.

6. The CAR T cell of claim 5, wherein the receptor polypeptide is a chimeric cytokine receptor (CCR) or a CD70 binding protein.

7. The CAR T cell of claim 5, wherein the nucleic acid inhibitory agent is an RNA interference agent.

8. The CAR T cell of claim 5, wherein the CAR nucleic acid sequence and the additional nucleic acid sequence are linked by a P2A peptide.

9. The CAR T cell of any one of claims 1-4, wherein the truncated PGK promoter comprises one or more deletions in the sequence shown as SEQ ID NO: 34.

10. The CAR T cell of any one of claims 1-4, wherein the truncated PGK promoter comprises one or more 5’ region deletions and / or 3’ region deletions.

11. The CAR T cell of claim 9 or 10, wherein the truncated PGK promoter comprises a nucleotide sequence:(a) comprising about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, or about 390 nucleotides deletion from the 5’ end of SEQ ID NO: 34 and having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 35;(b) comprising about 210, about 220, about 230 about 240, about 250, about 260, about 270, about 280, or about 290 nucleotides deletion from the 5’ end of SEQ ID NO: 34 and having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 36;(c) comprising about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, or about 190 nucleotides deletion from the 5’ end of SEQ ID NO: 34 and having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 37; or(d) comprising about 50, about 60, about 70, about 80, about 90 or about 100 nucleotides deletion from the 5’ end of SEQ ID NO: 34 and having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 38.

12. The CAR T cell of claim 9 or 10, wherein the truncated PGK promoter comprises SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38.

13. The CAR T cell of any one of claims 1-10, wherein the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain.

14. The CAR T cell of claim 13, wherein the extracellular domain comprises an antigen binding domain and / or wherein the intracellular domain comprises at least one costimulatory domain.

15. The CAR T cell of claim 14, wherein the costimulatory domain is a signaling region of CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell co-stimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1 (CD1 la / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC classI molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins,Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha., CD8beta, IL-2Rbeta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 lb, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAMI (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, a ligand that specifically binds with CD83, or any combination thereof.

16. The CAR T cell of claim 13 or 14, wherein the intracellular domain comprises at least one activating domain.

17. The CAR T cell of claim 16, wherein the activating domain comprises CD3.

18. The CAR T cell of claim 17, wherein the CD3 comprises CD3 zeta.

19. The CAR T cell of any one of claims 1-18, wherein the CAR nucleic acid sequence expresses a CAR that binds to BCMA, EGFRvIII, WT-1, CD20, CD23, CD30, CD38, CD33, CD133, MHC-WT1, TSPAN10, MHC-PRAME, Livl, ADAM10, CHRNA2, LeY, NKGD2D, CS1, CD44v6, R0R1, Claudin-18.2, Mucl7, FAP alpha, Ly6G6D, c6orf23, G6D, MEGT1, NG25, CD19, FLT3, CD70, DLL3, CD52 or CD34.

20. The CAR T cell of any one of claim 1-19, wherein the CAR T cell is an autologous or allogeneic CAR T cell.

21. A method of manufacturing a CAR T cell comprising introducing into a cell, a recombinant nucleic acid sequence that comprises in a 5’ to 3’ direction:(a) a 5’ region of the T cell receptor (TCR) alpha chain constant region,(b) a truncated PGK promoter that controls expression of the CAR nucleic acid sequence,(c) a CAR nucleic acid sequence, and(d) a 3' region of the human TCR alpha constant region gene,wherein the introducing is under conditions sufficient to allow integration of the recombinant nucleic acid sequence at the human T cell receptor (TCR) alpha chain constant region gene, thereby providing a CAR T cell.

22. The method of claim 21, wherein the integration is such that the expression of the TCR alpha constant region gene is prevented in the CAR T cell.

23. The method of claim 21, wherein the integrated recombinant nucleic acid sequence prevents expression of the TCR alpha constant region gene in the CAR T cell.

24. The method of claim 21, further comprising performing an expansion assay using the CAR T cell.

25. The method of claim 21, further comprising performing a cytotoxicity assay using the CAR T cell.

26. The method of claim 23, wherein the expansion assay is an in vitro or an in vivo expansion assay and / or wherein the cytotoxicity assay is an in vitro or an in vivo cytotoxicity assay.

27. The method of any one of claims 21-26, wherein the truncated PGK promoter comprises one or more deletions in a wild-type promoter shown as SEQ ID NO: 34.

28. The method of claim 27, wherein the one or more deletions comprises a 5’ region deletion and / or a 3’ region deletion.

29. The method of claim 28, wherein the truncated PGK promoter comprises a nucleotide sequence:(a) comprising about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, or about 390 nucleotides deletion from the 5’ end of SEQ ID NO: 34 and having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 35;(b) comprising about 210, about 220, about 230 about 240, about 250, about 260, about 270, about 280, or about 290 nucleotides deletion from the 5’ end of SEQ ID NO: 34 and having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 36;(c) comprising about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, or about 190 nucleotides deletion from the 5’ end of SEQ IDNO: 34 and having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 37; or(d) comprising about 50, about 60, about 70, about 80, about 90 or about 100 nucleotides deletion from the 5’ end of SEQ ID NO: 34 and having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 38.

30. The method of claim 28 or 29, wherein the truncated PGK promoter comprises a sequence selected from the group consisting of SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37, and SEQ ID NO: 38.

31. The method of any one of claims 21-30, wherein the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain.

32. The method of claim 31, wherein the extracellular domain comprises an antigen binding domain.

33. The method of claim 31 or 32, wherein the intracellular domain comprises at least one co-stimulatory domain.

34. The method of claim 33, wherein the costimulatory domain is a signaling region of CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD- 1), inducible T cell co-stimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1 (CD1 la / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha., CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 lb, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAMI (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT,GADS, SLP-76, PAG / Cbp, CD19a, a ligand that specifically binds with CD83, or any combination thereof.

35. The method of claim 31 or 33, wherein the intracellular domain comprises at least one activating domain.

36. The method of claim 35, wherein the activating domain comprises CD3.

37. The method of claim 36, wherein the CD3 comprises CD3 zeta.

38. The method of any one of claims 20-37, wherein the CAR nucleic acid sequence expresses a CAR that binds to BCMA, EGFRvIII, WT-1, CD20, CD23, CD30, CD38, CD33, CD133, MHC-WT1, TSPAN10, MHC-PRAME, Livl, ADAM10, CHRNA2, LeY, NKGD2D, CS1, CD44v6, ROR1, Claudin-18.2, Mucl7, FAP alpha, Ly6G6D, c6orf23, G6D, MEGT1, NG25, CD19, FLT3, CD70, DLL3, CD52 or CD34.

39. The method of any one of claims 20-38, wherein the CAR T cell is an autologous or allogeneic CAR T cell.

40. An isolated population of CAR T cells comprising a CAR T cell according to any one of claims 1-20.

41. A nucleic acid molecule comprising a recombinant nucleic acid sequence encoding a CAR, wherein the recombinant nucleic acid sequence comprises in a 5’ to 3’ direction:(a) a 5’ region of the T cell receptor (TCR) alpha chain constant region,(b) a truncated PGK promoter that controls expression of the CAR nucleic acid sequence,(c) a CAR nucleic acid sequence, and(d) a 3' region of the human TCR alpha constant region gene.

42. The nucleic acid molecule of claim 41, wherein the recombinant nucleic acid sequence further comprises an additional nucleic acid sequence encoding a receptor polypeptide or a nucleic acid inhibitory agent.

43. The nucleic acid molecule of claim 42, wherein the receptor polypeptide is a CCR or a CD70 binding protein.

44. The nucleic acid molecule of claim 42, wherein the nucleic acid inhibitory agent is an RNA interference agent.

45. The nucleic acid molecule of claim 42, wherein the CAR nucleic acid sequence and the additional nucleic acid sequence are linked by a P2A peptide.

46. The nucleic acid molecule of any one of claims 41-45, wherein the truncated PGK promoter comprises one or more deletions in the sequence shown as SEQ ID NO: 34.

47. The nucleic acid molecule of any one of claims 41-45, wherein the truncated PGK promoter comprises one or more 5’ region deletions and / or 3’ region deletions.

48. The nucleic acid molecule of claim 46 or 47, wherein the truncated PGK promoter comprises a nucleotide sequence:(a) comprising about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, or about 390 nucleotides deletion from the 5’ end of SEQ ID NO: 34 and having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 35;(b) comprising about 210, about 220, about 230 about 240, about 250, about 260, about 270, about 280, or about 290 nucleotides deletion from the 5’ end of SEQ ID NO: 34 and having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 36;(c) comprising about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, or about 190 nucleotides deletion from the 5’ end of SEQ ID NO: 34 and having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 37; or(d) comprising about 50, about 60, about 70, about 80, about 90 or about 100 nucleotides deletion from the 5’ end of SEQ ID NO: 34 and having at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of SEQ ID NO: 38.

49. The nucleic acid molecule of claim 41, wherein the truncated PGK promoter comprises SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, or SEQ ID NO: 38.

50. The nucleic acid molecule of any one of claims 41-49, wherein the CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain.

51. The nucleic acid molecule of claim 50, wherein the extracellular domain comprises an antigen binding domain and / or wherein the intracellular domain comprises at least one co-stimulatory domain.

52. The nucleic acid molecule of claim 51, wherein the costimulatory domain is a signaling region of CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell co-stimulator (ICOS), lymphocyte function- associated antigen-1 (LFA-1 (CD1 la / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha., CD8beta, IL-2Rbeta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 lb, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAMI (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD 19a, a ligand that specifically binds with CD83, or any combination thereof.

53. The nucleic acid molecule of claim 51 or 52, wherein the intracellular domain comprises at least one activating domain.

54. The nucleic acid molecule of claim 53, wherein the activating domain comprises CD3.

55. The nucleic acid molecule of claim 54, wherein the CD3 comprises CD3 zeta.

56. The nucleic acid molecule of any one of claims 41-55, wherein the CAR nucleic acid sequence expresses a CAR that binds to BCMA, EGFRvIII, WT-1, CD20, CD23, CD30, CD38, CD33, CD133, MHC-WT1, TSPAN10, MHC-PRAME, Livl, ADAM10, CHRNA2, LeY, NKGD2D, CS1, CD44v6, ROR1, Claudin-18.2, Mucl7, FAP alpha, Ly6G6D, c6orf23, G6D, MEGT1, NG25, CD19, FLT3, CD70, DLL3, CD52 or CD34.

57. A vector comprising the nucleic acid according to any one of claims 41-56.

58. A cell comprising the nucleic acid molecule according to any one of claims 41-5659. A cell comprising the vector according to claim 57.

60. The cell according to claim 59, wherein the cell is an immune cell selected from the group consisting of a T-cell, a dendritic cell, a killer dendritic cell, a mast cell, a natural killer (NK-)cell, a macrophage, a monocyte, a B-cell, and an immune cell derived from a stem cell.

61. The cell according to claim 58 or 59, wherein the cell is an immune cell.

62. The cell according to claim 60 or 61, wherein the immune cell is an autologous immune cell or an allogeneic immune cell.

63. An isolated population of cells comprising the CAR T cell according to any one of claims 1-20 or the cell according to any one of claims 58-62.

64. A pharmaceutical composition comprising the CAR T cell according to any one of claims 1-20, the cell according to any one of claims 58-62, or the isolated population of CAR T cells according to claim 63.

65. A kit comprising the CAR T cell according to any one of claims 1-20, the cell according to claims 43-47, or the isolated population of CAR T cells according to claim 63, or the pharmaceutical composition of claim 64.

66. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the CAR T cell according to any one of claims 1-20, the cell according to any one of claims 58-62, the isolated population of cells according to claim 63, or the pharmaceutical composition of claim 64.

67. The method of claim 66, wherein the cancer comprises a solid tumor.

68. The method of claim 66, wherein the cancer comprises a liquid tumor.

69. The CAR T cell according to any one of claims 1-20, the cell according to any one of claims 58-62, or the nucleic acid molecule of any one of claims 41-56, wherein the truncated PGK promoter comprises a nucleic acid sequence that binds to or is predicted to bind to a transcription factor selected from the group consisting ofa) YY1, RFX7, and HIFl; b) YY1, RFX7, HIF1 A, and ETS2; or c) YY1, STAT3, STAT1, RFX7, and ETS2.

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