Compositions and methods for co-expression of transgenes

Abstract

The present invention provides expression constructs and modified immune cells or precursors thereof expressing two or more transgenes each encoding a polypeptide, in particular multiple chimeric antigen receptors (CARs) specific for a plurality of antigens. The present invention also provides compositions and methods for treating a disease or condition such as cancer using the expression constructs and modified immune cells disclosed herein.

Description

[0001] Attorney Docket No: 046483-7473W01(04009)

[0002] COMPOSITIONS AND METHODS FOR CO-EXPRESSION OF TRANSGENES

[0003] CROSS-REFERENCE TO RELATED APPLICATION

[0004] The present application is entitled to priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 692,931 filed on September 10, 2024, which is herein incorporated by reference in its entirety.

[0005] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0006] The Sequence Listing submitted herewith as an XML file named "046483- 7473xx_l.xml," created on September 9, 2025 and having a size of 213,109 bytes, is herein incorporated by reference in its entirety.

[0007] BACKGROUND OF THE INVENTION

[0008] The development of chimeric antigen receptor (CARs) and their successful clinical use to direct T cells against specific types of cancers has been an important advancement in cancer immunotherapy. Once confined to experimental laboratory proofs of concept, CAR T cell immunotherapies were first approved in certain leukemias, lymphoma, and myeloma for patients who had exhausted all available options. Since then, CAR T cell therapies have moved up and are approved as second line in non-Hodgkin lymphoma and third line in myeloma. However, a common mechanism of resistance is the emergence of tumors with loss or downregulation of target antigens. As one example, while many patients with refractory or relapsed (R / R) B cell acute lymphoblastic leukemia (B-ALL) achieve complete remission, the tumor cells of many patients manage to evade CAR T attack, resulting in a CD 19-negative relapse, which is the predominant cause of treatment failure in patients treated with anti-CD19 CAR T cells as a standalone therapy.

[0009] Thus, there is a need in the art for novel and effective treatment strategies for treating cancer. The present invention addresses this need.

[0010] SUMMARY OF THE INVENTION

[0011] In one aspect, the present invention provides a nucleic acid comprising two or more transgenes, where: (1) each transgene is operably linked to a first or a second regulatory nucleic Attorney Docket No: 046483-7473W01(04009) acid sequence; (2) the first regulatory nucleic acid sequence comprises a first promoter directing transcription of one or more transgenes from a plus-strand of DNA; and (3) the second regulatory nucleic acid sequence comprises a second promoter directing transcription of one or more transgenes from a plus-strand of DNA.

[0012] In one embodiment, each of the two or more transgenes encodes a chimeric antigen receptor (CAR), where each CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain and the CAR expressed from one transgene comprises an antigen binding specificity that is different than the antigen binding specificity of the CAR expressed from the other transgene(s). In other embodiments, the nucleic acid comprises three, four, or five transgenes, each comprising a polynucleotide sequence encoding a CAR. In one embodiment, at least one transgene encodes a marker protein. In another embodiment, the nucleic acid comprises five transgenes encoding five polynucleotides, including four polynucleotides encoding CARs and one polynucleotide encoding a marker protein. In some embodiment, the marker protein is a mutant low affinity nerve growth factor (mLNGFR) marker protein.

[0013] In some embodiments, the nucleic acid is less than 15 kilobases in length. In some embodiments, the nucleic acid comprises a long terminal repeat (LTR) comprising a U3 deletion, such as an LTR comprises the nucleotide sequence of SEQ ID NO: 76.

[0014] In some embodiments, each CAR comprises an antigen binding domain that specifically binds a different antigen. In some embodiments, each CAR comprises an antigen binding domain in the form of a Fab, single-chain variable fragment (scFv), or nanobody. In some embodiments, each CAR comprises an antigen binding domain that specifically binds a different B cell protein. In some embodiments, the B cell protein is selected from the group consisting of CD10, CD19, CD20, CD22, CD79b, CD34, CD52, CD123, FLT-3, ROR1, CD179b, and CD79a.

[0015] In some embodiments, each CAR comprises a different antigen binding domain specifically binding each of CD 19, CD20, CD22, and CD79b. In some embodiments, each antigen binding domain comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 16, 18, 20, 22, 24, and 26, or wherein each antigen binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 19, 21, 23, 25, and 27. Attorney Docket No: 046483-7473W01(04009)

[0016] In some embodiments, each CAR comprises a transmembrane domain from a protein selected from the group consisting of alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. In some embodiments, each CAR comprises a different transmembrane domain selected from the group consisting of CD8, CD28, ICOS, and 0X40. In some embodiments, each transmembrane domain is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 39, 41, 43, and 45 or wherein each transmembrane domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 40, 42, 44, and 46.

[0017] In some embodiments, each intracellular domain comprises a costimulatory domain and an intracellular signaling domain. In some embodiments, each costimulatory domain is from a protein selected from the group consisting of CD28, 4-1BB (CD137), ICOS (CD278), 0X40, CD5, CD27, LFA-1 (CD1 la / CD18), ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD 18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP- 76, PAG / Cbp, BTLA, an MHC class I molecule, and a ligand that specifically binds with CD8.

[0018] In some embodiments, each costimulatory domain is from a protein selected from the group consisting of CD28, 4- IBB, ICOS, and 0X40. In some embodiments, each costimulatory domain is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 47, 49, 51, and 53. In some embodiments, each costimulatory domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 48, 50, 52, and 54.

[0019] In some embodiments, the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif. In some embodiments, each intracellular signaling domain is from a protein selected from the group consisting of CD3 zeta, common FcR gamma, FcyRIII Fc gamma Rlla, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD66d, DAP 10, and DAP 12. In some embodiments, each intracellular signaling domain is from Attorney Docket No: 046483-7473W01(04009)

[0020] CD3 zeta. In some embodiments, each intracellular signaling domain from CD3 zeta is encoded by a different codon-altered polynucleotide sharing no significant homology to one another.

[0021] In some embodiments, each intracellular signaling domain from CD3 zeta is encoded by a different nucleotide sequence selected from the group consisting of SEQ ID NOs: 55-58. In some embodiments, each intracellular signaling domain from CD3 zeta comprises an amino acid sequence of SEQ ID NO: 60.

[0022] In some embodiments, each antigen-binding domain is connected to the transmembrane domain by a hinge region. In some embodiments, the hinge region is from a protein selected from the group consisting of an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial hinge domain, a hinge comprising an amino acid sequence of CD8, or any combination thereof. In some embodiments, the hinge region is from CD8 or CD28. In some embodiments, the hinge region is from CD8 and is encoded by a different codon-altered polynucleotide sharing no significant homology to one another. In some embodiments, the hinge region from CD8 is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 35-37 and an amino acid sequence of SEQ ID NO: 38. In some embodiments, the hinge region from CD28 comprises a nucleotide sequence of SEQ ID NO: 33 and / or an amino acid sequence of SEQ ID NO: 34.

[0023] In some embodiments, each CAR comprises a leader sequence encoding an N-terminal signal peptide. In some embodiments, each CAR-encoded polynucleotide comprises a different CD8a signal peptide encoded by a different codon-altered polynucleotide sharing no significant homology to one another. In some embodiments, each CD8a signal peptide is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 28-31 and comprises an amino acid sequence of SEQ ID NO: 32.

[0024] In one embodiment, the nucleic acid comprises a first polynucleotide encoding a first CAR comprising a CD 19 antigen binding domain, a CD28 hinge domain, a CD28 transmembrane domain, aa CD28 costimulatory domain, and a CD3 zeta intracellular signaling domain; a second polynucleotide encoding a second CAR comprising a CD20 antigen binding domain, CD20 antigen binding domain, a CD8 hinge domain, a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3 zeta intracellular signaling domain; a third polynucleotide encoding a third CAR comprising a CD22 antigen binding domain, a CD8 hinge domain, an ICOS transmembrane domain, ICOS costimulatory domain, and a CD3 zeta Attorney Docket No: 046483-7473W01(04009) intracellular signaling domain; and a fourth polynucleotide encoding a fourth CAR comprising a CD79b binding domain, a CD8 hinge domain, an 0X40 or CD8 transmembrane domain, an 0X40 costimulatory domain, and a CD3 zeta intracellular signaling domain. In another embodiment, the nucleic acid further comprises a fifth polynucleotide encoding a mutant low affinity nerve growth factor (mLNGFR) marker protein.

[0025] In another embodiment, each CAR is encoded by a different nucleotide sequence selected from the group consisting of SEQ ID NOs: 6, 8, 10, and 12 and / or encoded by a different amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 9, 11, and 13.

[0026] In some embodiments, the nucleic acid comprise a first transgene comprising a first polynucleotide sequence encoding a first chimeric antigen receptor (CAR); a second transgene comprising a second polynucleotide sequence encoding a second CAR; a third transgene comprising a third polynucleotide sequence encoding a third CAR; and a fourth transgene comprising a fourth polynucleotide sequence encoding a fourth CAR, where each CAR is ordered 5’-3’ in a particular way. For example, in one embodiment, the nucleic acid comprises, from 5 ’-3’, the fourth, first, second, and third polynucleotides (e.g., PM486). In another embodiment, the nucleic acid comprises, from 5’-3’, the second, first, fourth, and third polynucleotides (e.g., PM511). In another embodiment, the nucleic acid comprises, from 5’-3 the fourth first second, and third polynucleotides(e.g., PM518). In another embodiment, the nucleic acid comprises, from 5 ’-3’, the first, fourth, second, and third polynucleotides (e.g., PM523).

[0027] In some embodiment, one of the first and second promoters is an EF-1 alpha promoter and the other of the first and second promoters is human PGK1 promoter. In one embodiment, the first promoter is an EF-1 alpha promoter, and the second promoter is a human PGK-1 promoter. In an exemplary embodiment, the EF-1 alpha promoter comprises a nucleotide sequence of SEQ ID NO: 74 and the PGK-1 promoter comprises a nucleotide sequence of SEQ ID NO: 72.

[0028] In an embodiment, the nucleic acid encodes a plurality of self-cleaving 2A peptide domains. In some embodiments, the nucleic acid encodes three or four 2A self-cleaving 2A peptide domains. Exemplary self-cleaving peptide domains include those selected from the group consisting of T2A, P2A, E2A and F2A, such as those encoded by nucleotide sequences Attorney Docket No: 046483-7473W01(04009) selected from the group consisting of SEQ ID NOs: 61 , 62, and 64 and / or amino acid sequences selected from the group consisting of SEQ ID NOs: 63 or 65-67.

[0029] In some embodiments, the nucleic acid further encodes a furin cleavage site between the intracellular signaling domain and a self-cleaving 2A peptide domain in each CAR.

[0030] In other embodiments, the EF-1 alpha promoter drives expression of, from N-terminus to C-terminus, an anti-CD19 CAR, an anti-CD20 CAR, and an anti-CD22 CAR.

[0031] In some embodiments, the anti-CD19 CAR, the anti-CD20 CAR, and the anti-CD22 CAR are encoded by the nucleotide sequence of SEQ ID NO: 2 and / or the nucleotide sequence encodes the amino acid sequence of SEQ ID NO: 3.

[0032] In some embodiments, the hPGK-1 promoter drives expression of, from N-terminus to C- terminus, an anti-CD79b CAR and a mLNGFR marker protein.

[0033] In some embodiments, the anti-CD79b CAR and mLNGFR marker protein are encoded by the nucleotide sequence of SEQ ID NO: 4 or 113 and / or the nucleotide sequence encodes the amino acid sequence of SEQ ID NO: 5 or 114.

[0034] In some embodiments, the EF-1 alpha promoter drives expression of, from N-terminus to C-terminus, an anti-CD79b CAR, an anti-CD20 CAR, and an anti-CD22 CAR.

[0035] In some embodiments, the anti-CD79b CAR, an anti-CD20 CAR, and anti-CD22 CAR are encoded by the nucleotide sequence of SEQ ID NO: 115 and / or the nucleotide sequence encodes the amino acid sequence of SEQ ID NO: 116.

[0036] In some embodiments, the hPGK-1 promoter drives expression of, from N-terminus to C- terminus, a CD 19 CAR and a mLNGFR marker protein.

[0037] In some embodiments, the CD 19 CAR and a mLNGFR marker protein are encoded by the nucleotide sequence of SEQ ID NO: 117 and / or the nucleotide sequence encodes the amino acid sequence of SEQ ID NO: 118.

[0038] In another embodiment, the EF-1 alpha promoter drives expression of a first fusion protein comprising from N-terminus to C-terminus an anti-CD19 CAR, an anti-CD20 CAR, and an anti-CD22 CAR. In one embodiment, the first fusion protein is encoded by the nucleotide sequence of SEQ ID NO: 2 and / or the amino acid sequence of SEQ ID NO: 3 (e.g., PM486).

[0039] In another embodiment, the EF-1 alpha promoter drives expression of a first fusion protein comprising from N-terminus to C-terminus an anti-CD79a CAR, an anti-CD20 CAR, and an anti-CD22 CAR. In one embodiment, the first fusion protein is encoded by the nucleotide Attorney Docket No: 046483-7473W01(04009) sequence of SEQ ID NO: 115 and / or the amino acid sequence of SEQ ID NO: 116 (e.g., PM523).

[0040] In another embodiment, the hPGK-1 promoter drives expression of a second fusion protein comprising from N-terminus to C-terminus an anti-CD79b CAR and a mLNGFR marker protein. In one embodiment, the second fusion protein is encoded by the nucleotide sequence of SEQ ID NO: 4 or 113 and / or the amino acid sequence of SEQ ID NO: 5 or 114 (e.g., PM486, PM518, respectively).

[0041] In another embodiment, the hPGK-1 promoter drives expression of a second fusion protein comprising from N-terminus to C-terminus an anti-CD19 CAR and a mLNGFR marker protein. In one embodiment, the second fusion protein is encoded by the nucleotide sequence of SEQ ID NO: 117 and / or the amino acid sequence of SEQ ID NO: 118 (e.g., PM523).

[0042] In another aspect, the present invention provides an expression vector or construct comprising any one of the nucleic acids described herein. In some embodiments, the expression vector is a lentiviral vector, such as a self-inactivating HIV vector. In some embodiments, the HIV vector comprises a partial gag sequence exemplified by the nucleotide sequence of SEQ ID NO: 69. In some embodiments, the HIV vector further comprises a central polypurine tract (cPPT) as exemplified by the nucleotide sequence of SEQ ID NO: 70. In some embodiments, the nucleic acid further comprises a Woodchuck Hepatitis virus post-transcriptional regulatory element (WPRE) exemplified by the nucleic acid sequence of SEQ ID NO: 75. In some embodiments, an expression vector construct comprises the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 107, SEQ ID NO: 108, or SEQ ID NO: 109.

[0043] In another embodiment, the present invention provides a cell comprising a nucleic acid or expression construct described herein. In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), or a regulatory T cell (Treg). In some embodiments, the immune cell is an autologous cell.

[0044] In one embodiment, the cell expresses a first CAR comprising a CD 19 antigen binding domain, a second CAR comprising a CD20 antigen binding domain, a third CAR comprising a CD22 antigen binding domain, a fourth CAR comprising a CD79b antigen binding domain. In some embodiments, each transgene is integrated into the host genome in the form of a single expression cassette. Attorney Docket No: 046483-7473W01(04009)

[0045] In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of the cells described herein.

[0046] In another aspect, the present invention provides a method of treating a disease or disorder in a subject, comprising administering a pharmaceutical composition described herein to a subject in need thereof.

[0047] In some embodiments, the disease is associated with CD 19 expression and is a proliferative disease such as a cancer, tumor, or malignancy; or a precancerous condition such as a myelodysplasia; a myelodysplastic syndrome or a preleukemia; or is a non-cancer related indication associated with expression of CD19. In some embodiments, the disease is cancer. In some embodiments, the cancer is a hematological malignancy. In some embodiments, the hematological malignancy is acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia, myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, B-cell acute lymphoid leukemia (BALL), T-cell acute lymphoid leukemia (TALL), small lymphocytic leukemia (SLL), acute lymphoid leukemia (ALL), chronic leukemia, chronic myelocytic leukemia, chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia or a CD 19-negative relapsed cancer.

[0048] In some embodiments, the subject has a CD 19-positive cancer. In some embodiments, the subject has a CD 19-negative cancer and is positive for one or more of CD 10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a.

[0049] In another aspect, the present invention provides a method for generating the cells described herein. In an embodiment, the method comprises introducing into the cells a nucleic acid or the expression construct described herein. In some embodiments, the method comprises culturing the cells in the presence of IL-2, IL-7, IL- 15, IL-21, or a combination thereof.

[0050] BRIEF DESCRIPTION OF THE DRAWINGS

[0051] The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings. Attorney Docket No: 046483-7473W01(04009)

[0052] FIG. 1 is a schematic design of lentivirus expression constructs for co-expressing transgenes (designated as “cargol”, “cargo2”, “cargo3”, “cargo4” and “cargo5”) that according to some embodiments encode an anti-CD19 CAR (designated as “CD 19 CAR” or “CAR18”), an anti-CD20 CAR (designated as “CD20 CAR” or “CAR19ml”), an anti-CD22 CAR (designated as “CD22 CAR” or “CARB”), an anti-CD79b CAR (designated as “CD79b CAR” or “CAR14” or “CAR14ml”), and minimal Low-affinity Nerve Growth Factor Receptor (designated as “mLNGFR”).

[0053] FIG. 2 shows the killing of B cell lymphoma cells (Ramos) in vitro by T cells transduced with the PM486 lentivirus construct depicted in FIG. 1. FIG. 2 is a bar graph showing the % killing of single-antigen positive (CD19+, CD20+, CD22+, CD79b+), quadruple-positive (4+) or quadruple-knockout (4ko) Ramos tumor cells by PM486 T cells compared to untransduced (UTD) T cells after 66h of co-culture.

[0054] FIGs. 3A-3B show the killing of B cell lymphoma cells (Ramos) in vitro by PM486 T cells compared to CTL019 T cells and untransduced (UTD) T cells and the staining of tumor cells for CD19 / CD20 / CD22 / CD79b expression. FIG. 3 A is a bar graph showing the % killing of single-antigen positive (CD19+, CD20+, CD22+, CD79b+), quadruple-positive (4+), quadrupleknockout (4ko) or antigen-heterogeneous (Mix; 25% of each CD19+ / CD20+ / CD22+ / CD79b+) Ramos tumor cells by PM486 T cells, CTL019 T cells or UTD T cells after 48h of co-culture. FIG. 3B is a flow cytometry (FCM) analysis of residual tumors from the Mix condition in FIG. 3A (antigen-heterogeneous) that were treated with PM486 T cells, CTL019 T cells, or UTD T cells and stained for CD19 / CD20 / CD22 / CD79b antigen expression.

[0055] FIGs. 4A-4B show the killing of B cell lymphoma cells (Ramos) in vivo by PM486 T cells compared to CTL019 T cells and untransduced (UTD) T cells and the corresponding overall survival. NSG mice were engrafted with 0.5E6 quadruple-positive (4+) Ramos tumor cells expressing the click beetle green luciferase reporter by i.v. injection. After 4 days, mice were treated with 2E6 CTL019 T cells, PM486 T cells or UTD T cells by i.v. injection. FIG. 4A is a graph showing the killing of Ramos tumor cells by PM486 T cells compared to CTL019 T cells and UTD T cells. To quantify the degree of tumor cell killing, bioluminescent measurements were performed after i.p. injection of D-Luciferin using an IVIS spectrum imager. FIG. 4B is a graph showing the overall survival of mice from FIG. 4A. Attorney Docket No: 046483-7473W01(04009)

[0056] FIG. 5 shows the killing of B cell lymphoma cells (Ramos) in vitro by T cells transduced with the PM511 and PM518 lentivirus constructs depicted in FIG. 1. FIG. 5 is a bar graph showing the % killing of single-antigen positive (CD19+, CD20+, CD22+, CD79b+), quadruplepositive (4+), quadruple-knockout (4ko) Ramos tumor cells by PM511 T cells, PM518 T cells or untransduced (UTD) T cells after 44.5h of co-culture.

[0057] FIGs. 6A-6D depict representative Penta T cell variants and expression of CD 19 CAR therefrom. FIG. 6A is a schematic diagram showing the ability of Penta T-cells to simultaneously target multiple distinct tumor cell populations. FIG. 6B is a table summarizing the functional design of each receptor. FIG. 6C illustrates the receptor expression profiles under different promoters in the lentiviral vectors PM511, PM518 and PM523 (depicted in FIG. 1). Receptors driven by the strong EFl alpha (EFla) promoter exhibit high expression levels, enhancing functional activity, whereas those under the weaker human phosphoglycerate kinase-1 (hcPGKl) promoter display lower expression, reducing efficacy. FIG. 6D is a flow cytometry (FCM) analysis of Penta T-cells that were stained for CAR18 targeting CD19.

[0058] FIGs. 7A-7B show the in vitro killing of B cell lymphoma cells (Ramos) by T cells transduced with the PM511, PM518 and PM523 Penta lentivirus constructs depicted in FIG. 1. FIG. 7A is a bar graph showing the % killing of single-antigen positive (CD19+, CD20+, CD22+, CD79b+) and quadruple-positive (4+) Ramos tumor cells by PM511 T cells, PM518 T cells, PM523 T cells or untransduced (UTD) T cells after 50.5h of co-culture at an effector-to- target (E:T) ratio of 1 : 1. FIG. 7B summarizes the C ARs with low tumor-killing activity in each Penta T variant.

[0059] FIGs. 8A-8B show the in vitro killing of B cell lymphoma cells (Ramos) by PM523 T cells compared to PM504 T cells expressing only a single CAR against CD19 and untransduced (UTD) T cells at different effector-to-target (E:T) ratios. FIGs. 8A-8B are bar graphs showing the % killing of single-antigen positive (CD 19+ CD20+, CD22+, CD79b+) or quadruplepositive (4+) Ramos tumor cells by PM523 T cells, PM504 T cells or UTD T cells after 3 days of co-culture at E:T ratios of 0.5: 1 (Med ET Ratio) and 0.25:1 (Low ET Ratio), respectively.

[0060] FIGs. 9A-9B show the in vitro killing of B cell lymphoma cells (Ramos) by PM523 T cells compared to PM504 T cells expressing only a single CAR against CD19 and untransduced (UTD) T cells combined with the staining of tumor cells for CD19 / CD20 / CD22 / CD79b expression. FIG. 9A is a bar graph showing the % killing of antigen-heterogeneous (25% of Attorney Docket No: 046483-7473W01(04009) each CD19+ / CD20+ / CD22+ / CD79b+) Ramos tumor cells by PM523 T cells, PM504 T cells or UTD T cells over time at an effector-to-target (E:T) ratio of 1 : 1. FIG. 9B is a flow cytometry (FCM) analysis of residual tumors from FIG. 9A that were stained for CD19 / CD20 / CD22 / CD79b antigen expression.

[0061] FIGs. 10A-10B show the in vitro killing of Burkitt lymphoma (BL), chronic lymphocytic leukemia (CLL), B-cell acute lymphoblastic leukemia (B-ALL), and diffuse large B-cell lymphoma (DLBCL) cells by PM523 Penta T cells compared to conventional PM504 T cells expressing only a single CAR against CD 19 and untransduced (UTD) T cells. FIGs. 10A-10B are bar graphs showing the % killing of BL, CLL, B-ALL and DLBCL tumor cells by PM523 T cells, PM504 T cells or UTD T cells after 50h using an effector-to-target (ET) ratio 1 : 1 (High ET Ratio) or 0.25: 1 / 0.5: 1 (Low ET Ratio), respectively.

[0062] DETAILED DESCRIPTION

[0063] It is to be understood that the methods described in this disclosure are not limited to particular methods and experimental conditions disclosed herein as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0064] Furthermore, the experiments described herein, unless otherwise indicated, use conventional molecular and cellular biological and immunological techniques within the skill of the art. Such techniques are well known to the skilled worker and are explained fully in the literature. See, e.g., Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2008), including all supplements, Molecular Cloning: A Laboratory Manual (Fourth Edition) by MR Green and J. Sambrook and Harlow et al., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (2013, 2nd edition).

[0065] Definitions

[0066] Unless otherwise defined, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art to which the invention pertains. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall Attorney Docket No: 046483-7473W01(04009) include pluralities and plural terms shall include the singular. The use of “or” means “and / or” unless stated otherwise. The use of the term “including,” as well as other forms, such as “includes” and “included,” is not limiting.

[0067] Generally, nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein is well-known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art, or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well- known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0068] That the disclosure may be more readily understood, select terms are defined below. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0069] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0070] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0071] “Activation,” as used herein, refers to the state of a T cell that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with induced cytokine production, and detectable effector functions. The term “activated T cells” refers to, among other things, T cells that are undergoing cell division. Attorney Docket No: 046483-7473W01(04009)

[0072] As used herein, to “alleviate” a disease means reducing the severity of one or more symptoms of the disease.

[0073] The term “antigen” as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen.

[0074] Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell, or a biological fluid.

[0075] The term “anti-tumor effect” as used herein, refers to a biological effect which can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or amelioration of various physiological symptoms associated with the cancerous condition. An “anti-tumor effect” can also be manifested by the ability of the peptides, polynucleotides, cells, and antibodies of the invention in prevention of the occurrence of tumor in the first place.

[0076] As used herein, the term “autologous” is meant to refer to any material derived from the same individual to which it is later to be re-introduced into the individual.

[0077] The term “chimeric intracellular signaling molecule” refers to recombinant receptor comprising one or more intracellular domains of one or more co-stimulatory molecules. The chimeric intracellular signaling molecule substantially lacks an extracellular domain. In some embodiments, the chimeric intracellular signaling molecule comprises additional domains, such as a transmembrane domain, a detectable tag, and a spacer domain. Attorney Docket No: 046483-7473W01(04009)

[0078] “Co-stimulatory ligand,” as the term is used herein, includes a molecule on an antigen presenting cell (e.g., an aAPC, dendritic cell, B cell, and the like) that specifically binds a cognate co-stimulatory 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-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, an agonist or antibody that binds Toll ligand receptor and a ligand that specifically binds with B7-H3. A co-stimulatory ligand also encompasses, 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, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83.

[0079] A “co-stimulatory molecule” refers to the cognate binding partner on a T cell that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the T cell, such as, but not limited to, proliferation. Co-stimulatory molecules include, but are not limited to an MHC class I molecule, BTLA and a Toll ligand receptor.

[0080] A “co-stimulatory signal”, as used herein, refers to a signal, which in combination with a primary signal, such as TCR / CD3 ligation, leads to T cell proliferation and / or upregulation or downregulation of key molecules.

[0081] A “disease” is a state of health of a human or non-human animal wherein the human or non-human animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the human’s or non-human animal’s health continues to deteriorate. In contrast, a “disorder” in a human or non-human animal is a state of health in which the human or non- human animal is able to maintain homeostasis, but in which the human’s or non-human animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the human or non-human animal’s state of health.

[0082] The term “downregulation” as used herein refers to the decrease or elimination of gene expression of one or more genes. Attorney Docket No: 046483-7473W01(04009)

[0083] “Effective amount” or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result or provides a therapeutic or prophylactic benefit. Such results may include, but are not limited to an amount that when administered to a mammal, causes a detectable level of immune suppression or tolerance compared to the immune response detected in the absence of the composition of the invention. The immune response can be readily assessed by a plethora of art-recognized methods. The skilled artisan would understand that the amount of the composition administered herein varies and can be readily determined based on a number of factors such as the disease or condition being treated, the age and health and physical condition of the mammal being treated, the severity of the disease, the particular compound being administered, and the like.

[0084] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0085] As used herein “endogenous” refers to any material from or produced inside an organism, cell, tissue, or system.

[0086] The term “epitope” as used herein is defined as a small chemical molecule on an antigen that can elicit an immune response, inducing B and / or T cell responses. An antigen can have one or more epitopes. Most antigens have many epitopes; i.e., they are multivalent. In general, an epitope is roughly about 10 amino acids and / or sugars in size. Preferably, the epitope is about 4- 18 amino acids, more preferably about 5-16 amino acids, and even more most preferably 6-14 amino acids, more preferably about 7-12, and most preferably about 8-10 amino acids. One skilled in the art understands that generally the overall three-dimensional structure, rather than the specific linear sequence of the molecule, is the main criterion of antigenic specificity and Attorney Docket No: 046483-7473W01(04009) therefore distinguishes one epitope from another. Based on the present disclosure, a peptide used in the present invention can be an epitope.

[0087] As used herein, the term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue, or system.

[0088] The term “expand” as used herein refers to increasing in number, as in an increase in the number of T cells. In one embodiment, the T cells that are expanded ex vivo increase in number relative to the number originally present in the culture. In another embodiment, the T cells that are expanded ex vivo increase in number relative to other cell types in the culture. The term "ex vivo," as used herein, refers to cells that have been removed from a living organism, (e.g., a human) and propagated outside the organism (e.g., in a culture dish, test tube, or bioreactor).

[0089] The term “expression” as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0090] “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., Sendai viruses, lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0091] “Identity” as used herein refers to the subunit sequence identity between two polymeric molecules particularly between two amino acid molecules, such as, between two polypeptide molecules. When two amino acid sequences have the same residues at the same positions; e.g., if a position in each of two polypeptide molecules is occupied by an arginine, then they are identical at that position. The identity or extent to which two amino acid sequences have the same residues at the same positions in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions; e.g., if half (e g., five positions in a polymer ten amino acids in length) of the positions in two sequences are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 of 10), are matched or identical, the two amino acids sequences are 90% identical. Attorney Docket No: 046483-7473W01(04009)

[0092] The term “immune response” as used herein is defined as a cellular response to an antigen that occurs when lymphocytes identify antigenic molecules as foreign and induce the formation of antibodies and / or activate lymphocytes to remove the antigen.

[0093] The term “immunosuppressive” is used herein to refer to reducing overall immune response.

[0094] “Insertion / deletion,” commonly abbreviated “indel,” is a type of genetic polymorphism in which a specific nucleotide sequence is present (insertion) or absent (deletion) in a genome.

[0095] “Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0096] A “lentivirus” as used herein refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses offer the means to achieve significant levels of gene transfer in vivo.

[0097] By the term “modified” as used herein, is meant a changed state or structure of a molecule or cell of the invention. Molecules may be modified in many ways, including chemically, structurally, and functionally. Cells may be modified through the introduction of nucleic acids.

[0098] By the term “modulating,” as used herein, is meant mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and / or compared with the level of a response in an otherwise identical but untreated subject. The term encompasses perturbing and / or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a human.

[0099] In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine. Attorney Docket No: 046483-7473W01(04009)

[0100] The term “nanobody” is used with reference to a single-domain antibody fragment consisting of a single monomeric variable antibody domain, which are also referred to as VHH fragments. Like a whole antibody, it is able to bind selectively to a specific antigen.

[0101] The term “oligonucleotide” typically refers to short polynucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, C, G), this also includes an RNA sequence (i.e., A, U, C, G) in which “U” replaces “T.”

[0102] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some versions contain an intron(s).

[0103] The term “operably linked” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.

[0104] The term “overexpressed” tumor antigen or “overexpression” of a tumor antigen is intended to indicate an abnormal level of expression of a tumor antigen in a cell from a disease area like a solid tumor within a specific tissue or organ of the patient relative to the level of expression in a normal cell from that tissue or organ. Patients having solid tumors, or a hematological malignancy characterized by overexpression of the tumor antigen can be determined by standard assays known in the art.

[0105] “Parenteral” administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrastemal injection, or infusion techniques.

[0106] The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means Attorney Docket No: 046483-7473W01(04009) available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.

[0107] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides, and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0108] The term “promoter” as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.

[0109] As used herein, the term “promoter / regulatory sequence” means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter / regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter / regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.

[0110] A “constitutive” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0111] An “inducible” promoter is a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be Attorney Docket No: 046483-7473W01(04009) produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell.

[0112] A “tissue-specific” promoter is a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0113] A "self-inactivating" (SIN) long terminal repeat (LTR) refers to a retroviral or lentiviral LTR in which a portion of the U3 region has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication from the LTR, but not any internal promoters.

[0114] The terms “single-chain antibody” and “single-chain fragment variable” (scFv) are used interchangeably with reference to a complete antigen-binding domain of a whole antibody comprised of variable domains of the heavy and light chains (Vn and VL) regions joined together in a single chain or variable fragment (Fv), typically by a peptide linker.

[0115] By the term “specifically binds,” as used herein with respect to an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.

[0116] By the term “stimulation,” is meant a primary response induced by binding of a stimulatory molecule (e.g., a TCR / CD3 complex) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR / CD3 Attorney Docket No: 046483-7473W01(04009) complex. Stimulation can mediate altered expression of certain molecules, such as upregulation of interferon-gamma, and / or reorganization of cytoskeletal structures, and the like.

[0117] A “stimulatory molecule,” as the term is used herein, means a molecule on a T cell that specifically binds with a cognate stimulatory ligand present on an antigen presenting cell.

[0118] A “stimulatory ligand,” as used herein, means a ligand that when present on an antigen presenting cell (e.g., an aAPC, a dendritic cell, a B-cell, and the like) can specifically bind with a cognate binding partner (referred to herein as a “stimulatory molecule”) on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, and the like. Stimulatory ligands are well-known in the art and encompass, inter alia, an MHC Class I molecule loaded with a peptide, an anti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonist anti-CD2 antibody.

[0119] The term “subject” is intended to include living organisms in which an immune response can be elicited (e.g., mammals). A “subject” or “patient,” as used therein, may be a human or non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals. Preferably, the subject is human.

[0120] As used herein, the term “T cell receptor” or “TCR” refers to a complex of membrane proteins that participate in the activation of T cells in response to the presentation of antigen. The TCR is responsible for recognizing antigens bound to major histocompatibility complex molecules. TCR is composed of a heterodimer of an alpha (a) and beta (0) chain, although in some cells the TCR consists of gamma and delta (y / 5) chains. TCRs may exist in alpha / beta and gamma / delta forms, which are structurally similar but have distinct anatomical locations and functions. Each chain is composed of two extracellular domains, a variable and constant domain. In some embodiments, the TCR may be modified on any cell comprising a TCR, including, for example, a helper T cell, a cytotoxic T cell, a memory T cell, regulatory T cell, natural killer T cell, and gamma delta T cell.

[0121] The term “therapeutic” as used herein means a treatment and / or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state.

[0122] The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, Attorney Docket No: 046483-7473W01(04009) transformed, or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0123] “Transplant” refers to a biocompatible lattice or a donor tissue, organ, or cell, to be transplanted. An example of a transplant may include but is not limited to skin cells or tissue, bone marrow, and solid organs such as heart, pancreas, kidney, lung, and liver. A transplant can also refer to any material that is to be administered to a host. For example, a transplant can refer to a nucleic acid or a protein.

[0124] The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed, or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0125] To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0126] The term “tumor” as used herein, refers to an abnormal growth of tissue that may be benign, pre-cancerous, malignant, or metastatic.

[0127] A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, Sendai viral vectors, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.

[0128] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically Attorney Docket No: 046483-7473W01(04009) disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0129] In some aspects, the present invention provides nucleic acids, expression constructs and modified immune cells or precursors thereof expressing multiple polypeptides such as, for example, chimeric antigen receptors (CARs) specific for a plurality of B cell antigens; cytokines; and / or switch receptors. In other aspects, the present invention provides pharmaceutical compositions and methods of treatment, along with methods and source materials for making modified immune cells (e.g., T cells) expressing the multiple polypeptides.

[0130] Nucleic Acids and Expression Vectors

[0131] In some aspects, the present invention provides a nucleic acid comprising two or more transgenes, wherein each transgene is operably linked to a first or a second regulatory nucleic acid sequence; wherein the first regulatory nucleic acid sequence comprises a first promoter directing transcription of one or more transgenes from a plus-strand of DNA; and wherein the second regulatory nucleic acid sequence comprises a second promoter directing transcription of one or more transgenes from a plus-strand of DNA.

[0132] In some aspects, the present invention provides a nucleic acid comprising two or more transgenes, wherein each transgene is operably linked to a first or a second regulatory nucleic acid sequence; wherein the first regulatory nucleic acid sequence comprises a first promoter directing transcription of one or more transgenes from a plus-strand of DNA; and wherein the second regulatory nucleic acid sequence comprises a second promoter directing transcription of one or more transgenes from a plus-strand of DNA.

[0133] In other embodiments, the two or more transgenes encode two or more polypeptides that may include a set of polypeptides, linked with or without a linker or unlinked.

[0134] In some embodiments, the two or more transgenes can encode any combination of two or more polypeptides, in particular to enable the preparation of engineered immune cells (e.g., T or natural killer (NK) cells) modified to express the nucleic acid for use in adoptive immunotherapy to prevent or treat a disease or disorder (e.g., for use in adoptive cancer immunotherapies to provide enhanced anticancer immune functions). Attorney Docket No: 046483-7473W01(04009)

[0135] In some embodiments, the two or more transgenes can encode any combination of two or more polypeptides that may include, without limitation, immunomodulatory polypeptides e.g., a polypeptide agonist of a costimulatory signal of an immune cell (e.g., an agonist of a T effector cell) and / or antagonist of an inhibitory signal of an immune cell such as a regulatory T cell.

[0136] In some embodiments, the immunomodulatory polypeptide is a cytokine such as, for example, a pro-inflammatory cytokine, e.g., interleukin- 12 (IL-12), interleukin-7 (IL-7), or granulocyte-macrophage colony-stimulating factor (GM-CSF); or is an inhibitor (e.g., an interleukin- 10 (IL- 10) trap) of an inhibitory cytokine (e.g., IL- 10) that decrease T effector cell function.

[0137] In one embodiment, each of the two or more transgenes encodes a chimeric antigen receptor (CAR).

[0138] In some embodiment, the two or more transgenes can encode any combination of two or more polypeptides comprising a CAR and an antitumor cytokine (e.g., IL-12), for example for use as T cells redirected for universal cytokine killing (TRUCK).

[0139] In some embodiment, the two or more transgenes can encode any combination of two or more polypeptides comprising a CAR and a chemokine receptor, which binds to a tumor ligand (for example, C-C motif chemokine receptor 2 (CCR2)-C-C motif chemokine ligand 2 (CCL2)), thereby enhancing tumor homing.

[0140] In some embodiment, the two or more transgenes can encode any combination of two or more polypeptides comprising a CAR to provide for universal, allogeneic CAR T cells engineered to no longer express endogenous T cell receptor (TCR) and / or major histocompatibility complex (MHC) molecules, thereby preventing graft-versus-host disease (GVHD) or rejection, respectively.

[0141] In some embodiment, the two or more transgenes can encode any combination of two or more polypeptides comprising a CAR to provide for CAR T cells engineered to be resistant to immunosuppression (armored CARs), wherein the CAR T cells may be genetically modified to no longer express various immune checkpoint molecules (for example, cytotoxic T lymphocyte- associated antigen 4 (CTLA4) or programmed cell death protein 1 (PD 1 )).

[0142] In other embodiments, the two or more transgenes can encode any combination of two or more polypeptides comprising a CAR and a tumor epitope to which an existing monoclonal antibody agent binds. Attorney Docket No: 046483-7473W01(04009)

[0143] In other embodiments, the two or more transgenes can encode any combination of two or more polypeptides comprising a tandem CAR (TanCAR) comprising two linked single-chain variable fragments (scFvs) that have different affinities fused to intracellular co-stimulatory domain(s) and a CD3^ domain.

[0144] In other embodiments, the two or more transgenes can encode any combination of two or more polypeptides comprising two separate CARs with different ligand binding targets; one CAR includes only the CD3(^ domain and the other CAR includes only the co-stimulatory domain(s). Dual CAR T cell activation requires co-expression of both targets on the tumor.

[0145] In other embodiments, the two or more transgenes can encode any combination of two or more polypeptides comprising a safety CAR (sCAR) comrpising an extracellular scFv fused to an intracellular inhibitory domain (for example, CTLA4 or PD1). sCAR T cells co-expressing a standard CAR become activated only when encountering target cells that possess the standard CAR target but lack the sCAR target.

[0146] In some embodiment, the CAR expressed from one transgene comprises an antigen binding specificity that is different than the antigen binding specificity of the CAR expressed from the other transgene(s).

[0147] In some embodiments, each of the two or more transgenes encodes a CAR, wherein the CAR expressed from one transgene comprises an antigen binding specificity that is different than the antigen binding specificity of the CAR expressed from the other transgene(s), and wherein each CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain.

[0148] In one embodiment, the nucleic acid comprises a first transgene comprising a first polynucleotide sequence encoding a first chimeric antigen receptor (CAR); a second transgene comprising a second polynucleotide encoding a second CAR; a third transgene comprising a third polynucleotide encoding a third CAR; a fourth transgene comprising a fourth polynucleotide encoding a fourth CAR; and a fifth transgene comprising a fifth polynucleotide encoding a fifth CAR. In some embodiments, each of the first, second, third, and fourth CARs specifically binds to a different antigen than the antigen specifically bound by the other CARs.

[0149] In some embodiment, each of the first, second, third, and fourth CAR specifically binds to a different antigen than the antigen specifically bound by the other CARs. Attorney Docket No: 046483-7473W01(04009)

[0150] In another embodiment, the nucleic acid comprise four or more transgenes, including one or more polynucleotides encoding a CAR. In another embodiment, the nucleic acid comprises five transgenes, including one or more polynucleotides encoding a CAR.

[0151] In some embodiments, the nucleic acid comprises five transgenes, including at least three different CARs. In another embodiment, the nucleic acid comprises five transgenes, including at least four different CARs. In another embodiment, the nucleic acid comprises five transgenes, including four different CARs and a marker protein. In another embodiment, the five transgenes encode five different CARs.

[0152] In some embodiments, each CAR specifically binds a different antigen. In some embodiments, two or more CARs specifically bind the same antigen. In some embodiments, each CAR is capable of specifically binding an antigen expressed by a cancer cell (e.g., a tumor associated antigen (TAA).

[0153] In some embodiments, the nucleic acid further comprises one or more transgenes comprising polynucleotide sequences encoding one or more immune checkpoint inhibitor(s) and / or one or more cytokines.

[0154] In another embodiment, the nucleic acid comprises a first promoter directing transcription of one or more transgenes from a plus-strand of the nucleic acid; and a second promoter directing transcription of one or more transgenes from a minus-strand of the construct.

[0155] In some embodiments, an expression vector comprises the nucleic acid.

[0156] In one embodiment, the expression vector is a lentiviral vector.

[0157] In other embodiments, the nucleic acid further comprises a lentivirus LTR comprising a U3 deletion.

[0158] In another embodiment, the nucleic acid is less than 15 kilobases (kb).

[0159] In some embodiments, the nucleic acid is less 14.5 kb, less than 14 kb, or less than 13.5 kb in length.

[0160] In some embodiments, an expression cassette encoding the five transgenes is less than 9 kb, less than 8.5 kb, less than 8 kb, or less than 7.5 kb in length.

[0161] In one embodiment, each CAR comprises an antigen binding domain, a transmembrane domain, an intracellular domain. In some embodiments, the polynucleotide encoding each CAR further encodes a leader sequence encoding an N-terminal signal peptide and a hinge domain for connecting the antigen binding domain to the transmembrane domain. Attorney Docket No: 046483-7473W01(04009)

[0162] In some embodiments, each CAR specifically binds a B cell antigen, for example, as expressed on a noncancerous and / or cancer B cell. Exemplary B cell antigens include but are not limited to CD5, CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD30, CD33 (IL3Ra), CD34, CD37, CD38, CD40, CD52, CD53, CD69, CD72, CD73, CD74, CD75, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD123, CD135, CD138, CD179, CD269. Flt3, ROR1, BCMA, FcRn5, FcRn2, CS-L CXCR4, 5, 7, IL-7 / 3R, IL7 / 4 / 3R, and L4R, and variants thereof.

[0163] In one embodiment, the B cell antigen is selected from the group consisting of CD 10, CD19, CD20, CD22, CD33 (IL3Ra), CD34, CD79b, CD123, CD179b, FLT-3, and ROR1, or a variant thereof. In another embodiment, the B cell antigens are selected from the group consisting of CD 19, CD20, CD22, and CD79b, or a variant thereof.

[0164] In one embodiment, the nucleic acid encodes a first CAR specifically binding CD 19 (SEQ ID NOs: 6, 7), a second CAR specifically binding CD20 (SEQ ID NOs: 8, 9) a third CAR specifically binding CD22 (SEQ ID NOs: 10, 11), and a fourth CAR specifically binding CD79b (SEQ ID NOs: 12, 13). In another embodiment, the nucleic acid encodes a first CAR specifically binding CD 19, a second CAR specifically binding CD20, a third CAR specifically binding CD22, a fourth CAR specifically binding CD79b, and a minimal low affinity nerve growth factor receptor (mLNGFR) marker (SEQ ID NOs: 14, 15).

[0165] In some embodiments, one of the transgenes encodes a marker protein for monitoring CAR expression. Exemplary marker proteins include mLNGFR, truncated EGFR (tEGFR), and truncated CD34 (tCD34).

[0166] In some embodiments, the nucleic acid encodes a plurality of self-cleaving 2A peptides positioned between the CARs and / or marker proteins. As used herein, a “self-cleaving 2A peptide” or “2A peptide” refers to an oligopeptide that allow multiple proteins to be encoded as polyproteins, which dissociate into component proteins upon translation. Use of the term “selfcleaving” is not intended to imply a proteolytic cleavage reaction. Various self-cleaving or 2A peptides are known to those of skill in the art, including, without limitation, those found in members of the Picornaviridae virus family, e.g., foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV0, Thosea asigna virus (TaV), and porcine tescho virus-1 (PTV-1); and carioviruses such as Theilovirus and encephalomyocarditis viruses. 2A peptides derived from Attorney Docket No: 046483-7473W01(04009)

[0167] FMDV, ERAV, PTV-1, and TaV are referred to herein as “F2A,” “E2A,” “P2A,” and “T2A,” respectively.

[0168] In one embodiment, the nucleic acid of the present disclosure comprises the nucleotide sequence of SEQ ID NOs: 61 or 62, which encodes the T2A self-cleaving peptide of SEQ ID NO: 63. In another embodiment, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 64, which encodes the P2A self-cleaving peptide of SEQ ID NO: 65. Those of skill in the art would be able to select the appropriate self-cleaving peptide for use in the present invention.

[0169] In some embodiments, one or more transgenes are separated or additionally separated from one another by a nucleotide sequence encoding a furin cleavage site. Furin is a ubiquitously expressed protease that resides in the trans-golgi and processes protein precursors before their secretion. Furin cleaves at the COOH- terminus of its consensus recognition sequence. Various furin consensus recognition sequences (or “furin cleavage sites”) are known to those of skill in the art, including, without limitation, R(X1)KR (SEQ ID NO: 78) or R(X1)RR (SEQ ID NO: 79), (X2)R(X1)(X2)R (SEQ ID NO: 80) and R(X1)(X1)R (SEQ ID NO: 81), such as RRKR (SEQ ID NO: 82), RQKR (SEQ ID NO: 83), where XI is any naturally occurring amino acid, X2 is Lys or Arg. Those of skill in the art would be able to select the appropriate Furin cleavage site for use in the present invention.

[0170] In some embodiments, the nucleic acid comprises a plurality of linkers encoding a combination of a Furin cleavage site upstream of a 2A peptide. Examples include, without limitation, a linker comprising a nucleic acid sequence encoding a Furin cleavage site and an F2A peptide, a linker comprising a nucleic acid sequence encoding a Furin cleavage site and an E2A peptide, a linker comprising a nucleic acid sequence encoding a Furin cleavage site and a P2A peptide, a linker comprising a nucleic acid sequence encoding a Furin cleavage site and a T2A peptide, or a combination thereof. Those of skill in the art would be able to select the appropriate combination for use in the present invention.

[0171] In some embodiments, the linker may further comprise a spacer sequence between the Furin cleavage site and the 2A peptide. Various spacer sequences are known in the art, including, without limitation, glycine serine (GS) spacers such as (GS)n, GSG, (GSGGS)n (SEQ ID NO: 83), (GGGS)n (SEQ ID NO: 84), and (GGGGS)n (SEQ ID NO: 85), where n represents an integer of at least 1. Exemplary spacer sequences can comprise amino acid sequences including, without limitation, GGSG (SEQ ID NO: 86), GGSGG (SEQ ID NO: 87), GSGSG Attorney Docket No: 046483-7473W01(04009)

[0172] (SEQ ID NO:88), GSGGG (SEQ ID NO:89), GGGSG (SEQ ID NO:90), GSSSG (SEQ ID NO:91), GGGGS (SEQ ID NO: 92), (G4S)3 (SEQ ID NO: 93), (G4S)4 (SEQ ID NO: 94), and the like. Those of skill in the art would be able to select the appropriate spacer sequence for use in the present invention.

[0173] In some embodiments, the linker comprises a nucleic acid sequence that encodes for an internal ribosome entry site (IRES; SEQ ID NO: 92). As used herein, “an internal ribosome entry site” or “IRES” refers to an element that promotes direct internal ribosome entry to the initiation codon, such as ATG, of a protein coding region, thereby leading to cap-independent translation of the gene. Various internal ribosome entry sites are known to those of skill in the art, including, without limitation, IRES obtainable from viral or cellular mRNA sources, e.g., immunoglobulin heavy-chain binding protein (BiP); vascular endothelial growth factor (VEGF); fibroblast growth factor 2; insulin-like growth factor; translational initiation factor eIF4G; yeast transcription factors TFIID and HAP4; and IRES obtainable from, e.g., cardiovirus, rhinovirus, aphthovirus, HCV, Friend murine leukemia virus (FrMLV), and Moloney murine leukemia virus (MoMLV). An exemplary IRES, encephalomyocarditis virus (EMCV) IRES, comprises the nucleotide sequence of SEQ ID NO: 95. Those of skill in the art would be able to select the appropriate IRES for use in the present invention.

[0174] In some embodiments, a nucleic acid of the present disclosure is provided for the production of a chimeric receptor as described herein, e.g., in a mammalian cell. In some embodiments, a nucleic acid of the present disclosure provides for amplification of the chimeric receptor-encoding nucleic acid.

[0175] In some embodiments, a nucleic acid of the present disclosure further comprises a leader sequence encoding a signal peptide at the N-terminal end of the CAR and / or marker protein, which is cleaved from the antigen binding domain during cellular processing and localization of the CAR and / or marker protein to the cellular membrane. Suitable leader sequences are known to those of skill in the art. In one embodiment, each CAR is encoded by a codon-altered polynucleotide encoding a CD8a signal peptide, which shares no significant homology to one another. In a particular embodiment, each CD8a signal peptide is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 28-31 and comprises an amino acid sequence of SEQ ID NO: 32. Attorney Docket No: 046483-7473W01(04009)

[0176] In embodiments, the nucleic acid of the present disclosure is operably linked to a transcriptional control element, e.g., a promoter, enhancer, etc. Suitable promoter and enhancer elements are known to those of skill in the art. In certain embodiments, the promoter is a phosphoglycerate kinase- 1 (PGK) promoter. An exemplary PGK promoter comprises the nucleotide sequence of SEQ ID NO: 72. In other embodiments, the promoter is an EF-1 alpha promoter. An exemplary EF-1 alpha comprises the nucleotide sequence of SEQ ID NO: 74. In other embodiments, the promoter is a Rous sarcoma virus (RSV) promoter. An exemplary RSV promoter comprises the nucleotide sequence of SEQ ID NO: 75. Other constitutive promoter sequences may also be used, including, but not limited to a cytomegalovirus (CMV) immediate early promoter, a simian virus 40 (SV40) early promoter, a mouse mammary tumor virus (MMTV) or human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, a MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, as well as human gene promoters such as, but not limited to, an actin promoter, a myosin promoter, a hemoglobin promoter, and a creatine kinase promoter.

[0177] Other suitable promoters include, but are not limited to cell-type specific promoters, including light and / or heavy chain immunoglobulin gene promoters, a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, an NK cell-specific promoter, and various art-known tissue specific promoters.

[0178] In certain embodiments, the nucleic acid of the present disclosure comprises a first promoter driving transcription of one or more transgenes from the plus-strand of DNA and a second promoter driving transcription of one or more transgenes from the negative- strand of DNA. In some embodiments, the first and second promoters are adjacent to one another. In some embodiments, the first and second promoters are separated from one another by a spacer, such as a spacer comprising the nucleotide sequence of SEQ ID NO: 73.

[0179] Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to tetracycline regulated promoters, (e.g., promoter systems including Tet Activators, TetON, TetOFF, etc.), steroid regulated promoters (e.g., rat glucocorticoid receptor promoter systems, human estrogen receptor promoter systems, retinoid promoter Attorney Docket No: 046483-7473W01(04009) systems, glucocorticoid promoters, progesterone promoters, and thyroid promoter systems, ecdysone promoter systems, mifepristone promoter systems, etc.), metal regulated promoters (e.g., metallothionein promoter systems, etc.), pathogenesis-related regulated promoters (e.g., salicylic acid regulated promoters, ethylene regulated promoters, benzothiadiazole regulated promoters, etc.), temperature regulated promoters (e.g., heat shock inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.), light regulated promoters, synthetic inducible promoters, and the like.

[0180] In some embodiments, the locus or construct or transgene containing the suitable promoter is irreversibly switched through the induction of an inducible system. Suitable systems for induction of an irreversible switch are well known in the art, e.g., induction of an irreversible switch may make use of a Cre-lox-mediated recombination (see, e.g., Fuhrmann-Benzakein, et al., Proc. Natl. Acad. Sci. USA (2000) 28:e99, the disclosure of which is incorporated herein by reference). Any suitable combination of recombinase, endonuclease, ligase, recombination sites, etc. known to the art may be used in generating an irreversibly switchable promoter. Methods, mechanisms, and requirements for performing site-specific recombination, described elsewhere herein, find use in generating irreversibly switched promoters and are well known in the art, see, e.g., Grindley et al. Annual Review of Biochemistry (2006) 567-605; and Tropp, Molecular Biology (2012) (Jones & Bartlett Publishers, Sudbury, Mass.), the disclosures of which are incorporated herein by reference.

[0181] In some embodiments, a nucleic acid of the present disclosure further comprises a nucleic acid sequence encoding a chimeric receptor inducible expression cassette. In one embodiment, the chimeric receptor inducible expression cassette is for the production of a transgenic polypeptide product that is released upon chimeric receptor signaling. See, e.g., Chmielewski and Abken, Expert Opin. Biol. Ther. (2015) 15(8): 1145-1154; and Abken, Immunotherapy (2015) 7(5): 535-544. In some embodiments, a nucleic acid of the present disclosure comprises one or more transgenes operably linked to a T-cell activation responsive promoter (e.g., NF AT).

[0182] A nucleic acid of the present disclosure may be present within an expression vector and / or a cloning vector. An expression vector can include a selectable marker, an origin of replication, and other features that provide for replication and / or maintenance of the vector. Suitable expression vectors include, e.g., plasmids, viral vectors, and the like. Large numbers of suitable vectors and promoters are known to those of skill in the art; many are commercially Attorney Docket No: 046483-7473W01(04009) available for generating a subject recombinant construct. The following vectors are provided by way of example, and should not be construed in anyway as limiting: Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif, USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).

[0183] Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. A selectable marker operative in the expression host may be present. Suitable expression vectors include, but are not limited to, e.g., viral vectors based on lentiviruses, such as human immunodeficiency virus (see, e.g., Miyoshi et al., Proc. Natl. Acad. Sci. USA (1997) 94: 10319- 23; Takahashi et al., J. Virol. (1999) 73: 7812-7816); other retroviruses, including gammaretrovirus vectors derived from Moloney Murine Leukemia Virus (MoMLV, MMLV, MuLV, or MLV) or Murine Stem Cell Virus (MSCV), spleen necrosis virus Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, myeloproliferative sarcoma virus, and mammary tumor virus); adenovirus (see, e.g., Li et al., Invest. Opthalmol. Vis. Sci. (1994) 35: 2543-2549; Borras et al., Gene Ther. (1999) 6: 515-524; Li and Davidson, Proc. Natl. Acad. Sci. USA (1995) 92: 7700-7704; Sakamoto et al., H. Gene Ther. (1999) 5: 1088-1097; WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655); adeno-associated virus (see, e.g., Ali et al., Hum. Gene Ther. (1998) 9: 81-86, Flannery et al., Proc. Natl. Acad. Sci. USA (1997) 94: 6916-6921; Bennett et al., Invest. Opthalmol. Vis. Sci. (1997) 38: 2857-2863; Jomary et al., Gene Ther. (1997) 4:683 690, Rolling et al., Hum. Gene Ther. (1999) 10: 641-648; Ali et al., Hum. Mol. Genet. (1996) 5: 591-594; Srivastava in WO 93 / 09239, Samulski et al., J. Vir. (1989) 63: 3822-3828; Mendelson et al., Virol. (1988) 166: 154-165; and Flotte et al., Proc. Natl. Acad. Sci. USA (1993) 90: 10613-10617); vaccinia virus; poliovirus; SV40; herpes simplex virus; and the like.

[0184] Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno- associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of Attorney Docket No: 046483-7473W01(04009) replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No. 6,326,193).

[0185] In some embodiments, an expression vector (e.g., a lentiviral vector) may be used to introduce the chimeric receptor into an immune cell or precursor thereof (e.g., a T cell). Accordingly, an expression vector (e.g., a lentiviral vector) of the present invention may comprise a nucleic acid encoding for a chimeric receptor. In some embodiments, the expression vector (e.g., lentiviral vector) will comprise additional elements that will aid in the functional expression of the chimeric receptor encoded therein. In some embodiments, an expression vector comprising a nucleic acid encoding for a chimeric receptor further comprises a mammalian promoter. In one embodiment, the vector further comprises an elongation-factor- 1- alpha promoter (EF-la promoter). Use of an EF-la promoter may increase the efficiency in expression of downstream transgenes (e.g., a chimeric receptor encoding nucleic acid sequence). Physiologic promoters (e.g., an EF-la promoter) may be less likely to induce integration mediated genotoxicity, and may abrogate the ability of the retroviral vector to transform stem cells. Other physiological promoters suitable for use in a vector (e.g., lentiviral vector) are known to those of skill in the art and may be incorporated into a vector of the present invention. In some embodiments, the vector (e.g., lentiviral vector) further comprises a non-requisite cis acting sequence that may improve titers and gene expression. One non-limiting example of a non-requisite cis acting sequence is the central polypurine tract and central termination sequence (cPPT / CTS) which is important for efficient reverse transcription and nuclear import. Other non-requisite cis acting sequences are known to those of skill in the art and may be incorporated into a vector (e.g., lentiviral vector) of the present invention.

[0186] In some embodiments, the vector further comprises a posttranscriptional regulatory element. Posttranscriptional regulatory elements may improve RNA translation, improve transgene expression, and stabilize RNA transcripts. One example of a posttranscriptional regulatory element is the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). In one embodiment, the WPRE comprises the nucleotide sequence of SEQ ID NO: 76. Accordingly, in some embodiments a vector for the present invention further comprises a WPRE sequence. Various posttranscriptional regulator elements are known to those of skill in the art and may be incorporated into a vector (e.g., lentiviral vector) of the present invention. Attorney Docket No: 046483-7473W01(04009)

[0187] A vector of the present invention may further comprise additional elements such as a rev response element (RRE) for RNA transport, packaging sequences, and 5’ and 3’ long terminal repeats (LTRs). The term “long terminal repeat” or “LTR” refers to domains of base pairs located at the ends of retroviral DNAs which comprise U3, R and U5 regions. LTRs generally provide functions required for the expression of retroviral genes (e.g., promotion, initiation, and polyadenylation of gene transcripts) and to viral replication. In one embodiment, a vector (e.g., lentiviral vector) of the present invention includes a 3’ U3 deleted LTR. Accordingly, a vector (e.g., lentiviral vector) of the present invention may comprise any combination of the elements described herein to enhance the efficiency of functional expression of transgenes. For example, a vector (e.g., lentiviral vector) of the present invention may comprise a WPRE sequence, an RRE sequence, a packaging signal, a central polypurine tract (cPPT) sequence, a 5’LTR, and a 3’ U3 deleted LTR in addition to the polynucleotides encoding the transgenes of the present disclosure.

[0188] Vectors of the present invention may be self-inactivating vectors. As used herein, the term “self-inactivating vector” refers to vectors in which the 3’ LTR enhancer promoter region (U3 region) has been modified (e.g., by deletion or substitution). See Zufferey, R. et al., J. Virol. 73(12):9873-9880, Dec. 1998; and Dull et al., J. Virol. 72(11):8463-8471, Nov. 1998). A selfinactivating vector is engineered to prevent viral transcription beyond the first round of viral replication. Consequently, a self-inactivating vector may be capable of infecting and then integrating into a host genome (e.g., a mammalian genome) only once, and cannot be passed further. Accordingly, self-inactivating vectors virtually eliminate the risk of creating a replication-competent virus.

[0189] In one embodiment, the vector of the present invention is a self-inactivating lentivirus comprising a 5’ U3 deleted LTR (e.g., SEQ ID NO: 77), a 3’ U3 deleted LTR (e.g., SEQ ID NO: 77), a partial HIV gag sequence comprising an RRE sequence and packaging signal (t|i) (e.g., SEQ ID NO: 69), and an HIV central polypurine tract (cPPT) sequence (SEQ ID NO: 70).

[0190] In some embodiments, a nucleic acid of the present invention may be RNA, e.g., in vitro synthesized RNA. Methods for in vitro synthesis of RNA are known to those of skill in the art; any known method can be used to synthesize RNA comprising a sequence encoding a chimeric receptor of the present disclosure. Methods for introducing RNA into a host cell are known in the art. See, e.g., Zhao et al. Cancer Res. (2010) 15: 9053. Introducing RNA comprising a Attorney Docket No: 046483-7473W01(04009) nucleotide sequence encoding a chimeric receptor of the present disclosure into a host cell can be carried out in vitro, ex vivo or in vivo. For example, a host cell (e.g., an NK cell, a cytotoxic T lymphocyte, etc.) can be electroporated in vitro or ex vivo with RNA comprising a nucleotide sequence encoding a chimeric receptor of the present disclosure.

[0191] In order to assess the expression of a polypeptide or portions thereof, the expression vector to be introduced into a cell may also contain either a selectable marker gene or a reporter gene, or both, to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In some embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, without limitation, antibiotic-resistance genes.

[0192] Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assessed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include, without limitation, genes encoding a luciferase, betagalactosidase, chloramphenicol acetyl transferase, or secreted alkaline phosphatase. In certain embodiments, the luciferase gene encodes a blue fluorescent protein (BFP) or green fluorescent protein (GFP). In an embodiment, the blue fluorescent protein comprises the amino acid sequence of SEQ ID NO: 121 and / or is encoded by the nucleotide sequence of SEQ ID NO: 120.

[0193] One aspect of the invention provides a nucleic acid comprising:

[0194] (a) a first polynucleotide sequence encoding a first chimeric antigen receptor (CAR) comprising a first antigen binding domain, a first hinge domain, a first transmembrane domain, a first costimulatory domain, and an intracellular signaling domain;

[0195] (b) a second polynucleotide sequence encoding a second CAR comprising a second antigen binding domain, a second hinge domain, a second transmembrane domain, a second costimulatory domain that confers enhanced effector function, and a second intracellular signaling domain; Attorney Docket No: 046483-7473W01(04009)

[0196] (c) a third polynucleotide sequence encoding a third CAR comprising a third antigen binding domain, a third hinge domain, a third transmembrane domain, a third costimulatory domain that confers enhanced effector function, and a third intracellular signaling domain;

[0197] (d) a fourth polynucleotide sequence encoding a fourth CAR comprising a fourth antigen binding domain, a fourth hinge domain, a fourth transmembrane domain, a fourth costimulatory domain that confers enhanced effector function, and a fourth intracellular signaling domain; and optionally

[0198] (e) a fifth polynucleotide sequence encoding a marker protein or a fifth CAR.

[0199] In some embodiments, one or more of the B-cell antigen specifically binds are the same. In other embodiments, one or more (or all) of the B-cell antigen specifically binds are different.

[0200] In certain embodiments, the first, second, third and fourth antigen binding domains specifically bind a B-cell surface antigen selected from the group consisting of CD 10, CD 19, CD20, CD22, CD33 (IL3Ra), CD34, CD52, CD79b, CD123, CD179b, FLT-3, and ROR1.

[0201] In certain embodiments, the first, second, third and fourth antigen binding domains specifically bind a cell surface antigen selected from the group consisting of CD 19, CD20, CD22, and CD72b.

[0202] The five polynucleotides in the nucleic acid may be arranged in any order in a 5’ to 3’ direction. In one embodiment, the polynucleotides in the nucleic acid are arranged from 5 ’-3’, as first polynucleotide, second polynucleotide, third polynucleotide, fourth polynucleotide, and optionally fifth polynucleotide.

[0203] In certain embodiments, each of the first, second, third, and fourth polynucleotides encodes a CAR. For example, in one embodiment:

[0204] (a) the first CAR comprises a CD19 antigen binding domain (e.g., SEQ ID NOs: 16-21);

[0205] (b) the second CAR comprises a CD20 antigen binding domain (e.g., SEQ ID NOs: 22- 23);

[0206] (c) the third CAR comprises a CD22 antigen binding domain (e.g., SEQ ID NOs: 24-25);

[0207] (d) the fourth CAR comprises a CD79b antigen binding domain (e.g., SEQ ID NOs: 26- 27).

[0208] The foregoing CARs may be arranged in any order in the 5’ to 3’ direction.

[0209] In some embodiments, each of the first, second, third, and fourth polynucleotides encoding a CAR comprises a leader sequence encoding a signal peptide at the N-terminal end of Attorney Docket No: 046483-7473W01(04009) the CAR. In certain embodiments, each of the first, second, third, and fourth polynucleotides encoding a CAR comprises a leader sequence encoding a signal peptide from CD8 alpha. In one embodiment, a polynucleotide encoding a CD8 alpha leader peptide comprises the nucleotide sequence of any one of SEQ ID NOs: 28-31 and the amino acid sequence of SEQ ID NO: 32.

[0210] In certain embodiments of the nucleic acid, the first, second, third, or fourth CARs comprise a hinge domain from CD8 (e.g., SEQ ID NOs: 35-38) or CD28 (e.g., SEQ ID NOs: 33- 34).

[0211] In certain embodiments of the nucleic acid, the first, second, third, or fourth CARs comprise a transmembrane domain selected from the group consisting of CD28 (e.g., SEQ ID NOs: 39-40), CD8 (e.g., SEQ ID NOs: 41-42), ICOS (e.g., SEQ ID NOs: 43-44, and 0X40 (e.g., SEQ ID NOs: 45-46)..

[0212] In certain embodiments of the nucleic acid, the first, second, third, or fourth CARs comprise a costimulatory domain selected from the group consisting of CD28 (e g., SEQ ID NOs: 47-48), 4-1BB (e.g., SEQ ID NOs: 49-50), ICOS (e.g., SEQ ID NOs: 50-51), and 0X40 (e.g., SEQ ID NOs: 52-53).

[0213] In certain embodiments of the nucleic acid, one or all of the first, second, third, or fourth CARs comprise a CD3z intracellular signaling domain, where each signaling domain is encoded by a codon-altered polynucleotide sharing no significant homology to prevent or reduce recombination between otherwise identical nucleotide sequences (see e.g., SEQ ID NOs: 55-58).

[0214] In certain embodiments:

[0215] (a) the first antigen binding domain comprises a CD 19 binding domain, the first hinge domain comprises a CD28 hinge domain, the first transmembrane domain comprises a CD28 transmembrane domain, the first costimulatory domain comprises a CD28 costimulatory domain, and the first intracellular signaling domain comprises a CD3z intracellular signaling domain, as exemplified by the nucleotide sequence of SEQ ID NO: 6 and / or the amino acid sequence of SEQ ID NO: 7;

[0216] (b) the second antigen binding domain comprises a CD20 binding domain, the second hinge domain comprises CD8 hinge domain, the second transmembrane domain comprises a CD8 transmembrane domain, the second costimulatory domain comprises a 4- IBB costimulatory domain, and the second intracellular signaling domain comprises a CD3z intracellular signaling Attorney Docket No: 046483-7473W01(04009) domain, as exemplified by the nucleotide sequence of SEQ ID NO: 8 and / or the amino acid sequence of SEQ ID NO: 9;

[0217] (c) the third antigen binding domain comprises a CD22 binding domain, the third hinge domain comprises a CD8 hinge domain, the third transmembrane domain comprises an ICOS transmembrane domain, the third costimulatory domain comprises an ICOS costimulatory domain, and the third intracellular signaling domain comprises a CD3z intracellular signaling domain, as exemplified by the nucleotide sequence of SEQ ID NO: 10 and / or the amino acid sequence of SEQ ID NO: 11;

[0218] (d) the fourth antigen binding domain comprises a CD79b binding domain, the fourth hinge domain comprises a CD8 hinge domain, the fourth transmembrane domain comprises a CD8 transmembrane domain, the fourth costimulatory domain comprises an 0X40 costimulatory domain, and the fourth intracellular signaling domain comprises a CD3z intracellular signaling domain, as exemplified by the nucleotide sequence of SEQ ID NO: 12 or 111 and / or the amino acid sequence of SEQ ID NO: 13 or 112.

[0219] In certain embodiments, multiple CARs share common functional domains, as exemplified above. In certain embodiments, each shared functional domain is encoded by a codon-altered polynucleotide sharing no significant homology with one another to prevent or reduce recombination between otherwise identical nucleotide sequences. For example, in certain embodiments, each CD3z intracellular signaling domain (see e.g., SEQ ID NOs: 55-58), each CD8 hinge region (see e.g., SEQ ID NOs: 35-36), each CD8 transmembrane domain (see e.g., SEQ ID NOs: 41 or 110), and each CD8 alpha signal peptide is encoded by a different codon- altered polynucleotide sharing no significant homology therebetween (see e.g., SEQ ID NOs: 28- 31).

[0220] Chimeric Antigen Receptors (CARs)

[0221] In one aspect, the present invention provides compositions and methods for modified immune cells or precursor cells thereof, e.g., modified T cells, comprising multiple chimeric antigen receptors (CARs), each having specific binding affinity for a different antibody. A subject CAR of the invention comprises an antigen binding domain, a transmembrane domain, a costimulatory signaling domain, and an intracellular signaling domain. A subject CAR of the invention may additionally comprise a signal peptide domain and / or a hinge domain. Attorney Docket No: 046483-7473W01(04009)

[0222] Accordingly, in an embodiment, a subject CAR of the invention comprises (N-terminal to C terminal) a signal peptide domain, an antigen binding domain, a hinge domain, a transmembrane domain, a costimulatory signaling domains, and an intracellular signaling domain, such as the T cell antigen receptor complex zeta chain (e.g., CD3 zeta). In some embodiments, the CAR comprises two costimulatory domains, such as two costimulatory domains back-to-back between the transmembrane domain and the intracellular signaling domain. In some embodiments, one or more of the CAR functional domains are separated by a linker or spacer.

[0223] The antigen binding domain may be operably linked to another domain of the CAR, such as the transmembrane domain, the costimulatory signaling domain or the intracellular signaling domain, each described elsewhere herein, for expression in the cell. In one embodiment, a first nucleic acid sequence encoding the antigen binding domain is operably linked to a second nucleic acid encoding a transmembrane domain, and further operably linked to a third a nucleic acid sequence encoding a costimulatory signaling domain.

[0224] The antigen binding domains described herein can be combined with any of the transmembrane domains, any of the costimulatory signaling domains, any of the intracellular signaling domains, or any of the other domains described herein that may be included in a CAR of the present invention.

[0225] The antigen binding domains described herein can be combined with any of the transmembrane domains, any of the costimulatory signaling domains, any of the intracellular signaling domains as described above, or any of the other domains described herein that may be included in a CAR of the present invention

[0226] In one embodiment, the present invention provides a nucleic acid encoding- and a cell comprising a CD 19 CAR with a CD 19 antigen binding domain having e.g., the nucleotide sequence of SEQ ID NO: 6 and / or amino acid sequence of SEQ ID NO: 7.

[0227] In another embodiment, the present invention provides a nucleic acid encoding- and a cell comprising a CD20 CAR with a CD20 antigen binding domain having e.g., the nucleotide sequence of SEQ ID NO: 8 and / or amino acid sequence of SEQ ID NO: 9.

[0228] In another embodiment, the present invention provides a nucleic acid encoding- and a cell comprising a CD22 CAR with a CD22 antigen binding domain having e.g., the nucleotide sequence of SEQ ID NO: 10 and / or amino acid sequence of SEQ ID NO: 11. Attorney Docket No: 046483-7473W01(04009)

[0229] In another embodiment, the present invention provides a nucleic acid encoding- and a cell comprising a CD79b CAR with a CD79b antigen binding domain having e.g., the nucleotide sequence of SEQ ID NO: 12 or 111 and / or amino acid sequence of SEQ ID NO: 13 or 112.

[0230] Antigen Binding Domain

[0231] The antigen binding domain of a CAR is an extracellular region of the CAR for binding to a specific target antigen including proteins, carbohydrates, and glycolipids. In some embodiments, the CAR comprises affinity to a target antigen (e.g., a tumor associated antigen) on a target cell (e.g., a cancer cell). The target antigen may include any type of protein, or epitope thereof, associated with the target cell. For example, the CAR may comprise affinity to a target antigen on a target cell that indicates a particular status of the target cell.

[0232] The choice of antigen binding domain depends upon the type and number of antigens that are present on the surface of a target cell. For example, the antigen binding domain may be chosen to recognize an antigen that acts as a cell surface marker on a target cell associated with a particular status of the target cell.

[0233] In one embodiment, a CAR of the invention comprises an extracellular domain having an antigen binding domain that targets a tumor antigen. In some embodiments, the tumor antigen is a B cell antigen. Exemplary B cell antigens include but are not limited to CD5, CD 10, CD 19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD30, CD33 (IL3Ra), CD34, CD37, CD38, CD40, CD52, CD53, CD69, CD72, CD73, CD74, CD75, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD123, CD135, CD138, CD179, CD269. Flt3, ROR1, BCMA, FcRn5, FcRn2, CS-L CXCR4, 5, 7, IL-7 / 3R, IL7 / 4 / 3R, and L4R, and variants thereof.

[0234] In one embodiment, the B cell antigens are selected from the group consisting of CD 10, CD19, CD20, CD22, CD33 (IL3Ra), CD34, CD52, CD79b, CD123, CD179b, FLT-3, ROR1, and any variant thereof.

[0235] In an exemplary embodiment, the nucleic acid encodes four CARs, each comprising a different B cell antigen selected from the group consisting of CD19, CD20, CD22, and CD79b.

[0236] As described herein, a CAR of the present disclosure having affinity for a specific target antigen on a target cell may comprise a target-specific binding domain. In some embodiments, the target-specific binding domain is a human target-specific binding domain, e.g., the targetspecific binding domain is of human origin. Attorney Docket No: 046483-7473W01(04009)

[0237] Depending on the desired antigen to be targeted, the CAR of the invention can be engineered to include the appropriate antigen binding moiety that is specific to the desired antigen target. For example, if CD 19 is the desired antigen that is to be targeted, an antibody for CD 19 can be used as the antigen binding moiety for incorporation into the CAR of the invention.

[0238] For example, in an exemplary embodiment, a CAR of the present disclosure having affinity for CD 19 on a target cell may comprise a human CD 19 binding domain in the form of an scFv. In some embodiments, the binding domain is a murine binding domain. In some embodiment, the binding domain is a chimeric or partially humanized binding domain. In some embodiments, the binding domain is a human binding domain.

[0239] In an embodiment, the CD 19 binding domain is an scFv expressed from a polynucleotide comprising a nucleic acid selected from the group consisting of SEQ ID NOs: 16, 18, and 20, and / or an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 19, and 21, respectively.

[0240] In an embodiment, the CD20 binding domain is a nanobody expressed from a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 22 and / or the amino acid sequence of SEQ ID NO: 23.

[0241] In an embodiment, the CD22 binding domain is a nanobody expressed from a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 24 and / or the amino acid sequence of SEQ ID NO: 25.

[0242] In an embodiment, the CD79b binding domain is an scFv expressed from a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 24 and / or the amino acid sequence of SEQ ID NO: 25.

[0243] The antigen binding domain can include any domain that binds to the antigen and may include, but is not limited to, a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a non-human antibody, a nanobody, or any fragment thereof. Thus, in one embodiment, the antigen binding domain portion comprises a mammalian antibody or a fragment thereof. In some embodiments, the antigen binding domain is selected from the group consisting of an antibody, an antigen binding fragment (Fab), a single-chain variable fragment (scFv), and a nanobody. In some embodiments, CD 19 and CD79B binding domains of the present invention are CD19-specific and CD79b-specific scFv. In some Attorney Docket No: 046483-7473W01(04009) embodiments, CD20 and CD20 binding domains of the present invention are CD20-specific and CD22-specific nanobodies.

[0244] As used herein, the term “single-chain variable fragment” or “scFv” is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin (e.g., mouse or human) covalently linked to form a VH:VL heterodimer. The heavy (VH) and light chains (VL) are either joined directly or joined by a peptide-encoding linker or spacer, which connects the N-terminus of the VH with the C-terminus of the VL, or the C-terminus of the VH with the N-terminus of the VL. The terms “linker” and “spacer” are used interchangeably herein. In some embodiments, the antigen binding domain comprises an scFv having the configuration from N- terminus to C-terminus, VH - linker - VL. In some embodiments, the antigen binding domain comprises an scFv having the configuration from N-terminus to C-terminus, VL - linker - VH. Those of skill in the art would be able to select the appropriate configuration for use in the present invention.

[0245] The linker is typically rich in glycine for flexibility, as well as serine or threonine for solubility. The linker can link the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain. Non-limiting examples of linkers are disclosed in Shen et al., Anal. Chem. 80(6): 1910-1917 (2008) and WO 2014 / 087010, the contents of which are hereby incorporated by reference in their entireties. Various linker sequences are known in the art, including, without limitation, glycine serine (GS) linkers such as (GS)n, GSG, (GSGGS)n (SEQ ID NO: 83), (GGGS)n (SEQ ID NO: 84), and (GGGGS)n (SEQ ID NO: 85), where n represents an integer of at least 1. Exemplary spacer sequences can comprise amino acid sequences including, without limitation, GGSG (SEQ ID NO: 86), GGSGG (SEQ ID NO:87), GSGSG (SEQ ID NO:88), GSGGG (SEQ ID NO:89), GGGSG (SEQ ID NO:90), GSSSG (SEQ ID NO:91), GGGGS (SEQ ID NO: 92), (G4S)3 (SEQ ID NO: 93), (G4S)4 (SEQ ID NO: 94), and the like. Those of skill in the art would be able to select the appropriate linker sequence for use in the present invention.

[0246] Despite removal of the constant regions and the introduction of a linker, scFv proteins retain the specificity of the original immunoglobulin. Single chain Fv polypeptide antibodies can be expressed from a nucleic acid comprising VH- and VL-encoding sequences as described by Huston, et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). See, also, U.S. Patent Nos. 5,091,513, 5,132,405 and 4,956,778; and U.S. Patent Publication Nos. 20050196754 and Attorney Docket No: 046483-7473W01(04009)

[0247] 20050196754. Antagonistic scFvs having inhibitory activity have been described (see, e.g., Zhao et al., Hybridoma (Larchmt) 2008 27(6):455-51; Peter et al., J Cachexia Sarcopenia Muscle 2012 August 12; Shieh et al., J hnunol 2009 183(4):2277-85; Giomarelli et al., Thromb Haemost 2007 97(6):955-63; Fife eta., J Clin Invest 2 6 116(8):2252-61; Brocks et al., Immunotechnology 1997 3(3):173-84; Moosmayer et al., Ther Immunol 1995 2(10:31-40). Agonistic scFvs having stimulatory activity have been described (see, e.g., Peter et al., J Biol Chem 2003 25278(38):36740-7; Xie et al., Nat Biotech 1997 15(8):768-71 ; Ledbetter et al., Crit Rev Immunol 1997 17(5-6):427-55; Ho et al., Biochim Biophys Acta 2003 1638(3):257-66).

[0248] As used herein, “Fab” refers to a fragment of an antibody structure that binds to an antigen but is monovalent and does not have a Fc portion, for example, an antibody digested by the enzyme papain yields two Fab fragments and an Fc fragment (e.g., a heavy (H) chain constant region; Fc region that does not bind to an antigen).

[0249] As used herein, “F(ab')2” refers to an antibody fragment generated by pepsin digestion of whole IgG antibodies, wherein this fragment has two antigen binding (ab’) (bivalent) regions, wherein each (ab') region comprises two separate amino acid chains, a part of a H chain and a light (L) chain linked by an S — S bond for binding an antigen and where the remaining H chain portions are linked together. A “F(ab')2” fragment can be split into two individual Fab' fragments.

[0250] In some instances, the antigen binding domain may be derived from the same species in which the CAR will ultimately be used. For example, for use in humans, the antigen binding domain of the CAR may comprise a human antibody as described elsewhere herein, or a fragment thereof.

[0251] Tolerable variations of the antigen binding domain will be known to those of skill in the art, while maintaining specific binding to its target. For example, in some embodiments the an antigen binding domain comprises an amino acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%>, at least 82%, at least 83%, at least 84%, 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%, at least 99% sequence identity to another binding domain corresponding to the same target..

[0252] In some embodiments, the binding domain is encoded by a nucleic acid sequence comprising a nucleotide sequence that has at least 60%, at least 65%, at least 70%, at least 75%, Attorney Docket No: 046483-7473W01(04009) at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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%, at least 99% sequence identity to another binding domain corresponding to the same target.

[0253] The antigen binding domain may be operably linked to another domain of the CAR, such as the signal peptide domain or transmembrane domain both described elsewhere herein. In one embodiment, a nucleic acid encoding the antigen binding domain is operably linked to a nucleic acid encoding a signal peptide domain and a nucleic acid encoding a transmembrane domain.

[0254] The antigen binding domains described herein, such as the antibody or fragment thereof can be combined and operatively linked to any of the transmembrane domains described herein, any of the costimulatory domains, any of the intracellular signaling domains described herein, or any of the other domains described herein that may be included in the CAR.

[0255] Transmembrane Domain

[0256] With respect to the transmembrane domain, a CAR of the present invention can be designed to comprise a transmembrane domain that connects the antigen binding domain of the CAR to the intracellular domain. The transmembrane domain of a subject CAR is a region that is capable of spanning the plasma membrane of a cell (e.g., an immune cell or precursor thereof). The transmembrane domain is for insertion into a cell membrane, e.g., a eukaryotic cell membrane. In some embodiments, the transmembrane domain is interposed between the antigen binding domain and the intracellular domain of a CAR.

[0257] In one embodiment, the transmembrane domain is naturally associated with one or more of the domains in 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.

[0258] The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein, e.g., a Type I transmembrane protein. Where the source is synthetic, the transmembrane domain may be any artificial sequence that facilitates insertion of the CAR into a cell membrane, e.g., an artificial hydrophobic sequence. Examples of the transmembrane regions Attorney Docket No: 046483-7473W01(04009) of particular use in this invention include, without limitation, transmembrane domains derived from (i.e., comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD2, CD3 epsilon, CD45, CD4, CD5, CD7, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), CD278 (ICOS), CD357 (GITR), Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9.

[0259] A transmembrane domain may be combined with any of the antigen binding domains described herein, any of the costimulatory signaling domains or intracellular domains or cytoplasmic domains described herein, or any of the other domains described herein that may be included in the CAR. In some embodiments, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In certain exemplary embodiments, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.

[0260] In some embodiments, the transmembrane domain comprises a CD8ot transmembrane domain. In one embodiment, a subject CAR comprises a CD8a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 42 (such as encoded by e.g., SEQ ID NOs: 41 or 110.

[0261] In some embodiments, the transmembrane domain comprises a CD28 transmembrane domain. In one embodiment, a subject CAR comprises a CD28 transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 40 (such as encoded by e.g, SEQ ID NO: 39).

[0262] In some embodiments, a subject CAR of the present disclosure includes a hinge region that connects the antigen binding domain with the transmembrane domain, which, in turn, connects to the intracellular domain. The hinge region is capable of supporting the antigen binding domain to recognize and bind to the target antigen on the target cells and facilitating proper protein folding for the CAR (see, e.g., Hudecek et al., Cancer Immunol. Res. (2015) 3(2): 125-135). In some embodiments, the hinge region is a flexible, hydrophilic domain, thus allowing the antigen binding domain to have a structure to optimally recognize the specific structure and density of the target antigens on a cell such as tumor cell. The flexibility of the hinge region permits the hinge region to adopt many different conformations. Attorney Docket No: 046483-7473W01(04009)

[0263] The hinge region is an optional component for the CAR. In some embodiments, the CAR may include a hinge region selected from Fc fragments of antibodies, hinge regions of antibodies, CH2 regions of antibodies, CH3 regions of antibodies, artificial hinge sequences or combinations thereof. Examples of hinge regions include, without limitation, a CD8a hinge, artificial hinges made of polypeptides which may be as small as, three glycines (Gly), as well as CHI and CH3 domains of IgGs (such as human IgG4). In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. In some embodiments, the hinge region is a hinge region polypeptide derived from a receptor (e.g., a CD8-derived hinge region).

[0264] In some embodiments, the hinge region can comprise an amino acid sequence of a human IgGl, IgG2, IgG3, or IgG4, hinge region. In one embodiment, the hinge region can include one or more amino acid substitutions and / or insertions and / or deletions compared to a wild-type (naturally occurring) hinge region. For example, His229 of human IgGl hinge can be substituted with Tyr, so that the hinge region comprises the sequence EPKSCDKTYTCPPCP (SEQ ID NO: 103); see, e.g., Yan et al., J. Biol. Chem. (2012) 287: 5891-5897. In one embodiment, the hinge region can comprise an amino acid sequence derived from human CD8, or a variant thereof.

[0265] The hinge region can have a length of from about 4 amino acids to about 50 amino acids, e.g., from about 4 amino acids to about 10 amino acids, from about 10 amino acids to about 15 amino acids, from about 15 amino acids to about 20 amino acids, from about 20 amino acids to about 25 amino acids, from about 25 amino acids to about 30 amino acids, from about 30 amino acids to about 40 amino acids, or from about 40 amino acids to about 50 amino acids.

[0266] Suitable hinge regions can be readily selected and can be of any of a number of suitable lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0267] In some embodiments, the hinge region may include glycine polymers (G)n, glycineserine polymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO: 83) and (GGGS)n (SEQ ID NO: 84), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured, and therefore can serve as a neutral tether between components. Glycine polymers can be used; glycine accesses significantly more phi-psi Attorney Docket No: 046483-7473W01(04009) space than even alanine, and is much less restricted than residues with longer side chains (see, e.g., Scheraga, Rev. Computational. Chem. (1992) 2: 73-142). Exemplary hinge regions can comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 86), GGSGG (SEQ ID NO: 87), GSGSG (SEQ ID NO: 88), GSGGG (SEQ ID NO: 89), GGGSG (SEQ ID NO: 90), GSSSG (SEQ ID NO: 91), and the like.

[0268] In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. Immunoglobulin hinge region amino acid sequences are known in the art; see, e.g., Tan et al., Proc. Natl. Acad. Sci. USA (1990) 87(1): 162-166; and Huck et al., Nucleic Acids Res. (1986) 14(4): 1779-1789. As non-limiting examples, an immunoglobulin hinge region can include one of the following amino acid sequences: DKTHT (SEQ ID NO: 96); CPPC (SEQ ID NO: 97); CPEPKSCDTPPPCPR (SEQ ID NO: 98) (see, e g., Glaser et al., J. Biol. Chem. (2005) 280:41494-41503); ELKTPLGDTTHT (SEQ ID NO: 99); KSCDKTHTCP (SEQ ID NO: 100); KCCVDCP (SEQ ID NO: 101); KYGPPCP (SEQ ID NO: 102); EPKSCDKTHTCPPCP (SEQ ID NO: 103) (human IgGl hinge); ERKCCVECPPCP (SEQ ID NO: 104) (human IgG2 hinge); ELKTPLGDTTHTCPRCP (SEQ ID NO: 105) (human IgG3 hinge); SPNMVPHAHHAQ (SEQ ID NO: 106) (human IgG4 hinge); and the like.

[0269] In some embodiments, a subject CAR comprises a CD8a hinge domain and a CD8a transmembrane domain. In one embodiment, the CD8 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 42 the CD8a hinge domain comprises the amino acid sequence set forth in SEQ ID NO: 38.

[0270] Tolerable variations of the transmembrane and / or hinge domain will be known to those of skill in the art, while maintaining its intended function. For example, in some embodiments a transmembrane domain or hinge domain comprises an amino acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least

[0271] 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least

[0272] 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least

[0273] 97%, at least 98%, at least 99% sequence identity to any amino acid sequence corresponding to a particular transmembrane domain or hinge region.

[0274] The transmembrane domain may be combined with any hinge region and / or may comprise one or more transmembrane domains described herein. Attorney Docket No: 046483-7473W01(04009)

[0275] In some embodiments, a subject CAR may further comprise, between the extracellular domain and the transmembrane domain of the CAR, or between the intracellular domain and the transmembrane domain of the CAR, a spacer domain. As used herein, the term “spacer domain” generally means any oligo- or polypeptide that functions to link the transmembrane domain to, either the extracellular domain or, the intracellular domain in the polypeptide chain. A spacer domain may comprise up to 300 amino acids, e.g., 10 to 100 amino acids, or 25 to 50 amino acids. In some embodiments, the spacer domain may be a short oligo- or polypeptide linker, e.g., between 2 and 10 amino acids in length as described above. In some embodiments, a glycineserine doublet, a glycine-serine-glycine triplet, or any of the glycine-serine rich linkers described above can provide a suitable linker between the transmembrane domain and the intracellular signaling domain of the subject CAR.

[0276] Accordingly, a subject CAR of the present disclosure may comprise any of the transmembrane domains, hinge domains, or spacer domains described herein.

[0277] Intracellular Domain

[0278] A subject CAR of the present invention also includes an intracellular domain. The intracellular domain of the CAR is responsible for activation of at least one of the effector functions of the cell in which the CAR is expressed (e.g., immune cell). The intracellular domain transduces the effector function signal and directs the cell (e.g., immune cell) to perform its specialized function, e.g., harming and / or destroying a target cell.

[0279] The intracellular domain or otherwise the cytoplasmic domain of the CAR is responsible for activation of the cell in which the CAR is expressed. Examples of an intracellular domain for use in the invention include, but are not limited to, the cytoplasmic portion of a surface receptor, co-stimulatory molecule, and any molecule that acts in concert to initiate signal transduction in the T cell, as well as any derivative or variant of these elements and any synthetic sequence that has the same functional capability.

[0280] In certain embodiments, the intracellular domain comprises a costimulatory signaling domain. In certain embodiments, the intracellular domain comprises an intracellular signaling domain. In certain embodiments, the intracellular domain comprises a costimulatory signaling domain and an intracellular signaling domain. Attorney Docket No: 046483-7473W01(04009)

[0281] Examples of the intracellular signaling domain include, without limitation, the chain of the T cell receptor complex or any of its homologs, e.g., r| chain, FcsRIy and P chains, MB 1 (Iga) chain, B29 (Ig) chain, etc., human CD3 zeta chain, CD3 polypeptides (A, 8 and a), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lek, Fyn, Lyn, etc.), and other molecules involved in T cell transduction, such as CD2, CD5 and CD28. In one embodiment, the intracellular signaling domain may be human CD3 zeta chain, FcyRIII, FcsRI, cytoplasmic tails of Fc receptors, an immunoreceptor tyrosine-based activation motif (IT AM) bearing cytoplasmic receptors, and combinations thereof.

[0282] Other examples of the intracellular domain include a fragment or domain from one or more molecules or receptors including, but are not limited to, TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fc gamma R1 la, DAP 10, DAP 12, T cell receptor (TCR), CD8, CD27, CD28, 4- IBB (CD 137), OX9, 0X40, CD30, CD40, PD-1, ICOS, a KIR family protein, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CD5, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD 103, ITGAL, CDl la, LFA-1, ITGAM, CD lib, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, LFA- 1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, other co-stimulatory molecules described herein, any derivative, variant, or fragment thereof, any synthetic sequence of a co-stimulatory molecule that has the same functional capability, and any combination thereof.

[0283] Additional examples of intracellular domains include, without limitation, intracellular signaling domains of several types of various other immune signaling receptors, including, but not limited to, first, second, and third generation T cell signaling proteins including CD3, B7 family costimulatory, and Tumor Necrosis Factor Receptor (TNFR) superfamily receptors (see, e g., Park and Brentjens, J. Clin. Oncol. (2015) 33(6): 651-653). Additionally, intracellular Attorney Docket No: 046483-7473W01(04009) signaling domains may include signaling domains used by NK and NKT cells (see, e.g., Hermanson and Kaufman, Front. Immunol. (2015) 6: 195) such as signaling domains of NKp30 (B7-H6) (see, e.g., Zhang et al., J. Immunol. (2012) 189(5): 2290-2299), and DAP 12 (see, e.g., Topfer et al., J. Immunol. (2015) 194(7): 3201-3212), NKG2D, NKp44, NKp46, DAP10, and CD3z.

[0284] Intracellular signaling domains suitable for use in a subject CAR of the present invention include any desired signaling domain that provides a distinct and detectable signal (e.g., increased production of one or more cytokines by the cell; change in transcription of a target gene; change in activity of a protein; change in cell behavior, e.g., cell death; cellular proliferation; cellular differentiation; cell survival; modulation of cellular signaling responses; etc.) in response to activation of the CAR (i.e., activated by antigen and dimerizing agent). In some embodiments, the intracellular signaling domain includes at least one (e.g., one, two, three, four, five, six, etc.) IT AM motifs as described below. In some embodiments, the intracellular signaling domain includes DAP10 / CD28 type signaling chains. In some embodiments, the intracellular signaling domain is not covalently attached to the membrane bound CAR, but is instead diffused in the cytoplasm.

[0285] Intracellular signaling domains suitable for use in a subject CAR of the present invention include immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptides. In some embodiments, an IT AM motif is repeated twice in an intracellular signaling domain, where the first and second instances of the IT AM motif are separated from one another by 6 to 8 amino acids. In one embodiment, the intracellular signaling domain of a subject CAR comprises 3 IT AM motifs. In some embodiments, intracellular signaling domains includes the signaling domains of human immunoglobulin receptors that contain immunoreceptor tyrosine-based activation motifs (ITAMs) such as, but not limited to, Fc gamma RI, Fc gamma RIIA, Fc gamma RIIC, Fc gamma RIIIA, FcRL5 (see, e.g., Gillis et al., Front. (2014) Immunol. 5:254).

[0286] A suitable intracellular signaling domain can be an ITAM motif-containing portion that is derived from a polypeptide that contains an ITAM motif. For example, a suitable intracellular signaling domain can be an ITAM motif-containing domain from any ITAM motif-containing protein. Thus, a suitable intracellular signaling domain need not contain the entire sequence of the entire protein from which it is derived. Examples of suitable ITAM motif-containing Attorney Docket No: 046483-7473W01(04009) polypeptides include, but are not limited to: DAP12, FCER1G (Fc epsilon receptor I gamma chain), CD3D (CD3 delta), CD3E (CD3 epsilon), CD3G (CD3 gamma), CD3Z (CD3 zeta), and CD79A (antigen receptor complex-associated protein alpha chain).

[0287] In one embodiment, the intracellular signaling domain is derived from DAP 12 (also known as TYROBP; TYRO protein tyrosine kinase binding protein; KARAP; PLOSL; DNAX- activation protein 12; KAR-associated protein; TYRO protein tyrosine kinase-binding protein; killer activating receptor associated protein; killer-activating receptor-associated protein; etc.). In one embodiment, the intracellular signaling domain is derived from FCER1G (also known as FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma-chain; Fc-epsilon RLgamma; FcR gamma; FceRl gamma; high affinity immunoglobulin epsilon receptor subunit gamma; immunoglobulin E receptor, high affinity, gamma chain; etc.). In one embodiment, the intracellular signaling domain is derived from T-cell surface glycoprotein CD3 delta chain (also known as CD3D; CD3 -DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD3d antigen, delta polypeptide (TiT3 complex); OKT3, delta chain; T-cell receptor T3 delta chain; T-cell surface glycoprotein CD3 delta chain; etc.). In one embodiment, the intracellular signaling domain is derived from T-cell surface glycoprotein CD3 epsilon chain (also known as CD3e, T- cell surface antigen T3 / Leu-4 epsilon chain, T-cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3epsilon, T3e, etc.). In one embodiment, the intracellular signaling domain is derived from T-cell surface glycoprotein CD3 gamma chain (also known as CD3G, T-cell receptor T3 gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3 complex), etc.). In one embodiment, the intracellular signaling domain is derived from T-cell surface glycoprotein CD3 zeta chain (also known as CD3Z, T-cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.). In one embodiment, the intracellular signaling domain is derived from CD79A (also known as B-cell antigen receptor complex-associated protein alpha chain; CD79a antigen (immunoglobulin-associated alpha); MB-1 membrane glycoprotein; Ig- alpha; membrane-bound immunoglobulin-associated protein; surface IgM-associated protein; etc.). In one embodiment, an intracellular signaling domain suitable for use in a subject CAR of the present disclosure includes a DAP10 / CD28 type signaling chain. In one embodiment, an intracellular signaling domain suitable for use in a subject CAR of the present disclosure includes a ZAP70 polypeptide. In some embodiments, the intracellular signaling domain includes a cytoplasmic signaling domain of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 Attorney Docket No: 046483-7473W01(04009) delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, or CD66d. In one embodiment, the intracellular signaling domain in the CAR includes a cytoplasmic signaling domain of human CD3 zeta.

[0288] In one embodiment, the intracellular domain of a subject CAR comprises a CD3 zeta intracellular signaling domain. In some embodiments, the CD3z intracellular signaling domain is encoded by a nucleotide sequence of any one of SEQ ID NOs: 55-58 or an amino acid sequence of SEQ ID NO: 59 or 60. In some embodiments, one or all of the CARs comprise a CD3z intracellular signaling domain that is encoded by a different codon-altered polynucleotide sharing no significant homology therebetween to prevent or reduce recombination between otherwise identical nucleotide sequences.

[0289] While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The intracellular signaling domain includes any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

[0290] In some embodiments, the intracellular domain of a subject CAR comprises a costimulatory signaling domain which includes any portion of one or more co-stimulatory molecules, such as at least one signaling domain from CD2, CD3, CD8, CD27, CD28, 0X40, ICOS, 4- IBB, PD-1, any derivative or variant thereof, any synthetic sequence thereof that has the same functional capability, and any combination thereof.

[0291] In certain embodiments, the costimulatory domain is derived from CD28, 4- IBB, ICOS, or 0X40. In an embodiment, a polynucleotide encoding the CD28 costimulatory domain comprises the nucleotide sequence of SEQ ID NO: 47 and / or amino acid sequence of SEQ ID NO: 48. In an embodiment, a polynucleotide encoding the 4- IBB costimulatory domain comprises the nucleotide sequence of SEQ ID NO: 49 and / or amino acid sequence of SEQ ID NO: 50. In an embodiment, a polynucleotide encoding the IC0S8 costimulatory domain comprises the nucleotide sequence of SEQ ID NO: 51 and / or amino acid sequence of SEQ ID NO: 52. In an embodiment, a polynucleotide encoding the 0X40 costimulatory domain comprises the nucleotide sequence of SEQ ID NO: 53 and / or amino acid sequence of SEQ ID NO: 54. Attorney Docket No: 046483-7473W01(04009)

[0292] The intracellular signaling domains described herein can be combined with any of the costimulatory signaling domains described herein, any of the antigen binding domains described herein, any of the transmembrane domains described herein, or any of the other domains described herein that may be included in the CAR. Tolerable variations of the intracellular domain will be known to those of skill in the art, while maintaining specific activity. For example, in some embodiments the intracellular domain, costimulatory domain, and / or intracellular signaling domain comprises an amino acid sequence that has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, 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

[0293] 96%, at least 97%, at least 98%, at least 99% sequence identity to another corresponding amino acid sequence for the particular intracellular domain, costimulatory domain, and / or intracellular signaling domain.

[0294] Exemplary nucleotide and amino acid sequences corresponding to various CARs and their functional domains, including vector sequences, and expression constructs are shown in Table 1.

[0295] Table 1 : Nucleotide and Amino Acid Sequences Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009) Attorney Docket No: 046483-7473W01(04009)

[0296] Modified Immune Cells

[0297] The present invention provides a modified immune cell or precursor cell thereof (e.g., a modified T cell, a modified NK cell, a modified NKT cell), comprising an above-described nucleic acid or expression vector encoding one or more CARs, marker proteins, or Attorney Docket No: 046483-7473W01(04009) therapeutically active proteins. Accordingly, such modified cells generally possess the specificity directed by one or more CARs expressed therein. For example, a modified cell of the present invention comprising a CD 19 CAR, CD20 CAR, CD22 CAR, and CD79b CAR possesses specificity for CD19, CD20, CD22, and / or CD79b expressed on a target cell.

[0298] Any modified cell comprising a CAR comprising any antigen binding domain, any hinge, any transmembrane domain, any intracellular costimulatory domain, and any intracellular signaling domain described herein is envisioned, and can readily be understood and made by a person of skill in the art in view of the disclosure herein.

[0299] In some embodiments, the modified cell is an immune cell or precursor cell thereof. In an exemplary embodiment, the modified cell is a T cell. In an exemplary embodiment, the modified cell is an autologous cell. In an exemplary embodiment, the modified cell is an autologous immune cell or precursor cell thereof. In an exemplary embodiment, the modified cell is an autologous T cell. Additional examples of modified immune cells are further described below.

[0300] Pharmaceutical Compositions and Formulations

[0301] Also provided are populations of modified immune cells of the invention, compositions containing such cells and / or enriched for such cells, such as in which cells expressing dual chimeric receptors make up at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of the total cells in the composition or cells of a certain type such as T cells or CD8+ or CD4+ cells. Among the compositions are pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy. Also provided are therapeutic methods for administering the cells and compositions to subjects, e.g., patients.

[0302] Also provided are compositions including the cells for administration, including pharmaceutical compositions and formulations, such as unit dose form compositions including the number of cells for administration in a given dose or fraction thereof. The pharmaceutical compositions and formulations generally include one or more optional pharmaceutically acceptable carrier or excipient. In some embodiments, the composition includes at least one additional therapeutic agent.

[0303] The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the Attorney Docket No: 046483-7473W01(04009) formulation would be administered. A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative. In some aspects, the choice of carrier is determined in part by the particular cell and / or by the method of administration. Accordingly, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, e.g., by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0304] Buffering agents in some aspects are included in the compositions. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for Attorney Docket No: 046483-7473W01(04009) example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

[0305] The formulations can include aqueous solutions. The formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition being treated with the cells, preferably those with activities complementary to the cells, where the respective activities do not adversely affect one another. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended. Thus, in some embodiments, the pharmaceutical composition further includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and / or vincristine. The pharmaceutical composition in some embodiments contains the cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. The desired dosage can be delivered by a single bolus administration of the cells, by multiple bolus administrations of the cells, or by continuous infusion administration of the cells.

[0306] Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell populations are administered parenterally. The term "parenteral," as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the cells are administered to the subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection. Compositions in some embodiments are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, Attorney Docket No: 046483-7473W01(04009) saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.

[0307] Sterile injectable solutions can be prepared by incorporating the cells in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, and / or colors, depending upon the route of administration and the preparation desired. Standard texts may in some aspects be consulted to prepare suitable preparations.

[0308] Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, and sorbic acid. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0309] The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.

[0310] The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

[0311] In certain aspects, the invention provides a pharmaceutical composition comprising a therapeutically effective amount of any of the modified cells disclosed herein.

[0312] Methods of Treatment

[0313] The modified cells (e.g., T cells) described herein may be included in a composition for immunotherapy. The composition may include a pharmaceutical composition and further include a pharmaceutically acceptable carrier. A therapeutically effective amount of the pharmaceutical composition comprising the modified T cells may be administered. Attorney Docket No: 046483-7473W01(04009)

[0314] In one aspect, the present invention provides a method for adoptive cell transfer therapy comprising administering to a subject in need thereof a modified T cell of the present invention. In another aspect, the invention includes a method of treating a disease or condition in a subject comprising administering to a subject in need thereof a population of modified T cells as described herein.

[0315] In one aspect, the present invention provides a method of treating cancer by providing to the subject in need thereof a modified immune effector cell (e.g., T cells, NK cells), or a modified immune or precursor cell generated by the methods of the present invention, wherein the modified immune or precursor cells are engineered to express one or more CARs as described herein, and wherein the cancer cells express one or more cell surface antigens (e.g., CD 19, CD20, CD22, CD79b) targeted by the CARs of the present invention. In some embodiments, the method of treating cancer in a subject in need thereof comprises administering to the subject a modified cell comprising a three, four, or five exogenous CARs as described herein. In some embodiments, the modified precursor cell is selected from the group consisting of a bone marrow stem cell, a hematopoietic progenitor cell, or a cord blood stem cell.

[0316] Methods for administration of immune cells for adoptive cell therapy are known and may be used in connection with the provided methods and compositions. For example, adoptive T cell therapy methods are described, e.g., in US Patent Application Publication No. 2003 / 0170238 to Gruenberg et al; US Patent No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Common 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338. In some embodiments, the cell therapy, e.g., adoptive T cell therapy is carried out by autologous transfer, in which the cells are isolated and / or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, following isolation and processing are administered to the same subject.

[0317] In some embodiments, the cell therapy, e.g., adoptive T cell therapy, is carried out by allogeneic transfer, in which the cells are isolated and / or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject. In such embodiments, the cells then are administered to a different subject, e.g., a second subject of the same species. In some embodiments, the first and second subjects are genetically identical. Attorney Docket No: 046483-7473W01(04009)

[0318] In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.

[0319] In some embodiments, the subject has been treated with a therapeutic agent targeting the disease or condition, e.g., the tumor, prior to administration of the cells or composition containing the cells. In some aspects, the subject is refractory or non-responsive to the other therapeutic agent. In some embodiments, the subject has persistent or relapsed disease, e.g., following treatment with another therapeutic intervention, including chemotherapy, radiation, and / or hematopoietic stem cell transplantation (HSCT), e.g., allogenic HSCT. In some embodiments, the administration effectively treats the subject despite the subject having become resistant to another therapy.

[0320] In some embodiments, the subject is responsive to the other therapeutic agent, and treatment with the therapeutic agent reduces disease burden. In some aspects, the subject is initially responsive to the therapeutic agent, but exhibits a relapse of the disease or condition over time. In some embodiments, the subject has not relapsed. In some such embodiments, the subject is determined to be at risk for relapse, such as at a high risk of relapse, and thus the cells are administered prophylactically, e.g., to reduce the likelihood of or prevent relapse. In some aspects, the subject has not received prior treatment with another therapeutic agent.

[0321] In some embodiments, the subject has persistent or relapsed disease, e.g., following treatment with another therapeutic intervention, including chemotherapy, radiation, and / or hematopoietic stem cell transplantation (HSCT), e.g., allogenic HSCT. In some embodiments, the administration effectively treats the subject despite the subject having become resistant to another therapy.

[0322] The modified immune cells of the present invention can be administered to an animal, preferably a mammal, even more preferably a human, to treat a cancer. In addition, the cells of the present invention can be used for the treatment of any condition related to a cancer, especially a cell-mediated immune response against a tumor cell(s), where it is desirable to treat or alleviate the disease.

[0323] In one embodiment, the cancer to be treated is a hematological cancer. In one embodiment, the cancer is a leukemia. In another embodiment, the cancer is a myeloma. In another embodiment, the cancer is a lymphoma. Exemplary hematological cancers be treated with the compositions of the present invention include those selected from the group consisting Attorney Docket No: 046483-7473W01(04009) of chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), multiple myeloma, acute lymphoid leukemia (ALL), Hodgkin lymphoma, B-cell acute lymphoid leukemia (BALL), T-cell acute lymphoid leukemia (TALL), small lymphocytic leukemia (SLL), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B cell lymphoma (DLBCL), DLBCL associated with chronic inflammation, follicular lymphoma, pediatric follicular lymphoma, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma (extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue), Marginal zone lymphoma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, splenic marginal zone lymphoma, splenic lymphoma / leukemia, splenic diffuse red pulp small B-cell lymphoma, hairy cell leukemia-variant, lymphoplasmacytic lymphoma, a heavy chain disease, plasma cell myeloma, solitary plasmocytoma of bone, extraosseous plasmocytoma, nodal marginal zone lymphoma, pediatric nodal marginal zone lymphoma, primary cutaneous follicle center lymphoma, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK+ large B-cell lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, primary effusion lymphoma, B-cell lymphoma, or unclassifiable lymphoma.

[0324] In certain exemplary embodiments, the modified immune cells of the invention are used to treat a myeloma, or a condition related to myeloma. Examples of myeloma or conditions related thereto include, without limitation, light chain myeloma, non-secretory myeloma, monoclonal gamopathy of undetermined significance (MGUS), plasmacytoma (e.g., solitary, multiple solitary, extramedullary plasmacytoma), amyloidosis, and multiple myeloma. In one embodiment, a method of the present disclosure is used to treat multiple myeloma. In one embodiment, a method of the present disclosure is used to treat refractory myeloma. In one embodiment, a method of the present disclosure is used to treat relapsed myeloma.

[0325] The cells of the invention to be administered may be autologous, with respect to the subject undergoing therapy.

[0326] The administration of the cells of the invention may be carried out in any convenient manner known to those of skill in the art. The cells of the present invention may be administered to a subject by aerosol inhalation, injection, ingestion, transfusion, implantation, or Attorney Docket No: 046483-7473W01(04009) transplantation. The compositions described herein may be administered to a patient transarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In other instances, the cells of the invention are injected directly into a site of inflammation in the subject, a local disease site in the subject, a lymph node, an organ, a tumor, and the like.

[0327] For the prevention or treatment of disease, the appropriate dosage may depend on the type of disease to be treated, the type of cells or recombinant receptors, the severity and course of the disease, whether the cells are administered for preventive or therapeutic purposes, previous therapy, the subject's clinical history and response to the cells, and the discretion of the attending physician. The compositions and cells are in some embodiments suitably administered to the subject at one time or over a series of treatments.

[0328] In some embodiments, the cells are administered at a desired dosage, which in some aspects includes a desired dose or number of cells or cell type(s) and / or a desired ratio of cell types. Thus, the dosage of cells in some embodiments is based on a total number of cells (or number per kg body weight) and a desired ratio of the individual populations or sub-types, such as the CD4+ to CD8+ ratio. In some embodiments, the dosage of cells is based on a desired total number (or number per kg of body weight) of cells in the individual populations or of individual cell types. In some embodiments, the dosage is based on a combination of such features, such as a desired number of total cells, desired ratio, and desired total number of cells in the individual populations.

[0329] In some embodiments, the populations, or sub-types of cells, such as CD8+and CD4+T cells, are administered at or within a tolerated difference of a desired dose of total cells, such as a desired dose of T cells. In some aspects, the desired dose is a desired number of cells or a desired number of cells per unit of body weight of the subject to whom the cells are administered, e.g., cells / kg. In some aspects, the desired dose is at or above a minimum number of cells or minimum number of cells per unit of body weight. In some aspects, among the total cells, administered at the desired dose, the individual populations or sub-types are present at or near a desired output ratio (such as CD4+to CD8+ratio), e.g., within a certain tolerated difference or error of such a ratio.

[0330] In some embodiments, the cells are administered at or within a tolerated difference of a desired dose of one or more of the individual populations or sub-types of cells, such as a desired Attorney Docket No: 046483-7473W01(04009) dose of CD4+ cells and / or a desired dose of CD8+ cells. In some aspects, the desired dose is a desired number of cells of the sub-type or population, or a desired number of such cells per unit of body weight of the subject to whom the cells are administered, e.g., cells / kg. In some aspects, the desired dose is at or above a minimum number of cells of the population or subtype, or minimum number of cells of the population or sub-type per unit of body weight. Thus, in some embodiments, the dosage is based on a desired fixed dose of total cells and a desired ratio, and / or based on a desired fixed dose of one or more, e.g., each, of the individual sub-types or subpopulations. Thus, in some embodiments, the dosage is based on a desired fixed or minimum dose of T cells and a desired ratio of CD4+to CD8+cells, and / or is based on a desired fixed or minimum dose of CD4+and / or CD8+cells.

[0331] In certain embodiments, the cells, or individual populations of sub-types of cells, are administered to the subject at a range of about one million to about 100 billion cells, such as, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value in between these ranges.

[0332] In some embodiments, the dose of total cells and / or dose of individual sub-populations of cells is within a range of between at or about IxlO5cells / kg to about IxlO11cells / kg 104and at or about 1011cells / kilograms (kg) body weight, such as between 105and 106cells / kg body weight, for example, at or about 1 x 105cells / kg, 1.5 x 105cells / kg, 2 x 105cells / kg, or 1 x 106cells / kg body weight. For example, in some embodiments, the cells are administered at, or within a certain range of error of, between at or about 104and at or about 109T cells / kilograms (kg) body weight, such as between 105and 106T cells / kg body weight, for example, at or about 1 x 105T cells / kg, 1.5 x 1CP T cells / kg, 2 x 105T cells / kg, or 1 x 106T cells / kg body weight. In other Attorney Docket No: 046483-7473W01(04009) exemplary embodiments, a suitable dosage range of modified cells for use in a method of the present disclosure includes, without limitation, from about IxlO5cells / kg to about IxlO6cells / kg, from about IxlO6cells / kg to about IxlO7cells / kg, from about IxlO7cells / kg about IxlO8cells / kg, from about IxlO8cells / kg about IxlO9cells / kg, from about IxlO9cells / kg about IxlO10cells / kg, from about IxlO10cells / kg about IxlO11cells / kg. In an exemplary embodiment, a suitable dosage for use in a method of the present disclosure is about IxlO8cells / kg. In an exemplary embodiment, a suitable dosage for use in a method of the present disclosure is about IxlO7cells / kg. In other embodiments, a suitable dosage is from about IxlO7total cells to about 5x107total cells. In some embodiments, a suitable dosage is from about IxlO8total cells to about 5xl08total cells. In some embodiments, a suitable dosage is from about 1.4xl07total cells to about l.lxlO9total cells. In an exemplary embodiment, a suitable dosage for use in a method of the present disclosure is about 7xl09total cells.

[0333] In some embodiments, the cells are administered at or within a certain range of error of between at or about 104and at or about 109CD41and / or CD81cells / kilograms (kg) body weight, such as between 105and 106CD4+and / or CD8+cells / kg body weight, for example, at or about 1 x 105CD4+and / or CD8+cells / kg, 1.5 x 105CD4+and / or CD8+cells / kg, 2 x 105CD4+and / or CD8+cells / kg, or 1 x 106CD4+and / or CD8 cells / kg body weight. In some embodiments, the cells are administered at or within a certain range of error of, greater than, and / or at least about 1 x 106, about 2.5 x 106, about 5 x 106, about 7.5 x 106, or about 9 x 106CD4+cells, and / or at least about I x lO6, about 2.5 x 106, about 5 x 106, about 7.5 x 106, or about 9 x 106CD8+ cells, and / or at least about 1 x 106, about 2.5 x 106, about 5 x 106, about 7.5 x 106, or about 9 x 106T cells. In some embodiments, the cells are administered at or within a certain range of error of between about 108and 1012or between about IO10and 1011T cells, between about 108and 1012or between about IO10and 1011CD4+cells, and / or between about 108and 1012or between about 1010and 1011CD8+cells.

[0334] In some embodiments, the cells are administered at or within a tolerated range of a desired output ratio of multiple cell populations or sub-types, such as CD4+ and CD8+ cells or sub-types. In some aspects, the desired ratio can be a specific ratio or can be a range of ratios, for example, in some embodiments, the desired ratio (e.g., ratio of CD4+to CD8+cells) is between at or about 5: 1 and at or about 5: 1 (or greater than about 1:5 and less than about 5: 1), or between at or about 1 :3 and at or about 3 : 1 (or greater than about 1 :3 and less than about 3: 1), such as Attorney Docket No: 046483-7473W01(04009) between at or about 2: 1 and at or about 1 :5 (or greater than about 1 :5 and less than about 2: 1, such as at or about 5: 1, 4.5: 1, 4: 1, 3.5: 1, 3: 1, 2.5: 1, 2: 1, 1.9: 1, 1.8: 1, 1.7: 1, 1.6: 1, 1.5: 1, 1.4: 1, 1.3: 1, 1.2: 1, 1.1: 1, 1 : 1, 1 : 1.1, 1 : 1.2, 1 : 1.3, 1: 1.4, 1 : 1.5, 1 : 1.6, 1: 1.7, 1 : 1.8, 1 : 1.9: 1 :2, 1 :2.5, 1 :3, 1 :3.5, 1 :4, 1 :4.5, or 1 :5. In some aspects, the tolerated difference is within about 1%, about 2%, about 3%, about 4% about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50% of the desired ratio, including any value in between these ranges.

[0335] In some embodiments, a dose of modified cells is administered to a subject in need thereof, in a single dose or multiple doses. In some embodiments, a dose of modified cells is administered in multiple doses, e.g., once a week or every 7 days, once every 2 weeks or every 14 days, once every 3 weeks or every 21 days, once every 4 weeks or every 28 days. In an exemplary embodiment, a single dose of modified cells is administered to a subject in need thereof. In an exemplary embodiment, a single dose of modified cells is administered to a subject in need thereof by rapid intravenous infusion.

[0336] In certain embodiments, the subject’s blood comprises at least about 100 modified cells / pL of blood by at least week three after a single administration of the modified T cell.

[0337] In certain embodiments, the subject’s blood comprises at least about 100 modified cells / pL of blood by at least week three after a single administration of the modified T cell.

[0338] In certain embodiments, the modified cell binds to the first and second targets of a cell expressing the first and second targets and kills the cell via granule-mediated cytolysis.

[0339] In certain embodiments, the modified cell is an autologous cell. In certain embodiments, the modified cell is an autologous cell obtained from a human subject. In certain embodiments, the modified cell is a modified T cell.

[0340] Following administration of the cells, the biological activity of the engineered cell populations in some embodiments is measured, e.g., by any of a number of known methods. Parameters to assess include specific binding of an engineered or natural T cell or other immune cell to antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in, for example, Kochenderfer et al., I. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J. Immunological Methods, 285(1): 25-40 (2004). In certain embodiments, the biological activity Attorney Docket No: 046483-7473W01(04009) of the cells is measured by assaying expression and / or secretion of one or more cytokines, such as CD 107a, IFNy, IL-2, and TNF. In some aspects the biological activity is measured by assessing clinical outcome, such as reduction in tumor burden or load.

[0341] In some embodiments, the cells are administered as part of a combination treatment, such as simultaneously with or sequentially with, in any order, another therapeutic intervention, such as an antibody or engineered cell or receptor or agent, such as a cytotoxic or therapeutic agent. The cells in some embodiments are co-administered with one or more additional therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order. In some contexts, the cells are co-administered with another therapy sufficiently close in time such that the cell populations enhance the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the cells are administered prior to the one or more additional therapeutic agents. In some embodiments, the cells are administered after the one or more additional therapeutic agents. In some embodiments, the one or more additional agents includes a cytokine, such as IL-2, for example, to enhance persistence. In some embodiments, the methods comprise administration of a chemotherapeutic agent.

[0342] In certain embodiments, the modified cells of the invention may be administered to a subject in combination with an immune checkpoint antibody (e.g., an anti-PDl, anti-CTLA-4, or anti-PDLl antibody). For example, the modified cell may be administered in combination with an antibody or antibody fragment targeting, for example, PD-1 (programmed death 1 protein). Examples of anti -PD-1 antibodies include, but are not limited to, pembrolizumab (KEYTRUDA®, formerly lambrolizumab, also known as MK-3475), and nivolumab (BMS- 936558, MDX-1106, ONO-4538, OPDIVA®) or an antigen-binding fragment thereof. In certain embodiments, the modified cell may be administered in combination with an anti-PD-Ll antibody or antigen-binding fragment thereof. Examples of anti-PD-Ll antibodies include, but are not limited to, BMS-936559, MPDL3280A (TECENTRIQ®, Atezolizumab), and MEDI4736 (Durvalumab, Imfinzi). In certain embodiments, the modified cell may be administered in combination with an anti-CTLA-4 antibody or antigen-binding fragment thereof. An example of an anti- CTLA-4 antibody includes, but is not limited to, Ipilimumab (trade name Yervoy). Other types of immune checkpoint modulators may also be used including, but not limited to, small molecules, siRNA, miRNA, and CRISPR systems. Immune checkpoint modulators may be administered before, after, or concurrently with the modified cell comprising the CAR. In certain Attorney Docket No: 046483-7473W01(04009) embodiments, combination treatment comprising an immune checkpoint modulator may increase the therapeutic efficacy of a therapy comprising a modified cell of the present invention.

[0343] In certain embodiments, the subject is provided a secondary treatment. Secondary treatments include but are not limited to chemotherapy, radiation, surgery, and medications.

[0344] In some embodiments, the subject can be administered a conditioning therapy prior to CAR T cell therapy. In some embodiments, the conditioning therapy comprises administering an effective amount of cyclophosphamide to the subject. In some embodiments, the conditioning therapy comprises administering an effective amount of fludarabine to the subject. In preferred embodiments, the conditioning therapy comprises administering an effective amount of a combination of cyclophosphamide and fludarabine to the subject. Administration of a conditioning therapy prior to CAR T cell therapy may increase the efficacy of the CAR T cell therapy. Methods of conditioning patients for T cell therapy are described in U.S. Patent No. 9,855,298, which is incorporated herein by reference in its entirety.

[0345] In some embodiments, a specific dosage regimen of the present disclosure includes a lymphodepletion step prior to the administration of the modified T cells. In an exemplary embodiment, the lymphodepletion step includes administration of cyclophosphamide and / or fludarabine.

[0346] In some embodiments, the lymphodepletion step includes administration of cyclophosphamide at a dose of between about 200 mg / m2 / day and about 2000 mg / m2 / day (e.g., 200 mg / m2 / day, 300 mg / m2 / day, or 500 mg / m2 / day). In an exemplary embodiment, the dose of cyclophosphamide is about 300 mg / m2 / day. In some embodiments, the lymphodepletion step includes administration of fludarabine at a dose of between about 20 mg / m2 / day and about 900 mg / m2 / day (e.g., 20 mg / m2 / day, 25 mg / m2 / day, 30 mg / m2 / day, or 60 mg / m2 / day). In an exemplary embodiment, the dose of fludarabine is about 30 mg / m2 / day.

[0347] In some embodiment, the lymphodepletion step includes administration of cyclophosphamide at a dose of between about 200 mg / m2 / day and about 2000 mg / m2 / day (e.g., 200 mg / m2 / day, 300 mg / m2 / day, or 500 mg / m2 / day), and fludarabine at a dose of between about 20 mg / m2 / day and about 900 mg / m2 / day (e.g., 20 mg / m2 / day, 25 mg / m2 / day, 30 mg / m2 / day, or 60 mg / m2 / day). In an exemplary embodiment, the lymphodepletion step includes administration of cyclophosphamide at a dose of about 300 mg / m2 / day, and fludarabine at a dose of about 30 mg / m2 / day. Attorney Docket No: 046483-7473W01(04009)

[0348] In an exemplary embodiment, the dosing of cyclophosphamide is 300 mg / m2 / day over three days, and the dosing of fludarabine is 30 mg / m2 / day over three days.

[0349] Dosing of lymphodepletion chemotherapy may be scheduled on Days -6 to -4 (with a -1 day window, i.e., dosing on Days -7 to -5) relative to T cell (e.g., CAR-T, TCR-T, a modified T cell, etc.) infusion on Day 0.

[0350] In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including 300 mg / m2of cyclophosphamide by intravenous infusion 3 days prior to administration of the modified T cells. In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including 300 mg / m2of cyclophosphamide by intravenous infusion for 3 days prior to administration of the modified T cells.

[0351] In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including fludarabine at a dose of between about 20 mg / m2 / day and about 900 mg / m2 / day (e.g., 20 mg / m2 / day, 25 mg / m2 / day, 30 mg / m2 / day, or 60 mg / m2 / day). In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including fludarabine at a dose of 30 mg / m2for 3 days.

[0352] In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including cyclophosphamide at a dose of between about 200 mg / m2 / day and about 2000 mg / m2 / day (e.g., 200 mg / m2 / day, 300 mg / m2 / day, or 500 mg / m2 / day), and fludarabine at a dose of between about 20 mg / m2 / day and about 900 mg / m2 / day (e.g., 20 mg / m2 / day, 25 mg / m2 / day, 30 mg / m2 / day, or 60 mg / m2 / day). In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including cyclophosphamide at a dose of about 300 mg / m2 / day, and fludarabine at a dose of 30 mg / m2for 3 days.

[0353] Cells of the invention can be administered in dosages and routes and at times to be determined in appropriate pre-clinical and clinical experimentation and trials. Cell compositions may be administered multiple times at dosages within these ranges. Administration of the cells of the invention may be combined with other methods useful to treat the desired disease or condition as determined by those of skill in the art.

[0354] It is known in the art that one of the adverse effects following infusion of CAR T cells is the onset of immune activation, known as cytokine release syndrome (CRS). CRS is immune Attorney Docket No: 046483-7473W01(04009) activation resulting in elevated inflammatory cytokines. CRS is a known on-target toxicity, development of which likely correlates with efficacy. Clinical and laboratory measures range from mild CRS (constitutional symptoms and / or grade-2 organ toxicity) to severe CRS (sCRS; grade >3 organ toxicity, aggressive clinical intervention, and / or potentially life threatening). Clinical features include: high fever, malaise, fatigue, myalgia, nausea, anorexia, tachycardia / hypotension, capillary leak, cardiac dysfunction, renal impairment, hepatic failure, and disseminated intravascular coagulation. Dramatic elevations of cytokines including interferon-gamma, granulocyte macrophage colony-stimulating factor, IL- 10, and IL-6 have been shown following CAR T-cell infusion. One CRS signature is elevation of cytokines including IL-6 (severe elevation), IFN-gamma, TNF-alpha (moderate), and IL -2 (mild). Elevations in clinically available markers of inflammation including ferritin and C-reactive protein (CRP) have also been observed to correlate with the CRS syndrome. The presence of CRS generally correlates with expansion and progressive immune activation of adoptively transferred cells. It has been demonstrated that the degree of CRS severity is dictated by disease burden at the time of infusion as patients with high tumor burden experience a more sCRS.

[0355] Accordingly, the invention provides for, following the diagnosis of CRS, appropriate CRS management strategies to mitigate the physiological symptoms of uncontrolled inflammation without dampening the antitumor efficacy of the engineered cells (e.g., CAR T cells). CRS management strategies are known in the art. For example, systemic corticosteroids may be administered to rapidly reverse symptoms of sCRS (e g., grade 3 CRS) without compromising initial antitumor response.

[0356] In some embodiments, an anti-IL-6R antibody may be administered. An example of an anti-IL-6R antibody is the Food and Drug Administration-approved monoclonal antibody tocilizumab, also known as atlizumab (marketed as Actemra, or RoActemra). Tocilizumab is a humanized monoclonal antibody against the interleukin-6 receptor (IL-6R). Administration of tocilizumab has demonstrated near-immediate reversal of CRS.

[0357] CRS is generally managed based on the severity of the observed syndrome and interventions are tailored as such. CRS management decisions may be based upon clinical signs and symptoms and response to interventions, not solely on laboratory values alone.

[0358] Mild to moderate cases generally are treated with symptom management with fluid therapy, non-steroidal anti-inflammatory drug (NSAID) and antihistamines as needed for Attorney Docket No: 046483-7473W01(04009) adequate symptom relief. More severe cases include patients with any degree of hemodynamic instability; with any hemodynamic instability, the administration of tocilizumab is recommended. The first-line management of CRS may be tocilizumab, in some embodiments, at the labeled dose of 8 mg / kg IV over 60 minutes (not to exceed 800 mg / dose); tocilizumab can be repeated Q8 hours. If suboptimal response to the first dose of tocilizumab, additional doses of tocilizumab may be considered. Tocilizumab can be administered alone or in combination with corticosteroid therapy. Patients with continued or progressive CRS symptoms, inadequate clinical improvement in 12-18 hours or poor response to tocilizumab, may be treated with high- dose corticosteroid therapy, generally hydrocortisone 100 mg IV or methylprednisolone 1-2 mg / kg. In patients with more severe hemodynamic instability or more severe respiratory symptoms, patients may be administered high-dose corticosteroid therapy early in the course of the CRS. CRS management guidance may be based on published standards (Lee et al. (2019) Biol Blood Marrow Transplant, doi.org / 10.1016 / j.bbmt.2018.12.758; Neelapu et al. (2018) Nat Rev Clin Oncology / , 15:47; Teachey et al. (2016) Cancer Discov, 6(6):664-679).

[0359] Features consistent with Macrophage Activation Syndrome (MAS) or Hemophagocytic lymphohistiocytosis (HLH) have been observed in patients treated with CAR-T therapy (Henter, 2007), coincident with clinical manifestations of the CRS. MAS appears to be a reaction to immune activation that occurs from the CRS and should therefore be considered a manifestation of CRS. MAS is similar to HLH (also a reaction to immune stimulation). The clinical syndrome of MAS is characterized by high grade non-remitting fever, cytopenias affecting at least two of three lineages, and hepatosplenomegaly. It is associated with high serum ferritin, soluble interleukin-2 receptor, and triglycerides, and a decrease of circulating natural killer (NK) activity.

[0360] Methods of Generating Modified Immune Cells

[0361] The present invention provides methods for producing / generating a modified immune cell or precursor cell thereof (e.g., a T cell / NK cell / NKT cell). The cells are generally engineered by introducing a nucleic acid encoding a plurality of CARs (e.g., lentivirus expression construct). For example, when transducing the cells with a lentivirus vector comprising the nucleic acid of the present invention, the five transgenes are integrated as a single expression cassette into the host genome. Attorney Docket No: 046483-7473W01(04009)

[0362] Methods of introducing nucleic acids into a cell include physical, biological, and chemical methods. Physical methods for introducing a polynucleotide, such as RNA, into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. RNA can be introduced into target cells using commercially available methods which include electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, MA) or the Gene Pulser II (BioRad, Denver, CO), Multiporator (Eppendorf, Hamburg Germany). RNA can also be introduced into cells using cationic liposome mediated transfection using lipofection, using polymer encapsulation, using peptide mediated transfection, or using biolistic particle delivery systems such as “gene guns” (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8): 861 -70 (2001).

[0363] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, especially lentivirus vectors, retroviral vectors, and adeno-associated virus (AAV) vectors have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from poxviruses, herpes simplex virus I, adenoviruses and, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.

[0364] An expression construct of the present invention is generally introduced into a cell by an expression vector. Expression vectors comprising a nucleic acid of the present invention are provided herein. Suitable expression vectors include lentivirus vectors, retrovirus vectors, gamma retrovirus vectors, foamy virus vectors, AAV vectors, adenovirus vectors, engineered hybrid viruses, naked DNA, including but not limited to transposon mediated vectors, such as Sleeping Beauty, Piggyback, and Integrases such as Phi31. Other suitable expression vectors include herpes virus vector, including herpes simplex virus (HSV) and Epstein-Barr virus (EBV) expression vectors.

[0365] Retrovirus expression vectors are capable of integrating into the host genome, delivering a large amount of foreign genetic material, infecting a broad spectrum of species and cell types, and being packaged in special cell lines. The retrovirus vector is constructed by inserting a nucleic acid (e.g., a nucleic acid encoding a subject CAR) into the viral genome at certain locations to produce a virus that is replication defective. Though the retrovirus vectors are able Attorney Docket No: 046483-7473W01(04009) to infect a broad variety of cell types, integration and stable expression of the subject CAR requires the division of host cells.

[0366] Lentivirus vectors are derived from lentiviruses, which are complex retroviruses that, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function (see, e.g., U.S. Patent Nos. 6,013,516 and 5,994, 136). Some examples of lentiviruses include the human immunodeficiency viruses (HIV-1, HIV-2) and the simian immunodeficiency virus (SIV). Lentivirus vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted making the vector biologically safe. Lentivirus vectors are capable of infecting non-dividing cells and can be used for both in vivo and ex vivo gene transfer and expression, e.g., of a nucleic acid encoding a subject CAR (see, e.g., U.S. Patent No. 5,994,136).

[0367] Adenovirus expression vectors are based on adenoviruses, which have a low capacity for integration into genomic DNA but a high efficiency for transfecting host cells. Adenovirus expression vectors contain adenovirus sequences sufficient to: (a) support packaging of the expression vector and (b) to ultimately express the subject CAR in the host cell. In some embodiments, the adenovirus genome is a 36 kb, linear, double stranded DNA, where a foreign DNA sequence (e.g., a nucleic acid encoding a subject CAR) may be inserted to substitute large pieces of adenoviral DNA in order to make the expression vector of the present invention (see, e.g., Danthinne and Imperiale, Gene Therapy (2000) 7(20): 1707-1714).

[0368] Another expression vector is based on an adeno associated virus, which takes advantage of the adenovirus coupled systems. This AAV expression vector has a high frequency of integration into the host genome. It can infect non-dividing cells, thus making it useful for delivery of genes into mammalian cells, for example, in tissue cultures or in vivo. The AAV vector has a broad host range for infectivity. Details concerning the generation and use of AAV vectors are described in U.S. Patent Nos. 5,139,941 and 4,797,368.

[0369] Expression vectors including a nucleic acid of the present disclosure can be introduced into a host cell by any means known to persons skilled in the art. The expression vectors may include viral sequences for transfection, if desired. Alternatively, the expression vectors may be introduced by fusion, electroporation, biolistics, transfection, lipofection, or the like. The host cell may be grown and expanded in culture before introduction of the expression vectors, followed by the appropriate treatment for introduction and integration of the vectors. The host Attorney Docket No: 046483-7473W01(04009) cells are then expanded and may be screened by virtue of a marker present in the vectors. Various markers that may be used are known in the art, and may include hprt, neomycin resistance, thymidine kinase, hygromycin resistance, etc. As used herein, the terms “cell,” “cell line,” and “cell culture” may be used interchangeably. In some embodiments, the host cell is an immune cell or precursor thereof, e.g., a T cell, an NK cell, or an NKT cell.

[0370] The present invention also provides genetically engineered cells which include and stably express a plurality of CARs described herein. In some embodiments, the genetically engineered cells are genetically engineered T-lymphocytes (T cells), regulatory T cells (Tregs), naive T cells (TN), memory T cells (for example, central memory T cells (TCM), effector memory cells (TEM)), natural killer cells (NK cells), natural killer T cells (NKT cells) and macrophages capable of giving rise to therapeutically relevant progeny. In one embodiment, the genetically engineered cells are autologous cells.

[0371] Modified cells (e.g., comprising a subject CAR) may be produced by stably transfecting host cells with an expression vector including a nucleic acid of the present disclosure. Additional methods to generate a modified cell of the present disclosure include, without limitation, chemical transformation methods (e.g., using calcium phosphate, dendrimers, liposomes and / or cationic polymers), non-chemical transformation methods (e.g., electroporation, optical transformation, gene electrotransfer and / or hydrodynamic delivery) and / or particle-based methods (e.g., impalefection, using a gene gun and / or magnetofection). Transfected cells expressing a subject CAR of the present disclosure may be expanded ex vivo.

[0372] Physical methods for introducing an expression vector into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells including vectors and / or exogenous nucleic acids are well- known in the art. See, e.g., Sambrook et al. (2001), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.

[0373] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). Attorney Docket No: 046483-7473W01(04009)

[0374] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, MO; dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, NY); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as the only solvent since it is more readily evaporated than methanol. “Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine- nucleic acid complexes.

[0375] Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to the inhibitor of the present invention, in order to confirm the presence of the nucleic acids in the host cell, a variety of assays may be performed. Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention.

[0376] Moreover, the nucleic acids may be introduced by any means, such as transducing the expanded T cells, transfecting the expanded T cells, and electroporating the expanded T cells. One nucleic acid may be introduced by one method and another nucleic acid may be introduced into the T cell by a different method.

[0377] Sources of Immune Cells Attorney Docket No: 046483-7473W01(04009)

[0378] Prior to expansion, a source of immune cells is obtained from a subject for ex vivo manipulation. Sources of target cells for ex vivo manipulation may also include, e.g., autologous or heterologous donor blood, cord blood, or bone marrow. For example, the source of immune cells may be from the subject to be treated with the modified immune cells of the invention, e.g., the subject's blood, the subject's cord blood, or the subject’s bone marrow. Non-limiting examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. In certain exemplary embodiments, the subject is a human.

[0379] Immune cells can be obtained from a number of sources, including blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, lymph, or lymphoid organs. Immune cells are cells of the immune system, such as cells of the innate or adaptive immunity, e.g., myeloid or lymphoid cells, including lymphocytes, typically T cells and / or NK cells and / or NKT cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). In certain aspects, the cells are human cells. With reference to the subject to be treated, the cells may be allogeneic and / or autologous. The cells typically are primary cells, such as those isolated directly from a subject and / or isolated from a subject and frozen.

[0380] In certain embodiments, the immune cell is a T cell, e.g., a CD8+ T cell (e.g., a CD8+ naive T cell, central memory T cell, or effector memory T cell), a CD4+ T cell, a natural killer T cell (NKT cells), a regulatory T cell (Treg), a stem cell memory T cell, a lymphoid progenitor cell, a hematopoietic stem cell, a natural killer cell (NK cell), a natural killer T cell (NK cell) or a dendritic cell. In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and / or basophils. In an embodiment, the target cell is an induced pluripotent stem (iPS) cell or a cell derived from an iPS cell, e.g., an iPS cell generated from a subject, manipulated to alter (e.g., induce a mutation in) or manipulate the expression of one or more target genes, and differentiated into, e.g., a T cell, e.g., a CD8+ T cell (e.g., a CD8+ naive T cell, central memory T cell, or effector memory T cell), a CD4+ T cell, a stem cell memory T cell, a lymphoid progenitor cell or a hematopoietic stem cell.

[0381] In some embodiments, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and / or persistence capacities, antigen- specificity, type of antigen Attorney Docket No: 046483-7473W01(04009) receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. Among the sub-types and subpopulations of T cells and / or of CD4+ and / or of CD8+ T cells are naive T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa- associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and delta / gamma T cells. In certain embodiments, any number of T cell lines available in the art, may be used.

[0382] In some embodiments, the methods include isolating immune cells from the subject, preparing, processing, culturing, and / or engineering them. In some embodiments, preparation of the engineered cells includes one or more culture and / or preparation steps. The cells for engineering as described may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject. In some embodiments, the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered. The subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and / or engineered. Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells. The samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g., transduction with viral vector), washing, and / or incubation. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.

[0383] In certain aspects, the sample from which the cells are derived or isolated is blood or a blood-derived sample or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, Attorney Docket No: 046483-7473W01(04009) stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and / or cells derived therefrom. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.

[0384] In some embodiments, the cells are derived from cell lines, e.g., T cell lines. The cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, nonhuman primate, and pig. In some embodiments, isolation of the cells includes one or more preparation and / or non-affinity-based cell separation steps. In some examples, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and / or resistance to particular components.

[0385] In some examples, cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis. The samples, in certain aspects, contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and / or platelets, and in certain aspects contains cells other than red blood cells and platelets. In some embodiments, the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments , a washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In certain embodiments, the cells are resuspended in a variety of biocompatible buffers after washing. In certain embodiments, components of a blood cell sample are removed, and the cells directly resuspended in culture media. In some embodiments, the methods include density-based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient.

[0386] In one embodiment, immune cells are obtained from the circulating blood of an individual are obtained by apheresis or leukapheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. The cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media, such as phosphate buffered saline (PBS) or wash solution lacks calcium and may lack magnesium or may lack Attorney Docket No: 046483-7473W01(04009) many if not all divalent cations, for subsequent processing steps. As those of ordinary skill in the art would readily appreciate a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca2+-free, Mg2+-free PBS, PlasmaLyte A, or another saline solution with or without buffer. In some embodiments, the undesirable components of the apheresis sample may be removed, and the cells directly resuspended in culture media.

[0387] In some embodiments, the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation is affinity- or immunoaffinity-based separation. For example, the isolation in certain aspects includes separation of cells and cell populations based on the cells' expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner. Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and / or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In certain aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population. The separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a Attorney Docket No: 046483-7473W01(04009) marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.

[0388] In certain exemplary embodiments, multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In certain exemplary embodiments, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection. Likewise, multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types.

[0389] In some embodiments, one or more of the T cell populations is enriched for or depleted of cells that are positive for (marker+) or express high levels (marker111811) of one or more particular markers, such as surface markers, or that are negative for (marker ) or express relatively low levels (marker'0") of one or more markers. For example, in certain aspects, specific subpopulations of T cells, such as cells positive or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+ T cells, are isolated by positive or negative selection techniques. In some cases, such markers are those that are absent or expressed at relatively low levels on certain populations of T cells (such as non-memory cells) but are present or expressed at relatively higher levels on certain other populations of T cells (such as memory cells). In one embodiment, the cells (such as the CD8+ cells or the T cells, e.g., CD3+ cells) are enriched for (i.e., positively selected for) cells that are positive or expressing high surface levels of CD45RO, CCR7, CD28, CD27, CD44, CD127, and / or CD62L and / or depleted of (e.g., negatively selected for) cells that are positive for or express high surface levels of CD45RA. In some embodiments, cells are enriched for or depleted of cells positive or expressing high surface levels of CD122, CD95, CD25, CD27, and / or IL7-Ra (CD127). In certain exemplary embodiments, CD8+ T cells are enriched for cells positive for CD45RO (or negative for CD45RA) and for CD62L. For example, CD3+, CD28+ T cells can be positively selected using CD3 / CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander).

[0390] In some embodiments, T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such Attorney Docket No: 046483-7473W01(04009) as CD 14. In certain aspects, a CD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into subpopulations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and / or effector T cell subpopulations. In some embodiments, CD8+ cells are further enriched for or depleted of naive, central memory, effector memory, and / or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation. In some embodiments, enrichment for central memory T (TCM) cells is carried out to increase efficacy, such as to improve longterm survival, expansion, and / or engraftment following administration, which in certain aspects is particularly robust in such sub-populations. In some embodiments, combining TCM-enriched CD8+ T cells and CD4+ T cells further enhances efficacy.

[0391] In some embodiments, memory T cells are present in both CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMC can be enriched for or depleted of CD62L-CD8+ and / or CD62L+CD8+ fractions, such as using anti-CD8 and anti-CD62L antibodies. In some embodiments, a CD4+ T cell population and / or a CD8+ T population is enriched for central memory (TCM) cells. In some embodiments, the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD 127; in certain aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and / or granzyme B. In certain aspects, isolation of a CD8+ population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD 14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one aspect, enrichment for central memory T (TCM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD 14 and CD45RA, and a positive selection based on CD62L. Such selections in certain aspects are carried out simultaneously and in other aspects are carried out sequentially, in either order. In some embodiments, the same CD4 expression-based selection step used in preparing the CD8+ cell population or subpopulation, also is used to generate the CD4+ cell population or subpopulation, such that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the methods, optionally following one or more further positive or negative selection steps. Attorney Docket No: 046483-7473W01(04009)

[0392] CD4+ T helper cells are sorted into naive, central memory, and effector cells by identifying cell populations that have cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+, CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ cells are CD62L- and CD45RO. In one example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CDl lb, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow for separation of cells for positive and / or negative selection.

[0393] In some embodiments, the cells are incubated and / or cultured prior to or in connection with genetic engineering. The incubation steps can include culture, cultivation, stimulation, activation, and / or propagation. In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and / or survival of cells in the population, to mimic antigen exposure, and / or to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor. The conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and / or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells. In some embodiments, the stimulating conditions or agents include one or more agent, e.g., ligand, which is capable of activating an intracellular signaling domain of a TCR complex. In certain aspects, the agent turns on or initiates TCR / CD3 intracellular signaling cascade in a T cell. Such agents can include antibodies, such as those specific for a TCR component and / or costimulatory receptor, e.g., anti-CD3, anti-CD28, for example, bound to solid support such as a bead, and / or one or more cytokines. Optionally, the expansion method may further comprise the step of adding anti-CD3 and / or anti CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng / ml). In some embodiments, the stimulating agents include IL -2 and / or IL-15, for example, an IL-2 concentration of at least about 10 units / mL. Attorney Docket No: 046483-7473W01(04009)

[0394] In another embodiment, T cells are isolated from peripheral blood by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient. Alternatively, T cells can be isolated from an umbilical cord. In any event, a specific subpopulation of T cells can be further isolated by positive or negative selection techniques.

[0395] The cord blood mononuclear cells so isolated can be depleted of cells expressing certain antigens, including, but not limited to, CD34, CD8, CD14, CD19, and CD56. Depletion of these cells can be accomplished using an isolated antibody, a biological sample comprising an antibody, such as ascites, an antibody bound to a physical support, and a cell bound antibody.

[0396] Enrichment of a T cell population by negative selection can be accomplished using a combination of antibodies directed to surface markers unique to the negatively selected cells. An exemplary method is cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD 14, CD20, CD1 lb, CD 16, HLA-DR, and CD8.

[0397] For isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and beads. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In a further embodiment, greater than 100 million cells / ml is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In yet another embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells / ml is used. In further embodiments, concentrations of 125 or 150 million cells / ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion.

[0398] T cells can also be frozen after the washing step, which does not require the monocyteremoval step. While not wishing to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the Attorney Docket No: 046483-7473W01(04009) art and will be useful in this context, in a non-limiting example, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or other suitable cell freezing media. The cells are then frozen to -80°C at a rate of 1°C per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20°C or in liquid nitrogen.

[0399] In one embodiment, the population of T cells is comprised within cells such as peripheral blood mononuclear cells, cord blood cells, a purified population of T cells, and a T cell line. In another embodiment, peripheral blood mononuclear cells comprise the population of T cells. In yet another embodiment, purified T cells comprise the population of T cells.

[0400] Expansion of Immune Cells

[0401] Whether prior to or after modification of cells to express a subject CAR, the cells can be activated and expanded in number using methods as described, for example, in U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Publication No. 20060121005. For example, the immune cells of the invention may be expanded by contact with a surface having attached thereto an agent that stimulates a CD3 / TCR complex associated signal and a ligand that stimulates a co-stimulatory molecule on the surface of the immune cells. In particular, immune cell populations may be stimulated by contact with an anti-CD3 antibody, or an antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For co-stimulation of an accessory molecule on the surface of the immune cells, a ligand that binds the accessory molecule is used. For example, immune cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the immune cells. Examples of an anti- CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) and these can be used in the invention, as can other methods and reagents known in the art (see, e.g., ten Berge et al., Transplant Proc. (1998) 30(8): 3975-3977; Haanen et al., J. Exp. Med. (1999) 190(9): 1319- 1328; and Garland et al., J. Immunol. Methods (1999) 227(1-2): 53-63).

[0402] Expanding the immune cells by the methods disclosed herein can be multiplied by about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, Attorney Docket No: 046483-7473W01(04009)

[0403] 300-fold, 400-fold, 500-fold, 600-fold, 700 fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000- fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, 10,000-fold, 100,000- fold, 1,000,000-fold, 10,000,000-fold, or greater, and any and all whole or partial integers therebetween. In one embodiment, the immune cells expand in the range of about 20-fold to about 50-fold.

[0404] Following culturing, the immune cells can be incubated in cell medium in a culture apparatus for a period of time or until the cells reach confluency or high cell density for optimal passage before passing the cells to another culture apparatus. The culturing apparatus can be of any culture apparatus commonly used for culturing cells in vitro. In certain exemplary embodiments, the level of confluence is 70% or greater before passing the cells to another culture apparatus. In particularly exemplary embodiments, the level of confluence is 90% or greater. A period of time can be any time suitable for the culture of cells in vitro. The immune cell medium may be replaced during the culture of the immune cells at any time. In certain exemplary embodiments, the immune cell medium is replaced about every 2 to 3 days. The immune cells are then harvested from the culture apparatus whereupon the immune cells can be used immediately or cryopreserved to be stored for use at a later time. In one embodiment, the invention includes cry opreserving the expanded immune cells. The cryopreserved immune cells are thawed prior to introducing nucleic acids into the immune cell.

[0405] In another embodiment, the method comprises isolating immune cells and expanding the immune cells. In another embodiment, the invention further comprises cry opreserving the immune cells prior to expansion. In yet another embodiment, the cryopreserved immune cells are thawed for electroporation with the RNA encoding the chimeric membrane protein.

[0406] Another procedure for ex vivo expansion cells is described in U.S. Pat. No. 5,199,942 (incorporated herein by reference). Expansion, such as described in U.S. Pat. No. 5,199,942 can be an alternative or in addition to other methods of expansion described herein. Briefly, ex vivo culture and expansion of immune cells comprises the addition to the cellular growth factors, such as those described in U.S. Pat. No. 5,199,942, or other factors, such as flt3-L, IL-1, IL-3, and c- kit ligand. In one embodiment, expanding the immune cells comprises culturing the immune cells with a factor selected from the group consisting of flt3-L, IL-1, IL-3, and c-kit ligand.

[0407] The culturing step as described herein (contact with agents as described herein or after electroporation) can be very short, for example less than 24 hours such as 1, 2, 3, 4, 5, 6, 7, 8, 9, Attorney Docket No: 046483-7473W01(04009)

[0408] 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, or 23 hours. The culturing step as described further herein (contact with agents as described herein) can be longer, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days.

[0409] Various terms are used to describe cells in culture. Cell culture refers generally to cells taken from a living organism and grown under controlled condition. A primary cell culture is a culture of cells, tissues or organs taken directly from an organism and before the first subculture. Cells are expanded in culture when they are placed in a growth medium under conditions that facilitate cell growth and / or division, resulting in a larger population of the cells. When cells are expanded in culture, the rate of cell proliferation is typically measured by the amount of time required for the cells to double in number, otherwise known as the doubling time.

[0410] Each round of subculturing is referred to as a passage. When cells are subcultured, they are referred to as having been passaged. A specific population of cells, or a cell line, is sometimes referred to or characterized by the number of times it has been passaged. For example, a cultured cell population that has been passaged ten times may be referred to as a P10 culture. The primary culture, i.e., the first culture following the isolation of cells from tissue, is designated P0. Following the first subculture, the cells are described as a secondary culture (Pl or passage 1). After the second subculture, the cells become a tertiary culture (P2 or passage 2), and so on. It will be understood by those of skill in the art that there may be many population doublings during the period of passaging. Therefore, the number of population doublings of a culture is greater than the passage number. The expansion of cells (i.e., the number of population doublings) during the period between passaging depends on many factors, including but is not limited to the seeding density, substrate, medium, and time between passaging.

[0411] In one embodiment, the cells may be cultured for several hours (about 3 hours) to about 14 days or any hourly integer value in between. Conditions appropriate for immune 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-gamma, IL-4, IL-7, GM- CSF, IL-10, IL-12, IL-15, TGF-beta, and TNF-a or any other additives for the growth of cells known to the skilled artisan. Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. Media can include RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, and X-Vivo Attorney Docket No: 046483-7473W01(04009)

[0412] 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 immune cells. Antibiotics, e.g., penicillin and streptomycin, are included only in experimental cultures, not in cultures of cells that are to be infused into a subject. The target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37° C) and atmosphere (e.g., air plus 5% CO2).

[0413] The medium used to culture the immune cells may include an agent that can co-stimulate the immune cells. For example, an agent that can stimulate CD3 is an antibody to CD3, and an agent that can stimulate CD28 is an antibody to CD28. This is because, as demonstrated by the data disclosed herein, a cell isolated by the methods disclosed herein can be expanded approximately 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100- fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, 6000-fold, 7000-fold, 8000-fold, 9000-fold, 10,000- fold, 100,000-fold, 1,000,000-fold, 10,000,000-fold, or greater. In one embodiment, the immune cells expand in the range of about 2-fold to about 50-fold, or more by culturing the electroporated population. In one embodiment, human T regulatory cells are expanded via anti- CD3 antibody coated KT64.86 artificial antigen presenting cells (aAPCs). Methods for expanding and activating immune cells can be found in U.S. Patent Numbers 7,754,482, 8,722,400, and 9,555,105, the contents of which are incorporated herein in their entirety.

[0414] In one embodiment, the method of expanding the immune cells can further comprise isolating the expanded immune cells for further applications. In another embodiment, the method of expanding can further comprise a subsequent electroporation of the expanded immune cells followed by culturing. The subsequent electroporation may include introducing a nucleic acid encoding an agent, such as a transducing the expanded immune cells, transfecting the expanded immune cells, or electroporating the expanded immune cells with a nucleic acid, into the expanded population of immune cells, wherein the agent further stimulates the immune cell. The agent may stimulate the immune cells, such as by stimulating further expansion, effector function, or another immune cell function.

[0415] EXPERIMENTAL EXAMPLES Attorney Docket No: 046483-7473W01(04009)

[0416] The invention is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only, and the invention is not limited to these Examples, but rather encompasses all variations that are evident as a result of the teachings provided herein.

[0417] Example 1 : Construction of Lentivirus Constructs and Expression of CARs

[0418] To test the killing of B cell lymphoma cells by T cells co-expressing multiple CARs from a single expression vector, the four lentivirus constructs shown in FIG. 1 were prepared, namely PM486, PM511, PM518, and PM523. Each construct co-expresses a total of five individual receptors: anti-CD19 CAR (CAR18), anti-CD20 CAR (CAR19ml), anti-CD22 CAR (CARB), anti-CD79b CAR (CAR14 or CAR14ml), and minimal Low-affinity Nerve Growth Factor Receptor (mLNGFR).

[0419] Each individual CAR enables tumor killing by binding to its respective antigen on tumor cells. mLNGFR acts as a surface tag for cell monitoring upon downregulation of CARs during tumor killing. To enable co-expression of these five transgenes, EFl alpha (EFla) and human phosphoglycerate kinase-1 (hcPGKl) promoters were designed in a back-to-back orientation to generate two independent expression cassettes. Each cassette contains multiple transgenes separated by a furin cleavage site (F) and 2A self-cleaving peptide (T2A, P2A) to enable efficient co-expression. DNA sequences of CAR, F and 2A were silently mutated to minimize DNA homologies reducing the potential for recombinations. In addition, CARs were designed to utilize binding domains comprised of single-chain variable fragments (scFv) or nanobodies to reduce the potential of cross-interactions.

[0420] All lentiviral constructs were generated via restriction cloning of synthesized DNA fragments (IDT) and genes (Genscript) into pTRPE, a third-generation self-inactivating (SIN) lentiviral vector (pTRPE; PMID: 27332733) containing a 5’ Long-Terminal Repeat sequence (SIN 5’LTR), psi sequence for viral packaging (v| / ), viral Rev Response Element (RRE), central PolyPurine Tract (cPPT), Woodchuck hepatitis virus Post-transcriptional Regulatory Element (WPRE) and 3’ Long-Terminal Repeat sequence (SIN 3’LTR). Final constructs were validated by Sanger sequencing (Genewiz) and whole-plasmid sequencing (Plasmidsaurus).

[0421] Example 2: Killing of B Cell Lymphoma Cells by PM486 T Cells. Attorney Docket No: 046483-7473W01(04009)

[0422] To examine tumor cell killing by T cells transduced with the PM486 lentivirus construct depicted in FIG. 1, B cell lymphoma cells (Ramos) were co-cultured with PM486 T cells in vitro.

[0423] FIG. 2 is a bar graph showing the % killing of single-antigen positive (CD19+, CD20+, CD22+, CD79b+), quadruple-positive (4+) or quadruple-knockout (4ko) Ramos tumor cells by the PM486 T cells or untransduced (UTD) T cells after 66h of co-culture. FIG. 2 shows nearly 100% killing of each single-antigen positive and quadruple positive tumor cells validating that PM486 T cells are able to kill with each of their four CARs alone and in combination. No killing of the quadruple-knockout (4ko) tumor cells was observed, nor were any of the tumor cells killed by the untransduced (UTD) negative control T cells.

[0424] Example 3: Killing of B Cell Lymphoma Cells by PM486 T Cells Compared to CTL019 T Cells in vitro.

[0425] To compare tumor cell killing between PM486 T cells and conventional anti-CD19 CAR T cells (CTL019) in vitro, B cell lymphoma cells (Ramos) were co-cultured with PM486 T cells or CTL019 T cells.

[0426] FIG. 3 shows the killing of Ramos tumor cells by PM486 T cells compared to CTL019 T cells and untransduced (UTD) T cells and the staining of tumor cells for CD19 / CD20 / CD22 / CD79b expression.

[0427] FIG. 3 A is a bar graph showing the % killing of single-antigen positive (CD19+, CD20+, CD22+, CD79b+), quadruple-positive (4+), quadruple-knockout (4ko) or antigen-heterogeneous (Mix; 25% of each CD19+ / CD20+ / CD22+ / CD79b+) Ramos tumor cells by PM486 T cells, CTL019 T cells or untransduced (UTD) T cells after 48h of co-culture. The results in FIG. 3A show that PM486 T cells were highly effective at killing all four single-antigen positive (CD19+, CD20+, CD22+, CD79b+) and the 4+ Ramos tumor cells. In contrast, CTL019 T cells were only effective at killing CD19+ and 4+ Ramos tumor cells. Moreover, PM486 T cells were highly effective in killing the antigen-heterogeneous mix cells (99%) while CTL019 T cells only showed a weak tumor cell killing (44%). In addition, PM486 T cells showed low killing (22%) of 4ko Ramos tumor cells, while the CTL019 T cells showed no tumor cell killing beyond background levels. The negative control UTD T cells were ineffective in killing any of the tumor cells. Attorney Docket No: 046483-7473W01(04009)

[0428] FIG. 3B is a flow cytometry (FCM) analysis of residual tumors from the Mix condition in FIG. 3 A (antigen-heterogeneous) that were treated with PM486 T cells, CTL019 T cells, or UTD T cells and stained for CD19 / CD20 / CD22 / CD79b antigen expression. The results in FIG. 3B reveal that CTL019 T cells are only able to eliminate the CD 19+ fraction within the heterogeneous tumor leading to antigen escape. In contrast, PM486 T cells target and kill all four tumor fractions (CD19+ / CD20+ / CD22+ / CD79b+), thereby preventing antigen escape.

[0429] Example 4: Killing of B Cell Lymphoma Cells by PM486 T Cells Compared to CTL019 T Cells in vivo.

[0430] To compare tumor killing between PM486 T cells and conventional anti-CD19 CAR T cells (CTL019) in vivo, NSG mice were engrafted with B cell lymphoma cells (Ramos) and treated with PM486 T cells or CTL019 T cells.

[0431] FIG. 4 shows the killing of Ramos tumor cells by PM486 T cells compared to CTL019 T cells and untransduced (UTD) T cells and the corresponding overall survival. NSG mice were engrafted with 0.5E6 quadruple-positive (4+) Ramos tumor cells expressing the click beetle green luciferase reporter by i.v. injection. After 4 days, mice were treated with 2E6 CTL019 T cells, PM486 T cells or UTD T cells by i.v. injection.

[0432] FIG. 4A is a graph showing the killing of Ramos tumor cells by PM486 T cells compared to CTL019 T cells and UTD T cells. To quantify the degree of tumor cell killing, bioluminescent measurements were performed after i.p. injection of D-Luciferin using an IVIS spectrum imager. As shown in FIG. 4A, CTL019 T cells and PM486 T cells were able to control tumor growth compared to UTD cells. Past day 12, CTL019 T cells appeared more effective than PM486 T cells at killing 4+ Ramos cells.

[0433] FIG. 4B is a graph showing the overall survival of mice from FIG. 4A. Treatment with CTL019 T cells and PM486 T cells prolonged survival past 32 days. In contrast, mice treated with UTD T cells reached their endpoint by day 17.

[0434] Example 5 : Killing of B Cell Lymphoma Cells by PM511 T cells and PM518 T cells.

[0435] To compare tumor cell killing between T cells transduced with the PM511 and PM518 lentivirus constructs depicted in FIG. 1, B cell lymphoma cells (Ramos) were co-cultured with PM511 T cells and PM518 T cells in vitro. Attorney Docket No: 046483-7473W01(04009)

[0436] FIG. 5 is a bar graph showing the % killing of single-antigen positive (CD19+, CD20+, CD22+, CD79b+), quadruple-positive (4+) and quadruple-knockout (4ko) Ramos tumor cells by PM511 T cells, PM518 T cells or untransduced (UTD) T cells after 44.5h of co-culture.

[0437] The results in FIG. 5 reveal different levels of killing of the single-antigen positive Ramos tumor cells depending on the expression levels of the corresponding CAR in PM511 T cells and PM518 T cells. As depicted in FIG. 1, PM518 T cells express the anti-CD19 CAR (CAR18) and the anti-CD20 CAR (C ARI 9ml) under the strong EFl alpha (EFla) promoter leading to enhanced killing of the corresponding tumor cells (CD 19+ and CD20+) compared to PM511 where the weaker human phosphoglycerate kinase-1 (hcPGKl) promoter is expressing the same CARs. Similarly, the anti-CD79b CAR (CAR14ml) expressed under EF 1 a in PM511 T cells shows enhanced killing of CD79b+ tumor cells compared to PM518 where its expression is under hcPGKl. Killing of 4+ and 4ko Ramos tumor cells was similar between PM511 T cells and PM518 T cells. The negative control UTD T cells were ineffective in killing any of the tumor cells.

[0438] Example 6: CAR Receptor Expression by Different Penta T Cell Variants

[0439] A series of lentiviral vectors were designed, each expressing the same CARs but in different positions within the lentiviral vector to provide for an evaluation of distinct CAR expression profiles.

[0440] FIG. 6A is a schematic diagram showing the ability of Penta T cells to simultaneously target multiple distinct tumor cell populations. FIG. 6B is a table summarizing the functional design of each receptor. FIG. 6C illustrates receptor expression profiles under different promoters using the lentiviral vectors PM 11, PM518 and PM523 (depicted in FIG. 1). Receptors driven by the strong EFl alpha (EFla) promoter exhibit high expression levels, enhancing functional activity, whereas those under the weaker human phosphoglycerate kinase-1 (hcPGKl) promoter display lower expression, reducing efficacy. FIG. 6D is a flow cytometry (FCM) analysis of Penta T cells (top) and untransduced (UTD) T cells (bottom) that were stained for CAR18 targeting CD 19.

[0441] The results in FIGs. 6C-6D demonstrate that the different Penta T cell variants can be generated efficiently, with transduction efficiencies ranging between 16.5%-26.8%. Moreover, expression of the CD19-targeting CAR18 (FIG. 6C, red) under the strong EFla promoter in Attorney Docket No: 046483-7473W01(04009)

[0442] PM518 results in an increased mean fluorescence intensity (MFI), as evidenced by the upwards shift of the corresponding CAR+ population (FIG. 6D, top).

[0443] Example 7: Killing of B Lymphoma Cells by PM511 T cells, PM518 T cells and PM523 T cells.

[0444] To compare tumor cell killing between T cells transduced with the lentiviral Penta T constructs PM511, PM518 and PM523 depicted in FIGs. 1 and 6C, B cell lymphoma cells (Ramos) were co-cultured with PM511 T cells, PM518 T cells and PM523 T cells in vitro.

[0445] FIG. 7A is a bar graph showing the % killing of single-antigen positive (CD19+, CD20+, CD22+, CD79b+) and quadruple-positive (4+) Ramos tumor cells by PM511 T cells, PM518 T cells, PM523 T cells or untransduced (UTD) T cells after 50.5h of co-culture with an effector-to- target (ET) ratio of 1 : 1. FIG. 7B summarizes the CARs with low tumor-killing activity in each Penta T cell variant.

[0446] The results in FIG. 7A reveal different levels of tumor lysis corresponding to the position and expression level of each CAR in PM511 T cells, PM518 T cells and PM523 T cells. As illustrated in FIGs. 1 and 6C, PM511 / 518 / 523 T cells express the same CARs but in different positions within the lentiviral vector, resulting in distinct expression profdes. Among them, PM523 T cells (red) exhibited the highest potency, efficiently eliminating all the different Ramos cell lines. PM523 T cells were able to eliminate tumor cells using each receptor alone (singleantigen positive CD19+, CD20+, CD22+, CD79b+ Ramos) or all combined (4+Ramos). The negative control UTD T cells were ineffective in killing any of the tumor cells.

[0447] Example 8: Killing of B Cell Lymphoma Cells by PM523 T Cells Compared to PM5Q4 T Cells in vitro.

[0448] To compare tumor cell killing between PM523 T cells and conventional anti-CD19 CAR T cells (PM504) in vitro, B cell lymphoma cells (Ramos) were co-cultured with PM523 T cells or PM504 T cells.

[0449] FIGs. 8A-8B show the killing of B cell lymphoma cells (Ramos) in vitro by PM523 T cells compared to PM504 T cells and untransduced (UTD) T cells at different effector-to-target (ET) ratios. PM504 T cells comprise a single-CAR control vector expressing only the CD19- targeted CAR18 for comparison with Penta Ts having CAR18 and the other CARs as noted Attorney Docket No: 046483-7473W01(04009) herein. PM504 expresses CAR18 followed by a furin cleavage site, a P2A self-cleaving peptide domain, and a blue-fluorescent reporter protein (mTag-BFP2).

[0450] FIGs. 8A-8B are bar graphs showing the % killing of single-antigen positive (CD19+, CD20+, CD22+, CD79b+) or quadruple-positive (4+) Ramos tumor cells by PM523 T cells, PM504 T cells or UTD T cells after 3 days of co-culture at E:T ratios of 0.5: 1 (Med ET Ratio) and 0.25: 1 (Low ET Ratio), respectively.

[0451] The results in FIGs. 8A-8B demonstrate that PM523 T cells consistently outperform PM504 T cells across all tumor cell lines, including both CD 19+ Ramos and 4+ Ramos cells, at medium and low E:T ratios. The negative control UTD T cells were ineffective in killing any of the tumor cells.

[0452] Example 9: Killing of Antigen-Heterogeneous B Cell Lymphoma Cells by PM523 T Cells Compared to PM504 T Cells in vitro.

[0453] To compare the killing of antigen-heterogeneous tumors between PM523 T cells and conventional anti-CD19 CAR T cells (PM504) in vitro, B cell lymphoma cells (Ramos) were cocultured with PM523 T cells or PM504 T cells.

[0454] FIG. 9A shows the killing of B cell lymphoma cells (Ramos) in vitro by PM523 T cells compared to PM504 T cells expressing only the CAR18 targeting CD19 and untransduced (UTD) T cells and the staining of tumor cells for CD19 / CD20 / CD22 / CD79b expression. FIG. 9A is a bar graph showing the % killing of antigen-heterogeneous (25% of each CD19+ / CD20+ / CD22+ / CD79b+) Ramos tumor cells by PM523 T cells, PM504 T cells or UTD T cells over time at an effector-to-target (E:T) ratio of 1 : 1. FIG. 9B is a flow cytometry (FCM) analysis of residual tumors from FIG. 9A that were stained for CD19 / CD20 / CD22 / CD79b antigen expression.

[0455] The results in FIG. 9A show that PM523 T cells completely eliminated the antigen- heterogeneous mix cells (100%) while PM504 T cells only showed a weak tumor lysis (46%) followed by tumor relapse after 164h. The results in FIG. 9B reveal that PM504 T cells are only able to eliminate the CD 19+ fraction within the heterogeneous tumor leading to antigen escape. In contrast, PM523 T cells target and kill all four tumor fractions (CD19+ / CD20+ / CD22+ / CD79b+), thereby preventing antigen escape. The negative control UTD T cells were ineffective in killing any of the tumor cells. Attorney Docket No: 046483-7473W01(04009)

[0456] Example 10: Killing of Different B-Cell Cancers by PM523 T Cells Compared to PM504 T Cells in vitro.

[0457] To compare the killing of different B-cell cancers between PM523 Penta T cells and conventional anti-CD19 CAR T cells (PM504) in vitro, PM523 T cells or PM504 T cells were co-cultured with Burkitt lymphoma (BL), chronic lymphocytic leukemia (CLL), B-cell acute lymphoblastic leukemia (B-ALL), and diffuse large B-cell lymphoma (DLBCL) cancer cells.

[0458] FIGs. 10A-10B show the killing of BL (Ramos), CLL (OSU), B-ALL (Nalm6), and DLBCL (Farage) cancer cells in vitro by PM523 T cells compared to PM504 T cells expressing only the CAR18 targeting CD 19 and untransduced (UTD) T cells. FIG. 10A and FIG. 10B are bar graphs showing the % killing of BL, CLL, B-ALL and DLBCL tumor cells by PM523 T cells, PM504 T cells or UTD T cells after 50h using an effector-to-target (E:T) ratio 1 : 1 (High ET Ratio) or 0.25: 1 / 0.5:1 (Low ET Ratio), respectively.

[0459] The results in FIG. 10A show that at high E:T ratios, both PM523 T cells and PM504 T cells are highly effective at killing BL, CLL and B-ALL tumor cells. However, for DLBCL, only PM523 T cells achieve a high level of tumor lysis (82%) in comparison to PM504 T cells (21%). At low E:T ratios, PM523 T cells consistently outperform PM504 T cells across all tested cancer types (BL: 72% vs. 18%, CLL: 81% vs. 49%, B-ALL: 91% vs. 62%, DLBCL: 55% vs. 3%). The negative-control UTD T cells were ineffective in killing any of the tumor cells.

[0460] Enumerated Embodiments

[0461] The following enumerated embodiments are provided, the numbering of which is not to be construed as designating levels of importance.

[0462] Embodiment 1 provides a nucleic acid comprising two or more transgenes, wherein each transgene is operably linked to a first or a second regulatory nucleic acid sequence; wherein the first regulatory nucleic acid sequence comprises a first promoter directing transcription of one or more transgenes from a plus-strand of DNA; and wherein the second regulatory nucleic acid sequence comprises a second promoter directing transcription of one or more transgenes from a plus-strand of DNA.

[0463] Embodiment 2 provides the nucleic acid of embodiment 1, wherein each of the two or more transgenes encodes a chimeric antigen receptor (CAR), wherein the CAR expressed from Attorney Docket No: 046483-7473W01(04009) one transgene comprises an antigen binding specificity that is different than the antigen binding specificity of the CAR expressed from the other transgene(s), and wherein each CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain.

[0464] Embodiment 3 provides the nucleic acid of embodiment 1 or 2, wherein the nucleic acid further comprises: a first transgene comprising a first polynucleotide sequence encoding a first CAR; a second transgene comprising a second polynucleotide sequence encoding a second CAR; and a third transgene comprising a polynucleotide sequence encoding a third CAR.

[0465] Embodiment 4 provides the nucleic acid of embodiment 3, wherein the nucleic acid further comprises: a fourth transgene comprising a fourth polynucleotide encoding a fourth CAR.

[0466] Embodiment 5 provides the nucleic acid of any one of embodiments 1-4, wherein the nucleic acid further comprises: a fifth transgene comprising a fifth polynucleotide encoding a marker protein.

[0467] Embodiment 6 provides the nucleic acid of any one of embodiments 1-4, wherein the nucleic acid further comprises: a long terminal repeat (LTR) comprising a U3 deletion.

[0468] Embodiment 7 provides the nucleic acid of embodiment 1, wherein the nucleic acid further comprises: a first transgene comprising a first polynucleotide sequence encoding a first chimeric antigen receptor (CAR); a second transgene comprising a second polynucleotide sequence encoding a second CAR; a third transgene comprising a third polynucleotide sequence encoding a third CAR; a fourth transgene comprising a fourth polynucleotide sequence encoding a fourth CAR; a fifth transgene comprising a fifth polynucleotide sequence encoding a marker protein; a long terminal repeat (LTR) comprising a U3 deletion, wherein the first promoter directs transcription of one or more of the transgenes from a plus-strand of DNA; and the second promoter directs transcription of one or more of the transgenes from a minus-strand of DNA; wherein each CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain, and wherein each CAR comprises a different antigen binding specificity for a different antigen.

[0469] Embodiment 8 provides the nucleic acid of any one of embodiments 1-7, wherein the nucleic acid is about 15 kilobases or less in length.

[0470] Embodiment 9 provides the nucleic acid of any one of embodiments 6-8, wherein the LTR comprises the nucleotide sequence of SEQ ID NO: 76. Attorney Docket No: 046483-7473W01(04009)

[0471] Embodiment 10 provides the nucleic acid of any one of embodiments 2-9, wherein each CAR comprises an antigen binding domain selected from the group consisting of a Fab, singlechain variable fragment (scFv), and nanobody.

[0472] Embodiment 11 provides the nucleic acid of embodiment 10, wherein each CAR comprises an antigen binding domain that specifically binds a different B cell protein.

[0473] Embodiment 12 provides the nucleic acid of embodiment 11, wherein the B cell protein is selected from the group consisting of CD 10, CD 19, CD20, CD22, CD79b, CD34, CD52, CD123, FLT-3, ROR1, CD179b, and CD79a.

[0474] Embodiment 13 provides the nucleic acid of embodiment 12, wherein each CAR comprises a different antigen binding domain specifically binding each of CD 19, CD20, CD22, and CD79b.

[0475] Embodiment 14 provides the nucleic acid of embodiment 13, wherein each antigen binding domain comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 16, 18, 20, 22, 24, and 26, or wherein each antigen binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 19, 21, 23, 25, and 27.

[0476] Embodiment 15 provides the nucleic acid of any one of embodiments 2-14, wherein each CAR comprises a transmembrane domain from a protein selected from the group consisting of alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.

[0477] Embodiment 16 provides the nucleic acid of embodiment 15, wherein each CAR comprises a different transmembrane domain selected from the group consisting of CD8, CD28, ICOS, and 0X40.

[0478] Embodiment 17 provides the nucleic acid of embodiment 16, wherein each transmembrane domain is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 39, 41, 43, and 45 or wherein each transmembrane domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 40, 42, 44, and 46.

[0479] Embodiment 18 provides the nucleic acid of any one of embodiments 2-17, wherein each intracellular domain comprises a costimulatory domain and an intracellular signaling domain.

[0480] Embodiment 19 provides the nucleic acid of embodiment 18, wherein each costimulatory domain is from a protein selected from the group consisting of CD28, 4-1BB (CD137), ICOS (CD278), 0X40, CD5, CD27, LFA-1 (CD 11 a / CD 18), ICAM-1, GITR, BAFFR, HVEM Attorney Docket No: 046483-7473W01(04009)

[0481] (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD 160, CD 19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD103, ITGAL, ITGAM, CD1 lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, BTLA, an MHC class I molecule, and a ligand that specifically binds with CD8.

[0482] Embodiment 20 provides the nucleic acid of embodiment 18, wherein each costimulatory domain is from a protein selected from the group consisting of CD28, 4- IBB, ICOS, and 0X40.

[0483] Embodiment 21 provides the nucleic acid of embodiment 20, wherein each costimulatory domain is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 47, 49, 51, and 53.

[0484] Embodiment 22 provides the nucleic acid of embodiment 20 or 21, wherein each costimulatory domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 48, 50, 52, and 54.

[0485] Embodiment 23. The nucleic acid of any one of claims 18-22, wherein the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif.

[0486] Embodiment 24 provides the nucleic acid of embodiment 23, wherein each intracellular signaling domain is from a protein selected from the group consisting of CD3 zeta, common FcR gamma, FcyRIII Fc gamma Rlla, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD66d, DAP 10, and DAP 12.

[0487] Embodiment 25 provides the nucleic acid of embodiment 24, wherein each intracellular signaling domain is from CD3 zeta.

[0488] Embodiment 26 provides the nucleic acid of embodiment 25, wherein each intracellular signaling domain from CD3 zeta is encoded by a different codon-altered polynucleotide sharing no significant homology to one another.

[0489] Embodiment 27 provides the nucleic acid of embodiment 25 or 26, wherein each intracellular signaling domain from CD3 zeta is encoded by a different nucleotide sequence selected from the group consisting of SEQ ID NOs: 55-58. Attorney Docket No: 046483-7473W01(04009)

[0490] Embodiment 28. The nucleic acid of any one of claims 25-27, wherein each intracellular signaling domain from CD3 zeta comprises the amino acid sequence of SEQ ID NO: 60.

[0491] Embodiment 29. The nucleic acid of any one of claims 2-28, wherein each antigenbinding domain is connected to the transmembrane domain by a hinge region.

[0492] Embodiment 30 provides the nucleic acid of embodiment 29, wherein the hinge region is from a protein selected from the group consisting of an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial hinge domain, a hinge comprising an amino acid sequence of CD8, or any combination thereof.

[0493] Embodiment 31 provides the nucleic acid of embodiment 29 or 30, wherein the hinge region is from CD 8 or CD28.

[0494] Embodiment 32 provides the nucleic acid of embodiment 31, wherein the hinge region is from CD8 and is encoded by a different codon-altered polynucleotide sharing no significant homology to one another.

[0495] Embodiment 33 provides the nucleic acid of embodiment 32, wherein the hinge region from CD8 is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 35-37 and the amino acid sequence of SEQ ID NO: 38.

[0496] Embodiment 34 provides the nucleic acid of embodiment 31, wherein the hinge region from CD28 comprises the nucleotide sequence of SEQ ID NO: 33 and / or the amino acid sequence of SEQ ID NO: 34.

[0497] Embodiment 35. The nucleic acid of any one of claims 2-34, wherein each CAR comprises a leader sequence encoding an N-terminal signal peptide.

[0498] Embodiment 36 provides the nucleic acid of embodiment 35, wherein each CAR-encoded polynucleotide comprises a different CD8a signal peptide encoded by a different codon-altered polynucleotide sharing no significant homology to one another.

[0499] Embodiment 37 provides the nucleic acid of embodiment 36, wherein each CD8a signal peptide is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 28-31 and comprises the amino acid sequence of SEQ ID NO: 32.

[0500] Embodiment 38 provides the nucleic acid of any one of embodiments 4-37, wherein: the first polynucleotide encodes a first CAR comprising a CD 19 antigen binding domain, a CD28 hinge domain, a CD28 transmembrane domain, aa CD28 costimulatory domain, and a CD3 zeta intracellular signaling domain; the second polynucleotide encodes a second CAR comprising a Attorney Docket No: 046483-7473W01(04009)

[0501] CD20 antigen binding domain, CD20 antigen binding domain, a CD8 hinge domain, a CD8 transmembrane domain, a 4- IBB costimulatory domain, and a CD3 zeta intracellular signaling domain; the third polynucleotide encodes a third CAR comprising a CD22 antigen binding domain, a CD8 hinge domain, an ICOS transmembrane domain, ICOS costimulatory domain, and a CD3 zeta intracellular signaling domain; and the fourth polynucleotide encodes a fourth CAR comprising a CD79b binding domain, a CD8 hinge domain, an 0X40 or CD8 transmembrane domain, an 0X40 costimulatory domain, and a CD3 zeta intracellular signaling domain.

[0502] Embodiment 39 provides the nucleic acid of embodiment 38, wherein each CAR is encoded by a different nucleotide sequence selected from the group consisting of SEQ ID NOs:

[0503] 6, 8, 10, and 12.

[0504] Embodiment 40 provides the nucleic acid of embodiment 38 or 39, wherein each CAR is encoded by a different amino acid sequence selected from the group consisting of SEQ ID NOs:

[0505] 7, 9, 11, and 13.

[0506] Embodiment 41. The nucleic acid of any one of claims 4 or 38-40, wherein the nucleic acid further comprises a fifth transgene encoding a mutant low affinity nerve growth factor (mLNGFR) marker protein.

[0507] Embodiment 42. The nucleic acid of any one of claims 38-41, wherein the nucleic acid comprises, from 5 ’-3’, the fourth, first, second, and third polynucleotides.

[0508] Embodiment 43. The nucleic acid of any one of claims 38-41, wherein the nucleic acid comprises, from 5 ’-3’, the second, first, fourth, and third polynucleotides.

[0509] Embodiment 44. The nucleic acid of any one of claims 38-41, wherein the nucleic acid comprises, from 5 ’-3’, the fourth first second, and third polynucleotides.

[0510] Embodiment 45. The nucleic acid of any one of claims 38-41, wherein the nucleic acid comprises, from 5 ’-3’, the first, fourth, second, and third polynucleotides.

[0511] Embodiment 46. The nucleic acid of any one of claims 2-45, wherein one of the first and second promoters is an EF-1 alpha promoter and the other of the first and second promoters is human PGK1 promoter.

[0512] Embodiment 47 provides the nucleic acid of embodiment 46, wherein the first promoter is an EF-1 alpha promoter, and the second promoter is a human PGK-1 promoter. Attorney Docket No: 046483-7473W01(04009)

[0513] Embodiment 48 provides the nucleic acid of embodiment 46 or 47, wherein the EF-1 alpha promoter comprises the nucleotide sequence of SEQ ID NO: 74 and the PGK-1 promoter comprises the nucleotide sequence of SEQ ID NO: 72.

[0514] Embodiment 49. The nucleic acid of any one of claims 46-48, wherein the EF-1 alpha promoter drives expression of, from N-terminus to C-terminus, an anti-CD19 CAR, an anti- CD20 CAR, and an anti-CD22 CAR.

[0515] Embodiment 50 provides the nucleic acid of embodiment 49, wherein the anti-CD19 CAR, the anti-CD20 CAR, and the anti-CD22 CAR are encoded by the nucleotide sequence of SEQ ID NO: 2 and / or the nucleotide sequence encodes the amino acid sequence of SEQ ID NO: 3.

[0516] Embodiment 51. The nucleic acid of any one of claims 46-50, wherein the hPGK-1 promoter drives expression of, from N-terminus to C-terminus, an anti-CD79b CAR and a mLNGFR marker protein.

[0517] Embodiment 52 provides the nucleic acid of embodiment 51, wherein the CD79b CAR and a mLNGFR marker protein are encoded by the nucleotide sequence of SEQ ID NO: 4 or 113 and / or the nucleotide sequence encodes the amino acid sequence of SEQ ID NO: 5 or 114.

[0518] Embodiment 53. The nucleic acid of any one of claims 46-48, wherein the EF-1 alpha promoter drives expression of, from N-terminus to C-terminus, an anti-CD79b CAR, an anti- CD20 CAR, and an anti-CD22 CAR.

[0519] Embodiment 54 provides the nucleic acid of embodiment 49, wherein the anti-CD79b CAR, an anti-CD20 CAR, and anti-CD22 CAR are encoded by the nucleotide sequence of SEQ ID NO: 115 and / or the nucleotide sequence encodes the amino acid sequence of SEQ ID NO: 116.

[0520] Embodiment 55 provides the nucleic acid of embodiment 53 or 54, wherein the hPGK-1 promoter drives expression of, from N-terminus to C-terminus, a CD 19 CAR and a mLNGFR marker protein.

[0521] Embodiment 56 provides the nucleic acid of embodiment 55, wherein the CD 19 CAR and a mLNGFR marker protein are encoded by the nucleotide sequence of SEQ ID NO: 117 and / or the nucleotide sequence encodes the amino acid sequence of SEQ ID NO: 118.

[0522] Embodiment 57. The nucleic acid of any one of claims 1-56, wherein the nucleic acid encodes a plurality of self-cleaving 2A peptide domains. Attorney Docket No: 046483-7473W01(04009)

[0523] Embodiment 58 provides the nucleic acid of embodiment 57, wherein the nucleic acid encodes three 2A self-cleaving 2A peptide domains.

[0524] Embodiment 59 provides the nucleic acid of embodiment 58, wherein the self-cleaving peptide domains are selected from the group consisting of T2A, P2A, E2A and F2A.

[0525] Embodiment 60 provides the nucleic acid of any one of embodiments 57-59, wherein the self-cleaving peptide domains are encoded by nucleotide sequences selected from the group consisting of SEQ ID NOs: 61, 62, and 64 and / or amino acid sequences selected from the group consisting of SEQ ID NOs: 63 or 65-67.

[0526] Embodiment 61 provides the nucleic acid of any one of embodiments 57-60, wherein the nucleic acid further encodes a furin cleavage site between the intracellular signaling domain and a self-cleaving 2A peptide domain in each CAR.

[0527] Embodiment 62 provides the nucleic acid of any one of embodiments 1-61, wherein the nucleic acid further comprises a Woodchuck Hepatitis virus post-transcriptional regulatory element (WPRE).

[0528] Embodiment 63 provides an expression vector comprising the nucleic acid of any one of embodiments 1-62.

[0529] Embodiment 64 provides the expression vector of embodiment 63, wherein the expression vector is a lentiviral vector.

[0530] Embodiment 65 provides the expression vector of embodiment 64, wherein the lentiviral vector is a self-inactivating HIV vector.

[0531] Embodiment 66 provides the expression vector of embodiment 65, wherein the HIV vector comprises a partial gag sequence, optionally wherein the partial gag sequence comprises the nucleotide sequence of SEQ ID NO: 69.

[0532] Embodiment 67 provides the expression vector of embodiment 65 or 66, wherein the HIV vector further comprises a central polypurine tract (cPPT), optionally wherein the cPPT comprises the nucleotide sequence of SEQ ID NO: 70.

[0533] Embodiment 68 provides the expression vector any one of embodiments 63-67, comprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 107, SEQ ID NO: 108, or SEQ ID NO: 109.

[0534] Embodiment 69 provides a cell comprising the nucleic acid of any one of embodiments 1-62 or the expression construct of any one of embodiments 63-68. Attorney Docket No: 046483-7473W01(04009)

[0535] Embodiment 70 provides the cell of embodiment 69, wherein the cell is an immune cell.

[0536] Embodiment 71 provides the cell of embodiment 70, wherein the immune cell is a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), or a regulatory T cell (Treg).

[0537] Embodiment 72 provides the cell of embodiment 69 or 71, wherein the immune cell is an autologous cell.

[0538] Embodiment 73 provides the cell of any one of embodiments 69-72, wherein the cell expresses a first CAR comprising a CD19 antigen binding domain, a second CAR comprising a CD20 antigen binding domain, a third CAR comprising a CD22 antigen binding domain, a fourth CAR comprising a CD79b antigen binding domain.

[0539] Embodiment 74 provides the cell of any one of embodiments 69-73, wherein the five transgenes are integrated into the host genome in the form of a single expression cassette.

[0540] Embodiment 75 provides a pharmaceutical composition comprising a therapeutically effective amount of the cell of any one of embodiments 69-74.

[0541] Embodiment 76 provides a method of treating a disease or disorder in a subject, comprising administering the pharmaceutical composition of embodiment 75 to a subject in need thereof.

[0542] Embodiment 77 provides the method of embodiment 76, wherein the disease is associated with CD 19 expression and is a proliferative disease such as a cancer, tumor, or malignancy; or a precancerous condition such as a myelodysplasia; a myelodysplastic syndrome or a preleukemia; or is a non-cancer related indication associated with expression of CD 19.

[0543] Embodiment 78 provides the method of embodiment 76 or 77, wherein the disease is cancer.

[0544] Embodiment 79 provides the method of embodiment 78, wherein the cancer is a hematological malignancy.

[0545] Embodiment 80 provides the method of embodiment 79, wherein the hematological malignancy is acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia, myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, B-cell acute lymphoid leukemia (BALL), T- cell acute lymphoid leukemia (TALL), small lymphocytic leukemia (SLL), acute lymphoid leukemia (ALL), chronic leukemia, chronic myelocytic leukemia, chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), polycythemia vera, lymphoma, Attorney Docket No: 046483-7473W01(04009)

[0546] Hodgkin's disease, non-Hodgkin lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia or a CD 19-negative relapsed cancer.

[0547] Embodiment 81 provides the method of any one of embodiments 78-80, wherein the subject has a CD 19-positive cancer,

[0548] Embodiment 82 provides the method of any one of embodiments 78-80 wherein the subject has a CD 19-negative cancer and is positive for one or more of CD 10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b, CD179b, or CD79a.

[0549] Embodiment 83 provides a method for generating the cell of any one of embodiments 69- 74, comprising: introducing into the cell the nucleic acid of nucleic acid of any one of claims 1- 61 or the expression construct of any one of claims 62-67.

[0550] Embodiment 84 provides the method of embodiment 83, wherein the cell is cultured in the presence of IL-2, IL-7, IL- 15, IL-21, or a combination thereof.

[0551] Other Embodiments

[0552] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0553] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this disclosure has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this disclosure may be devised by others skilled in the art without departing from the true spirit and scope of the disclosure. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

Attorney Docket No: 046483-7473W01(04009)CLAIMSWhat is claimed:

1. A nucleic acid comprising two or more transgenes, wherein each transgene is operably linked to a first or a second regulatory nucleic acid sequence; wherein the first regulatory nucleic acid sequence comprises a first promoter directing transcription of one or more transgenes from a plus-strand of DNA; and wherein the second regulatory nucleic acid sequence comprises a second promoter directing transcription of one or more transgenes from a plus-strand of DNA.

2. The nucleic acid of claim 1, wherein each of the two or more transgenes encodes a chimeric antigen receptor (CAR), wherein the CAR expressed from one transgene comprises an antigen binding specificity that is different than the antigen binding specificity of the CAR expressed from the other transgene(s), and wherein each CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain.

3. The nucleic acid of claim 1 or 2, wherein the nucleic acid further comprises: a first transgene comprising a first polynucleotide sequence encoding a first CAR; a second transgene comprising a second polynucleotide sequence encoding a second CAR; and a third transgene comprising a polynucleotide sequence encoding a third CAR.

4. The nucleic acid of claim 3, wherein the nucleic acid further comprises: a fourth transgene comprising a fourth polynucleotide encoding a fourth CAR.

5. The nucleic acid of any one of claims 1-4, wherein the nucleic acid further comprises: a fifth transgene comprising a fifth polynucleotide encoding a marker protein.

6. The nucleic acid of any one of claims 1-4, wherein the nucleic acid further comprises: a long terminal repeat (LTR) comprising a U3 deletion.Attorney Docket No: 046483-7473W01(04009)7. The nucleic acid of claim 1, wherein the nucleic acid further comprises: a first transgene comprising a first polynucleotide sequence encoding a first chimeric antigen receptor (CAR); a second transgene comprising a second polynucleotide sequence encoding a second CAR; a third transgene comprising a third polynucleotide sequence encoding a third CAR; a fourth transgene comprising a fourth polynucleotide sequence encoding a fourth CAR; a fifth transgene comprising a fifth polynucleotide sequence encoding a marker protein; a long terminal repeat (LTR) comprising a U3 deletion, wherein the first promoter directs transcription of one or more of the transgenes from a plus-strand of DNA; and the second promoter directs transcription of one or more of the transgenes from a minus-strand of DNA; wherein each CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain, and wherein each CAR comprises a different antigen binding specificity for a different antigen.

8. The nucleic acid of any one of claims 1-7, wherein the nucleic acid is about 15 kilobases or less in length.

9. The nucleic acid of any one of claims 6-8, wherein the LTR comprises the nucleotide sequence of SEQ ID NO: 76.

10. The nucleic acid of any one of claims 2-9, wherein each CAR comprises an antigen binding domain selected from the group consisting of a Fab, single-chain variable fragment (scFv), and a nanobody.

11. The nucleic acid of claim 10, wherein each CAR comprises an antigen binding domain that specifically binds a different B cell protein.Attorney Docket No: 046483-7473W01(04009)12. The nucleic acid of claim 1 1, wherein the B cell protein is selected from the group consisting of CD10, CD19, CD20, CD22, CD79b, CD34, CD52, CD123, FLT-3, R0R1, CD179b, and CD79a.

13. The nucleic acid of claim 12, wherein each CAR comprises a different antigen binding domain specifically binding each of CD 19, CD20, CD22, and CD79b.

14. The nucleic acid of claim 13, wherein each antigen binding domain comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 16, 18, 20, 22, 24, and 26, or wherein each antigen binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 19, 21, 23, 25, and 27.

15. The nucleic acid of any one of claims 2-14, wherein each CAR comprises a transmembrane domain from a protein selected from the group consisting of alpha, beta or zeta chain of the T- cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.

16. The nucleic acid of claim 15, wherein each CAR comprises a different transmembrane domain selected from the group consisting of CD8, CD28, ICOS, and 0X40.

17. The nucleic acid of claim 16, wherein each transmembrane domain is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 39, 41, 43, and 45 or wherein each transmembrane domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 40, 42, 44, and 46.

18. The nucleic acid of any one of claims 2-17, wherein each intracellular domain comprises a costimulatory domain and an intracellular signaling domain.

19. The nucleic acid of claim 18, wherein each costimulatory domain is from a protein selected from the group consisting of CD28, 4- IBB (CD 137), ICOS (CD278), 0X40, CD5, CD27, LFA- 1 (CD1 la / CD18), ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2,Attorney Docket No: 046483-7473W01(04009)CD18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD 160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, LylO8), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, BTLA, an MHC class I molecule, and a ligand that specifically binds with CD8.

20. The nucleic acid of claim 18, wherein each costimulatory domain is from a protein selected from the group consisting of CD28, 4- IBB, ICOS, and 0X40.

21. The nucleic acid of claim 20, wherein each costimulatory domain is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 47, 49, 51, and 53.

22. The nucleic acid of claim 20 or 21, wherein each costimulatory domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 48, 50, 52, and 54.

23. The nucleic acid of any one of claims 18-22, wherein the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif.

24. The nucleic acid of claim 23, wherein each intracellular signaling domain is from a protein selected from the group consisting of CD3 zeta, common FcR gamma, FcyRIII Fc gamma Rlla, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD66d, DAP10, and DAP12.

25. The nucleic acid of claim 24, wherein each intracellular signaling domain is from CD3 zeta.

26. The nucleic acid of claim 25, wherein each intracellular signaling domain from CD3 zeta is encoded by a different codon-altered polynucleotide sharing no significant homology to one another.

27. The nucleic acid of claim 25 or 26, wherein each intracellular signaling domain from CD3 zeta is encoded by a different nucleotide sequence selected from the group consisting of SEQ ID NOs: 55-58.

28. The nucleic acid of any one of claims 25-27, wherein each intracellular signaling domain from CD3 zeta comprises the amino acid sequence of SEQ ID NO: 60.Attorney Docket No: 046483-7473W01(04009)29. The nucleic acid of any one of claims 2-28, wherein each antigen-binding domain is connected to the transmembrane domain by a hinge region.

30. The nucleic acid of claim 29, wherein the hinge region is from a protein selected from the group consisting of an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial hinge domain, a hinge comprising an amino acid sequence of CD8, or any combination thereof.

31. The nucleic acid of claim 29 or 30, wherein the hinge region is from CD8 or CD28.

32. The nucleic acid of claim 31, wherein the hinge region is from CD8 and is encoded by a different codon-altered polynucleotide sharing no significant homology to one another.

33. The nucleic acid of claim 32, wherein the hinge region from CD8 is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 35-37 and the amino acid sequence of SEQ ID NO: 38.

34. The nucleic acid of claim 31, wherein the hinge region from CD28 comprises the nucleotide sequence of SEQ ID NO: 33 and / or the amino acid sequence of SEQ ID NO: 34.

35. The nucleic acid of any one of claims 2-34, wherein each CAR comprises a leader sequence encoding an N-terminal signal peptide.

36. The nucleic acid of claim 35, wherein each CAR-encoded polynucleotide comprises a different CD8ot signal peptide encoded by a different codon-altered polynucleotide sharing no significant homology to one another.

37. The nucleic acid of claim 36, wherein each CD8a signal peptide is encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 28-31 and comprises the amino acid sequence of SEQ ID NO: 32.

38. The nucleic acid of any one of claims 4-37, wherein: the first polynucleotide encodes a first CAR comprising a CD 19 antigen binding domain, a CD28 hinge domain, a CD28 transmembrane domain, aa CD28 costimulatory domain, and a CD3 zeta intracellular signaling domain;Attorney Docket No: 046483-7473W01(04009) the second polynucleotide encodes a second CAR comprising a CD20 antigen binding domain, CD20 antigen binding domain, a CD8 hinge domain, a CD8 transmembrane domain, a 4- IBB costimulatory domain, and a CD3 zeta intracellular signaling domain; the third polynucleotide encodes a third CAR comprising a CD22 antigen binding domain, a CD8 hinge domain, an ICOS transmembrane domain, ICOS costimulatory domain, and a CD3 zeta intracellular signaling domain; and the fourth polynucleotide encodes a fourth CAR comprising a CD79b binding domain, a CD8 hinge domain, an 0X40 or CD8 transmembrane domain, an 0X40 costimulatory domain, and a CD3 zeta intracellular signaling domain.

39. The nucleic acid of claim 38, wherein each CAR is encoded by a different nucleotide sequence selected from the group consisting of SEQ ID NOs: 6, 8, 10, and 12.

40. The nucleic acid of claim 38 or 39, wherein each CAR is encoded by a different amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 9, 11, and 13.

41. The nucleic acid of any one of claims 4 or 38-40, wherein the nucleic acid further comprises a fifth transgene encoding a mutant low affinity nerve growth factor (mLNGFR) marker protein.

42. The nucleic acid of any one of claims 38-41, wherein the nucleic acid comprises, from 5’-3’, the fourth, first, second, and third polynucleotides.

43. The nucleic acid of any one of claims 38-41, wherein the nucleic acid comprises, from 5’-3’, the second, first, fourth, and third polynucleotides.

44. The nucleic acid of any one of claims 38-41, wherein the nucleic acid comprises, from 5’-3’, the fourth first second, and third polynucleotides.

45. The nucleic acid of any one of claims 38-41, wherein the nucleic acid comprises, from 5’-3’, the first, fourth, second, and third polynucleotides.

46. The nucleic acid of any one of claims 2-45, wherein one of the first and second promoters is an EF-1 alpha promoter and the other of the first and second promoters is human PGK1 promoter.Attorney Docket No: 046483-7473W01(04009)47. The nucleic acid of claim 46, wherein the first promoter is an EF-1 alpha promoter, and the second promoter is a human PGK-1 promoter.

48. The nucleic acid of claim 46 or 47, wherein the EF-1 alpha promoter comprises the nucleotide sequence of SEQ ID NO: 74 and the PGK-1 promoter comprises the nucleotide sequence of SEQ ID NO: 72.

49. The nucleic acid of any one of claims 46-48, wherein the EF-1 alpha promoter drives expression of, from N-terminus to C-terminus, an anti-CD19 CAR, an anti-CD20 CAR, and an anti-CD22 CAR.

50. The nucleic acid of claim 49, wherein the anti-CD19 CAR, the anti-CD20 CAR, and the anti- CD22 CAR are encoded by the nucleotide sequence of SEQ ID NO: 2 and / or the nucleotide sequence encodes the amino acid sequence of SEQ ID NO: 3.

51. The nucleic acid of any one of claims 46-50, wherein the hPGK-1 promoter drives expression of, from N-terminus to C-terminus, an anti-CD79b CAR and a mLNGFR marker protein.

52. The nucleic acid of claim 51, wherein the CD79b CAR and a mLNGFR marker protein are encoded by the nucleotide sequence of SEQ ID NO: 4 or 113 and / or the nucleotide sequence encodes the amino acid sequence of SEQ ID NO: 5 or 114.

53. The nucleic acid of any one of claims 46-48, wherein the EF-1 alpha promoter drives expression of, from N-terminus to C-terminus, an anti-CD79b CAR, an anti-CD20 CAR, and an anti-CD22 CAR.

54. The nucleic acid of claim 49, wherein the anti-CD79b CAR, an anti-CD20 CAR, and anti- CD22 CAR are encoded by the nucleotide sequence of SEQ ID NO: 115 and / or the nucleotide sequence encodes the amino acid sequence of SEQ ID NO: 116.

55. The nucleic acid of claim 53 or 54, wherein the hPGK-1 promoter drives expression of, from N-terminus to C-terminus, a CD 19 CAR and a mLNGFR marker protein.Attorney Docket No: 046483-7473W01(04009)56. The nucleic acid of claim 55, wherein the CD19 CAR and a mLNGFR marker protein are encoded by the nucleotide sequence of SEQ ID NO: 117 and / or the nucleotide sequence encodes the amino acid sequence of SEQ ID NO: 118.

57. The nucleic acid of any one of claims 1-56, wherein the nucleic acid encodes a plurality of self-cleaving 2A peptide domains.

58. The nucleic acid of claim 57, wherein the nucleic acid encodes three 2A self-cleaving 2A peptide domains.

59. The nucleic acid of claim 58, wherein the self-cleaving peptide domains are selected from the group consisting of T2A, P2A, E2A and F2A.

60. The nucleic acid of any one of claims 57-59, wherein the self-cleaving peptide domains are encoded by nucleotide sequences selected from the group consisting of SEQ ID NOs: 61, 62, and 64 and / or amino acid sequences selected from the group consisting of SEQ ID NOs: 63 or 65-67.

61. The nucleic acid of any one of claims 57-60, wherein the nucleic acid further encodes a furin cleavage site between the intracellular signaling domain and a self-cleaving 2A peptide domain in each CAR.

62. The nucleic acid of any one of claims 1-61, wherein the nucleic acid further comprises a Woodchuck Hepatitis virus post-transcriptional regulatory element (WPRE).

63. An expression vector comprising the nucleic acid of any one of claims 1-62.

64. The expression vector of claim 63, wherein the expression vector is a lentiviral vector.

65. The expression vector of claim 64, wherein the lentiviral vector is a self-inactivating HIV vector.

66. The expression vector of claim 65, wherein the HIV vector comprises a partial gag sequence, optionally wherein the partial gag sequence comprises the nucleotide sequence of SEQ ID NO: 69.Attorney Docket No: 046483-7473W01(04009)67. The expression vector of claim 65 or 66, wherein the HIV vector further comprises a central polypurine tract (cPPT), optionally wherein the cPPT comprises the nucleotide sequence of SEQ ID NO: 70.

68. The expression vector of any one of claims 63-67, comprising the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 107, SEQ ID NO: 108, or SEQ ID NO: 109.

69. A cell, comprising the nucleic acid of any one of claims 1-62 or the expression construct of any one of claims 63-68.

70. The cell of claim 69, wherein the cell is an immune cell.

71. The cell of claim 70, wherein the immune cell is a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), or a regulatory T cell (Treg).

72. The cell of any one of claim 69 or 71, wherein the immune cell is an autologous cell.

73. The cell of any one of claims 69-72, wherein the cell expresses a first CAR comprising a CD 19 antigen binding domain, a second CAR comprising a CD20 antigen binding domain, a third CAR comprising a CD22 antigen binding domain, a fourth CAR comprising a CD79b antigen binding domain.

74. The cell of any one of claims 69-73, wherein the five transgenes are integrated into the host genome in the form of a single expression cassette.

75. A pharmaceutical composition comprising a therapeutically effective amount of the cell of any one of claims 69-74.

76. A method of treating a disease or disorder in a subject, comprising administering the pharmaceutical composition of claim 75 to a subject in need thereof.

77. The method of claim 76, wherein the disease is associated with CD 19 expression and is a proliferative disease such as a cancer, tumor, or malignancy; or a precancerous condition such as a myelodysplasia; a myelodysplastic syndrome or a preleukemia; or is a non-cancer related indication associated with expression of CD 19.Attorney Docket No: 046483-7473W01(04009)78. The method of claim 76 or 77, wherein the disease is cancer.

79. The method of claim 78, wherein the cancer is a hematological malignancy.

80. The method of claim 79, wherein the hematological malignancy is acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia, myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, B-cell acute lymphoid leukemia (BALL), T-cell acute lymphoid leukemia (TALL), small lymphocytic leukemia (SLL), acute lymphoid leukemia (ALL), chronic leukemia, chronic myelocytic leukemia, chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia or a CD 19-negative relapsed cancer.

81. The method of any one of claims 78-80, wherein the subject has a CD 19-positive cancer,82. The method of any one of claims 78-80 wherein the subject has a CD 19-negative cancer and is positive for one or more of CD10, CD20, CD22, CD34, CD123, FLT-3, ROR1, CD79b,CD 179b, or CD79a.

83. A method for generating the cell of any one of claims 69-74, comprising: introducing into the cell the nucleic acid of nucleic acid of any one of claims 1-61 or the expression construct of any one of claims 62-67.

84. The method of claim 83, wherein the cell is cultured in the presence of IL-2, IL-7, IL- 15, IL- 21, or a combination thereof.

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