Chimeric antigen receptor and its use

Abstract

The present invention provides immune effector cells (e.g., T cells, NK cells) that express chimeric antigen receptors (CARs), as well as compositions and methods thereof.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Patent Application No. 62 / 940,509, filed on November 26, 2019, the entire content of which is incorporated herein by reference.

[0002] Sequence Listing This application is filed electronically in ASCII format and includes a sequence listing that is incorporated herein by reference in its entirety. The ASCII copy was created on November 24, 2020, has the name N2067 - 7166WO_SL.txt, and is 598,194 bytes in size.

[0003] The present invention generally relates to immune effector cells (e.g., T cells or NK cells) engineered to express a chimeric antigen receptor (CAR), as well as compositions and uses thereof.

Background Art

[0004] Adoptive cell transfer (ACT) therapy with T cells, particularly T cells transduced with a chimeric antigen receptor (CAR), has shown promise in several blood cancer trials. There is a need for methods and processes to improve the production of CAR - expressing cell therapy products, enhance the quality of the products, and maximize the therapeutic effects of the products.

Summary of the Invention

Means for Solving the Problems

[0005] In one aspect, the invention features a cell comprising a first antigen-binding domain and a second antigen-binding domain, such as an immune cell, such as a T cell or an NK cell. In some embodiments, the first antigen-binding domain is an anti-BCMA binding domain. In some embodiments, the anti-BCMA binding domain comprises an anti-BCMA binding sequence disclosed herein, such as a CDR, VH, VL, or scFv sequence disclosed in Tables 3-15, 19, 20, 26, and 31. In some embodiments, the second antigen-binding domain is an anti-CD19 binding domain. In some embodiments, the anti-CD19 binding domain comprises an anti-CD19 binding sequence disclosed herein, such as a CDR, VH, VL, or scFv sequence disclosed in Tables 2, 19, 22, and 31.

[0006] In some embodiments, the invention provides a cell comprising: (a) a first antigen-binding domain that is an anti-BCMA binding domain, wherein the anti-BCMA binding domain comprises a heavy-chain variable region (VH) comprising heavy-chain complementarity-determining region 1 (HC CDR1), heavy-chain complementarity-determining region 2 (HC CDR2), and heavy-chain complementarity-determining region 3 (HC CDR3), and a light-chain variable region (VL) comprising light-chain complementarity-determining region 1 (LC CDR1), light-chain complementarity-determining region 2 (LC CDR2), and light-chain complementarity-determining region 3 (LC CDR3), and wherein HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 comprise the amino acid sequences of (i) SEQ ID NOs: 86, 130, 88, 95, 131, and 132; (ii) SEQ ID NOs: 44, 45, 84, 54, 55, and 56; or (iii) SEQ ID NOs: 179, 180, 181, 147, 182, and 183, respectively; and (b) a second antigen-binding domain. In some embodiments, the first antigen-binding domain and the second antigen-binding domain are disposed in two chimeric antigen receptors (CARs). In some embodiments, the first antigen-binding domain and the second antigen-binding domain are disposed in one CAR.

[0007] In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 each comprise the amino acid sequences of SEQ ID NO: 86, 130, 88, 95, 131, and 132, respectively. In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 each comprise the amino acid sequences of SEQ ID NO: 86, 87, 88, 95, 96, and 97, respectively. In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 each comprise the amino acid sequences of SEQ ID NO: 86, 109, 88, 95, 114, and 115, respectively. In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 each comprise the amino acid sequences of SEQ ID NO: 86, 109, 88, 95, 114, and 97, respectively. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 93 or 112 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, VH is encoded by the nucleic acid sequence of SEQ ID NO: 260, 94, or 113 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO: 102, 118, or 124 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, VL is encoded by the nucleic acid sequence of SEQ ID NO: 261, 103, 119, or 125 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, VH and VL each comprise the amino acid sequences of SEQ ID NO: 93 and 102, respectively. In some embodiments, VH and VL each comprise the amino acid sequences of SEQ ID NO: 112 and 118, respectively. In some embodiments, VH and VL each comprise the amino acid sequences of SEQ ID NO: 112 and 124, respectively.In some embodiments, the first antigen-binding domain comprises a single-chain variable region fragment (scFv) comprising the amino acid sequence of SEQ ID NO: 105, 120, or 126, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the first antigen-binding domain is encoded by the nucleic acid sequence of SEQ ID NO: 253, 106, 121, or 127, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the first antigen-binding domain is disposed in the first CAR. In some embodiments, the first CAR comprises the amino acid sequence of SEQ ID NO: 107, 226, 122, or 128, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the first CAR is encoded by the nucleic acid sequence of SEQ ID NO: 259, 258, 108, 123, or 129, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0008] In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 each comprise the amino acid sequences of SEQ ID NOs: 44, 45, 84, 54, 55, and 56. In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 each comprise the amino acid sequences of SEQ ID NOs: 44, 45, 76, 54, 55, and 56. In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 each comprise the amino acid sequences of SEQ ID NOs: 44, 45, 46, 54, 55, and 56. In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 each comprise the amino acid sequences of SEQ ID NOs: 44, 45, 68, 54, 55, and 56. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 78, 52, or 70 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, VH is encoded by the nucleic acid sequence of SEQ ID NO: 79, 53, or 71 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO: 61 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, VL is encoded by the nucleic acid sequence of SEQ ID NO: 62 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, VH and VL each comprise the amino acid sequences of SEQ ID NOs: 78 and 61. In some embodiments, VH and VL each comprise the amino acid sequences of SEQ ID NOs: 52 and 61. In some embodiments, VH and VL each comprise the amino acid sequences of SEQ ID NOs: 70 and 61. In some embodiments, the first antigen-binding domain comprises a single-chain variable region fragment (scFv) comprising the amino acid sequence of SEQ ID NO: 80, 64, or 72 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.In some embodiments, the first antigen-binding domain is encoded by the nucleic acid sequence of SEQ ID NO: 81, 65, or 73, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the first antigen-binding domain is disposed in the first CAR. In some embodiments, the first CAR comprises the amino acid sequence of SEQ ID NO: 224, 82, 66, or 74, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the first CAR is encoded by the nucleic acid sequence of SEQ ID NO: 83, 67, or 75, or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0009] In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 each comprise the amino acid sequences of SEQ ID NO: 179, 180, 181, 147, 182, and 183, respectively. In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 each comprise the amino acid sequences of SEQ ID NO: 137, 138, 139, 147, 148, and 149, respectively. In some embodiments, HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 each comprise the amino acid sequences of SEQ ID NO: 160, 161, 162, 147, 170, and 171, respectively. In some embodiments, VH comprises the amino acid sequence of SEQ ID NO: 145 or 168 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, VH is encoded by the nucleic acid sequence of SEQ ID NO: 146 or 169 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, VL comprises the amino acid sequence of SEQ ID NO: 154 or 173 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, VL is encoded by the nucleic acid sequence of SEQ ID NO: 155 or 174 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, VH and VL each comprise the amino acid sequences of SEQ ID NO: 145 and 154, respectively. In some embodiments, VH and VL each comprise the amino acid sequences of SEQ ID NO: 168 and 173, respectively. In some embodiments, the first antigen-binding domain comprises a single-chain variable region fragment (scFv) comprising the amino acid sequence of SEQ ID NO: 156 or 175 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the first antigen-binding domain is encoded by the nucleic acid sequence of SEQ ID NO: 157 or 176 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the first antigen-binding domain is disposed in the first CAR.In some embodiments, the first CAR comprises the amino acid sequence of SEQ ID NO: 158 or 177, or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto. In some embodiments, the first CAR is encoded by the nucleic acid sequence of SEQ ID NO: 159 or 178, or a nucleic acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto.

[0010] In some embodiments, the invention provides a cell comprising: (a) a first antigen-binding domain that is an anti-BCMA binding domain, wherein the anti-BCMA binding domain comprises: (i) a VH comprising the HC CDR1, HC CDR2, and HC CDR3 of the anti-BCMA sequences listed in Table 20 or 26 and a VL comprising the LC CDR1, LC CDR2, and LC CDR3 of the anti-BCMA sequences listed in Table 20 or 26, wherein the VH and VL are linked by a linker comprising the amino acid sequence of SEQ ID NO: 243; (ii) a VH and a VL comprising the amino acid sequences of SEQ ID NO: 239 and 242, respectively, wherein the VH and VL are linked by a linker comprising the amino acid sequence of SEQ ID NO: 243; or (iii) an scFv comprising the amino acid sequence of SEQ ID NO: 200; and (b) a second antigen-binding domain. In some embodiments, the first antigen-binding domain and the second antigen-binding domain are disposed in two chimeric antigen receptors (CARs). In some embodiments, the first antigen-binding domain and the second antigen-binding domain are disposed in one CAR. In some embodiments, the second antigen-binding domain binds to an antigen selected from CD19, CD5, CD10, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD30, CD34, CD37, CD38, CD40, CD53, CD69, CD72, CD73, CD74, CD75, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD123, CD135, CD138, CD179, CD269, Flt3, ROR1, FcRn5, FcRn2, CS-1, CXCR4, 5, 7, IL-7 / 3R, IL7 / 4 / 3R, or IL4R, and optionally, the B cell antigen is selected from CD19, CD20, CD22, FcRn5, FcRn2, CS-1, CD138, CD123, CD33, CD34, CLL-1, folate receptor β, or FLT3. In some embodiments, the second antigen-binding domain binds to CD19.In some embodiments, the second antigen-binding domain binds to an antigen selected from the group consisting of EGFRvIII, mesothelin, GD2, Tn antigen, sTn antigen, Tn-O-glycopeptide, sTn-O-glycopeptide, PSMA, CD97, TAG72, CD44v6, CEA, EPCAM, KIT, IL-13Ra2, legumain, GD3, CD171, IL-11Ra, PSCA, MAD-CT-1, MAD-CT-2, VEGFR2, Lewis Y, CD24, PDGFR-β, SSEA-4, folate receptor alpha, ERBB (e.g., ERBB2), Her2 / neu, MUC1, EGFR, NCAM, Ephrin B2, CAIX, LMP2, sLe, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, FAP, legumain, HPV E6 or E7, ML-IAP, CLDN6, TSHR, GPRC5D, ALK, polysialic acid, Fos-related antigen, neutrophil elastase, TRP-2, CYP1B1, sperm protein 17, beta-human chorionic gonadotropin, AFP, thyroglobulin, PLAC1, globo H, RAGE1, MN-CA IX, human telomerase reverse transcriptase, intestinal carboxylesterase, mut hsp 70-2, NA-17, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, NY-ESO-1, GPR20, Ly6k, OR51E2, TARP, GFRα4 or a peptide of any of these antigens presented by MHC.

[0011] In some embodiments, the second antigen-binding domain binds to CD19. In some embodiments, the second antigen-binding domain comprises HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and / or LC CDR3 of an anti-CD19 sequence listed in Table 19 or Table 22, such as HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 comprising the sequences of SEQ ID NOs: 295 and 245-249, respectively. In some embodiments, the second antigen-binding domain comprises VH and / or VL of an anti-CD19 sequence listed in Table 19 or Table 22, such as VH and VL comprising the amino acid sequences of SEQ ID NOs: 250 and 251, respectively, or amino acid sequences having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the second antigen-binding domain comprises an scFv of an anti-CD19 sequence listed in Table 19 or Table 22, such as an scFv comprising the amino acid sequence of SEQ ID NO: 211 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the second antigen-binding domain is disposed in the second CAR, and the CAR comprises a CAR of an anti-CD19 sequence listed in Table 19 or Table 22, such as a CAR comprising the amino acid sequence of SEQ ID NO: 225 or 229, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0012] In some embodiments, the first antigen-binding domain comprises HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 having the amino acid sequences of (a) SEQ ID NOs: 86, 87, 88, 95, 96, and 97, respectively; (b) SEQ ID NOs: 44, 45, 76, 54, 55, and 56, respectively; or (c) SEQ ID NOs: 44, 45, 46, 54, 55, and 56, respectively. In some embodiments, the second antigen-binding domain comprises HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 having the amino acid sequences of SEQ ID NOs: 295 and 245-249, respectively. In some embodiments, the first antigen-binding domain comprises VH and VL having the amino acid sequences of (a) SEQ ID NOs: 93 and 102, respectively; (b) SEQ ID NOs: 78 and 61, respectively; or (c) SEQ ID NOs: 52 and 61, respectively. In some embodiments, the second antigen-binding domain comprises VH and VL having the amino acid sequences of SEQ ID NOs: 250 and 251, respectively. In some embodiments, the first antigen-binding domain comprises an scFv having the amino acid sequence of SEQ ID NO: 105, 80, or 64. In some embodiments, the second antigen-binding domain comprises an scFv having the amino acid sequence of SEQ ID NO: 211. In some embodiments, the first antigen-binding domain is encoded by the nucleic acid sequence of SEQ ID NO: 253, 106, 81, or 65. In some embodiments, the second antigen-binding domain is encoded by the nucleic acid sequence of SEQ ID NO: 212.

[0013] In some embodiments, the first antigen-binding domain is disposed in the first CAR, and the second antigen-binding domain is disposed in the second CAR. In some embodiments, the first CAR further comprises a first transmembrane domain and a first intracellular signaling domain. In some embodiments, the second CAR further comprises a second transmembrane domain and a second intracellular signaling domain.

[0014] In some embodiments, the first CAR is encoded by a first nucleic acid sequence, and the second CAR is encoded by a second nucleic acid sequence, and the first and second nucleic acid sequences are disposed on separate nucleic acid molecules.

[0015] In some embodiments, the first CAR is encoded by a first nucleic acid sequence, and the second CAR is encoded by a second nucleic acid sequence, and the first and second nucleic acid sequences are disposed on a single nucleic acid molecule. In some embodiments, the single nucleic acid molecule comprises the following configuration in the 5' to 3' direction: a nucleic acid sequence encoding a first antigen-binding domain - a nucleic acid sequence encoding a first transmembrane domain - a nucleic acid sequence encoding a first intracellular signaling domain - a nucleic acid sequence encoding a linker - a nucleic acid sequence encoding a second antigen-binding domain - a nucleic acid sequence encoding a second transmembrane domain - a nucleic acid sequence encoding a second intracellular signaling domain. In some embodiments, the single nucleic acid molecule comprises the following configuration in the 5' to 3' direction: a nucleic acid sequence encoding a second antigen-binding domain - a nucleic acid sequence encoding a second transmembrane domain - a nucleic acid sequence encoding a second intracellular signaling domain - a nucleic acid sequence encoding a linker - a nucleic acid sequence encoding a first antigen-binding domain - a nucleic acid sequence encoding a first transmembrane domain - a nucleic acid sequence encoding a first intracellular signaling domain. In some embodiments, the linker comprises a self-cleaving site. In some embodiments, the linker comprises a P2A site, a T2A site, an E2A site, or an F2A site. In some embodiments, the linker comprises a P2A site. In some embodiments, the linker is encoded by the nucleic acid sequence of SEQ ID NO: 209 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 208 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the single nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 215, 217, 219, 221, or 223 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the single nucleic acid molecule encodes the amino acid sequence of SEQ ID NO: 214, 216, 218, 220, or 222 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0016] In some embodiments, the first antigen-binding domain and the second antigen-binding domain are disposed in one CAR, and the CAR further includes a transmembrane domain and an intracellular signaling domain. In some embodiments, the first antigen-binding domain includes a first VH (VH1) and a first VL (VL1), and the second antigen-binding domain includes a second VH (VH2) and a second VL (VL2). In some embodiments, VH1, VL1, VH2, and VL2 are arranged in the following configuration from the N-terminus to the C-terminus: VH2 - optionally linker 1 (“L1”) - VL1 - optionally linker 2 (“L2”) - VH1 - optionally linker 3 (“L3”) - VL2. In some embodiments, VH1, VL1, VH2, and VL2 are arranged in the following configuration from the N-terminus to the C-terminus: VH1 - optionally L1 - VH2 - optionally L2 - VL2 - optionally L3 - VL1. In some embodiments, VH1, VL1, VH2, and VL2 are arranged in the following configuration from the N-terminus to the C-terminus: VL2 - optionally L1 - VL1 - optionally L2 - VH1 - optionally L3 - VH2. In some embodiments, VH1, VL1, VH2, and VL2 are arranged in the following configuration from the N-terminus to the C-terminus: VL2 - optionally L1 - VH1 - optionally L2 - VL1 - optionally L3 - VH2. In some embodiments, VH1, VL1, VH2, and VL2 are arranged in the following configuration from the N-terminus to the C-terminus: VH2 - optionally L1 - VH1 - optionally L2 - VL1 - optionally L3 - VL2. In some embodiments, VH1, VL1, VH2, and VL2 are arranged in the following configuration from the N-terminus to the C-terminus: VL1 - optionally L1 - VH2 - optionally L2 - VL2 - optionally L3 - VH1. In some embodiments, VH1, VL1, VH2, and VL2 are arranged in the following configuration from the N-terminus to the C-terminus: VL1 - optionally L1 - VL2 - optionally L2 - VH2 - optionally L3 - VH1. In some embodiments, VH1, VL1, VH2, and VL2 are arranged in the following configuration from the N-terminus to the C-terminus: VH1 - optionally L1 - VL2 - optionally L2 - VH2 - optionally L3 - VL1.In some embodiments, VH1 and VL1 each comprise the amino acid sequences of SEQ ID NOs: 93 and 102 (or amino acid sequences having at least about 85%, 90%, 95% or 99% sequence identity thereto). In some embodiments, VH1 and VL1 each comprise the amino acid sequences of SEQ ID NOs: 333 and 334 (or amino acid sequences having at least about 85%, 90%, 95% or 99% sequence identity thereto). In some embodiments, VH1 and VL1 each comprise the amino acid sequences of SEQ ID NOs: 78 and 61 (or amino acid sequences having at least about 85%, 90%, 95% or 99% sequence identity thereto). In some embodiments, VH1 and VL1 each comprise the amino acid sequences of SEQ ID NOs: 335 and 336 (or amino acid sequences having at least about 85%, 90%, 95% or 99% sequence identity thereto). In some embodiments, VH2 and VL2 each comprise the amino acid sequences of SEQ ID NOs: 250 and 251 (or amino acid sequences having at least about 85%, 90%, 95% or 99% sequence identity thereto). In some embodiments, VH2 and VL2 each comprise the amino acid sequences of SEQ ID NOs: 331 and 332 (or amino acid sequences having at least about 85%, 90%, 95% or 99% sequence identity thereto). In some embodiments, L1 or L3 comprises the amino acid sequence of SEQ ID NO: 5 (or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto). In some embodiments, L2 comprises the amino acid sequence of SEQ ID NO: 63 (or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto). In some embodiments, the CAR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 321 - 330 or an amino acid sequence having at least 80, 85, 90, 95 or 99% identity thereto. In some embodiments, the CAR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 339 - 348 or an amino acid sequence having at least 80, 85, 90, 95 or 99% identity thereto).

[0017] In some embodiments, the CAR is encoded by a nucleic acid molecule that, in the 5' to 3' direction, has the following configuration: a nucleic acid sequence encoding a first antigen-binding domain - optionally a nucleic acid sequence encoding a linker - a nucleic acid sequence encoding a second antigen-binding domain - a nucleic acid sequence encoding a transmembrane domain - a nucleic acid sequence encoding an intracellular signaling domain. In some embodiments, the CAR is encoded by a nucleic acid molecule that, in the 5' to 3' direction, has the following configuration: a nucleic acid sequence encoding a second antigen-binding domain - optionally a nucleic acid sequence encoding a linker - a nucleic acid sequence encoding a first antigen-binding domain - a nucleic acid sequence encoding a transmembrane domain - a nucleic acid sequence encoding an intracellular signaling domain.

[0018] In some embodiments, the CAR, in the N to C direction, has the following configuration: a first antigen-binding domain - optionally a linker - a second antigen-binding domain - a transmembrane domain - an intracellular signaling domain. In some embodiments, the CAR, in the N to C direction, has the following configuration: a second antigen-binding domain - optionally a linker - a first antigen-binding domain - a transmembrane domain - an intracellular signaling domain.

[0019] In some embodiments, the first antigen-binding domain or the second antigen-binding domain comprises a VH and a VL. In some embodiments, the VH and the VL are linked by a linker. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 5, 63, 104, or 243 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0020] In some embodiments, the transmembrane domain, the first transmembrane domain, or the second transmembrane domain comprises the transmembrane domain of a protein selected from the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. In some embodiments, the transmembrane domain, the first transmembrane domain, or the second transmembrane domain comprises the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the transmembrane domain, the first transmembrane domain, or the second transmembrane domain is encoded by the nucleic acid sequence of SEQ ID NO: 17 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0021] In some embodiments, the first antigen-binding domain or the second antigen-binding domain is linked to the transmembrane domain, the first transmembrane domain, or the second transmembrane domain by a hinge region (e.g., the first or second hinge region). In some embodiments, the hinge region comprises the amino acid sequence of SEQ ID NO: 2, 3, or 4 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the hinge region is encoded by the nucleic acid sequence of SEQ ID NO: 13, 14, or 15 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the hinge region and the transmembrane domain comprise the amino acid sequence of SEQ ID NO: 202 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the hinge region and the transmembrane domain are encoded by the nucleic acid sequence of SEQ ID NO: 203 or 213 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0022] In some embodiments, the intracellular signaling domain, the first intracellular signaling domain, or the second intracellular signaling domain comprises a primary signaling domain (e.g., the first or second primary signaling domain). In some embodiments, the primary signaling domain comprises a functional signaling domain derived from CD3ζ, TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (ICOS), FcεRI, DAP10, DAP12, or CD66d. In some embodiments, the primary signaling domain comprises the amino acid sequence of SEQ ID NO: 9 or 10 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the primary signaling domain is encoded by the nucleic acid sequence of SEQ ID NO: 20, 21, or 205 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the intracellular signaling domain, the first intracellular signaling domain, or the second intracellular signaling domain comprises a co-stimulatory signaling domain (e.g., the first or second co-stimulatory signaling domain).In some embodiments, the costimulatory signaling domain comprises a functional signaling domain derived from the following: MHC class I molecule, TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, 4-1BB (CD137), B7-H3, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD28-OX40, CD28-4-1BB or a ligand that specifically binds to CD83. In some embodiments, the costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto. In some embodiments, the costimulatory signaling domain is encoded by the nucleic acid sequence of SEQ ID NO: 18 or 204 or a nucleic acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto.In some embodiments, the intracellular signaling domain, the first intracellular signaling domain, or the second intracellular signaling domain comprises a functional signaling domain derived from 4-1BB and a functional signaling domain derived from CD3ζ. In some embodiments, the intracellular signaling domain, the first intracellular signaling domain, or the second intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 7 (or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto) and the amino acid sequence of SEQ ID NO: 9 or 10 (or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the intracellular signaling domain, the first intracellular signaling domain, or the second intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO: 9 or 10.

[0023] In some embodiments, the CAR, the first CAR, or the second CAR further comprises a leader sequence (e.g., the first or second leader sequence). In some embodiments, the leader sequence comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the leader sequence is encoded by the nucleic acid sequence of SEQ ID NO: 199 or 210 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0024] In some embodiments, the first leader sequence and the second leader sequence are encoded by different (e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100% different) nucleic acid sequences. In some embodiments, the first hinge region and the second hinge region are encoded by different (e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100% different) nucleic acid sequences. In some embodiments, the first transmembrane domain and the second transmembrane domain are encoded by different (e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100% different) nucleic acid sequences. In some embodiments, the first intracellular signaling domain and the second intracellular signaling domain are encoded by different (e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100% different) nucleic acid sequences. In some embodiments, the first primary signaling domain and the second primary signaling domain are encoded by different (e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100% different) nucleic acid sequences. In some embodiments, the first co-stimulatory signaling domain and the second co-stimulatory signaling domain are encoded by different (e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100% different) nucleic acid sequences. In some embodiments, the first leader sequence and the second leader sequence comprise the same amino acid sequence (e.g., the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto). In some embodiments, the first leader sequence and the second leader sequence comprise different amino acid sequences. In some embodiments, the first hinge region and the second hinge region comprise the same amino acid sequence (e.g., the first hinge region and the second hinge region comprise the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto).In some embodiments, the first hinge region and second hinge region sequences comprise different amino acid sequences. In some embodiments, the first transmembrane domain and second transmembrane domain comprise the same amino acid sequence (e.g., the first transmembrane domain and second transmembrane domain comprise the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto). In some embodiments, the first transmembrane domain and second transmembrane domain comprise different amino acid sequences. In some embodiments, the first intracellular signaling domain and second intracellular signaling domain comprise the same amino acid sequence. In some embodiments, the first intracellular signaling domain and second intracellular signaling domain comprise different amino acid sequences. In some embodiments, the first primary signaling domain and second primary signaling domain comprise the same amino acid sequence (e.g., the first primary signaling domain and second primary signaling domain comprise the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto). In some embodiments, the first primary signaling domain and second primary signaling domain comprise different amino acid sequences. In some embodiments, the first co-stimulatory signaling domain and second co-stimulatory signaling domain comprise the same amino acid sequence (e.g., the first co-stimulatory signaling domain and second co-stimulatory signaling domain comprise the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto). In some embodiments, the first co-stimulatory signaling domain and second co-stimulatory signaling domain comprise different amino acid sequences (e.g., the first and second co-stimulatory signaling domains comprise the 4-1BB co-stimulatory domain sequence and CD28 co-stimulatory domain sequence, respectively; or the CD28 co-stimulatory domain sequence and 4-1BB co-stimulatory domain sequence, respectively). In some embodiments, the first leader sequence and second leader sequence are each encoded by a nucleic acid sequence comprising SEQ ID NOs: 199 and 210 (or a nucleic acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto).In some embodiments, the first leader sequence and the second leader sequence are each encoded by a nucleic acid sequence comprising SEQ ID NO: 210 and 199 (or a nucleic acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto). In some embodiments, the first hinge region and the second hinge region are each encoded by a nucleic acid sequence comprising SEQ ID NO: 337 and 13 (or a nucleic acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto). In some embodiments, the first hinge region and the second hinge region sequences are each encoded by a nucleic acid sequence comprising SEQ ID NO: 13 and 337 (or a nucleic acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto). In some embodiments, the first transmembrane domain and the second transmembrane domain are each encoded by a nucleic acid sequence comprising SEQ ID NO: 338 and 17 (or a nucleic acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto). In some embodiments, the first transmembrane domain and the second transmembrane sequences are each encoded by a nucleic acid sequence comprising SEQ ID NO: 17 and 338 (or a nucleic acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto). In some embodiments, the first co-stimulatory signaling domain and the second co-stimulatory signaling domain are each encoded by a nucleic acid sequence comprising SEQ ID NO: 204 and 18 (or a nucleic acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto). In some embodiments, the first co-stimulatory signaling domain and the second co-stimulatory sequences are each encoded by a nucleic acid sequence comprising SEQ ID NO: 18 and 204 (or a nucleic acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto). In some embodiments, the first primary signaling domain and the second primary signaling domain are each encoded by a nucleic acid sequence comprising SEQ ID NO: 205 and 21 (or a nucleic acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto).In some embodiments, the first co-stimulatory signaling domain and the second co-stimulatory sequence are each encoded by a nucleic acid sequence comprising SEQ ID NO: 21 and 205 (or a nucleic acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto).

[0025] In some embodiments, the CAR, the first CAR or the second CAR is encoded by a nucleic acid sequence comprising the woodchuck hepatitis posttranscriptional regulatory element (WPRE).

[0026] In some embodiments, provided herein is a nucleic acid molecule comprising: (a) a first nucleic acid sequence encoding a first antigen-binding domain that is an anti-BCMA binding domain, the anti-BCMA binding domain comprising a heavy chain variable region (VH) comprising heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2) and heavy chain complementarity determining region 3 (HC CDR3), and a light chain variable region (VL) comprising light chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2) and light chain complementarity determining region 3 (LC CDR3), wherein HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3 comprise the amino acid sequences of (i) SEQ ID NOs: 86, 130, 88, 95, 131 and 132, respectively; (ii) SEQ ID NOs: 44, 45, 84, 54, 55 and 56, respectively; or (iii) SEQ ID NOs: 179, 180, 181, 147, 182 and 183, respectively; and (b) a second nucleic acid sequence encoding a second antigen-binding domain.

[0027] In some embodiments, the isolated nucleic acid molecule comprises a first nucleic acid molecule and a second nucleic acid molecule that are separate nucleic acid molecules, the first nucleic acid sequence is disposed on the first nucleic acid molecule, and the second nucleic acid sequence is disposed on the second nucleic acid molecule.

[0028] In some embodiments, provided herein are nucleic acid molecules comprising: (a) a first nucleic acid sequence encoding a first antigen binding domain that is an anti-BCMA binding domain, wherein the anti-BCMA binding domain comprises: (i) a VH comprising a HC CDR1, HC CDR2, and HC CDR3 of an anti-BCMA sequence listed in Table 20 or 26, and a VL comprising a LC CDR1, LC CDR2, and LC CDR3 of an anti-BCMA sequence listed in Table 20 or 26, wherein the VH and VL are linked by a linker comprising the amino acid sequence of SEQ ID NO: 243; (ii) a VH and VL comprising the amino acid sequences of SEQ ID NOs: 239 and 242, respectively, wherein the VH and VL are linked by a linker comprising the amino acid sequence of SEQ ID NO: 243; or (iii) an scFv comprising the amino acid sequence of SEQ ID NO: 200; and (b) a second nucleic acid sequence encoding a second antigen binding domain.

[0029] In some embodiments, provided herein is a nucleic acid molecule comprising a first nucleic acid sequence encoding a first CAR and a second nucleic acid sequence encoding a second CAR, wherein the first CAR comprises a first antigen-binding domain that is an anti-BCMA binding domain, a first transmembrane domain, and a first intracellular signaling domain, and the second CAR comprises a second antigen-binding domain that is an anti-CD19 binding domain, a second transmembrane domain, and a second intracellular signaling domain, (i) the first antigen-binding domain comprises HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR comprising the amino acid sequences of SEQ ID NOs: 86, 87, 88, 95, 96, and 97, respectively, and the second antigen-binding domain comprises HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR comprising the amino acid sequences of SEQ ID NOs: 295 and 245-249, respectively; (ii) the first antigen-binding domain comprises VH and VL comprising the amino acid sequences of SEQ ID NOs: 93 and 102, respectively, and the second antigen-binding domain comprises VH and VL comprising the amino acid sequences of SEQ ID NOs: 250 and 251, respectively; (iii) the first antigen-binding domain comprises an scFv comprising the amino acid sequence of SEQ ID NO: 105, and the second antigen-binding domain comprises an scFv comprising the amino acid sequence of SEQ ID NO: 211; (iv) the first CAR comprises the amino acid sequence of SEQ ID NO: 107 or 226, and the second CAR comprises the amino acid sequence of SEQ ID NO: 225 or 229; or (v) the isolated nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 271.

[0030] In some embodiments, provided herein is a nucleic acid molecule comprising a first nucleic acid sequence encoding a first CAR and a second nucleic acid sequence encoding a second CAR, wherein the first CAR comprises a first antigen-binding domain that is an anti-BCMA binding domain, a first transmembrane domain, and a first intracellular signaling domain, and the second CAR comprises a second antigen-binding domain that is an anti-CD19 binding domain, a second transmembrane domain, and a second intracellular signaling domain, and (i) the first antigen-binding domain comprises HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 comprising the amino acid sequences of SEQ ID NOs: 44, 45, 76, 54, 55, and 56, respectively, and the second antigen-binding domain comprises HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 comprising the amino acid sequences of SEQ ID NOs: 295 and 245-249, respectively; or (ii) the first antigen-binding domain comprises VH and VL comprising the amino acid sequences of SEQ ID NOs: 78 and 61, respectively, and the second antigen-binding domain comprises VH and VL comprising the amino acid sequences of SEQ ID NOs: 250 and 251, respectively; or (iii) the first antigen-binding domain comprises an scFv comprising the amino acid sequence of SEQ ID NO: 80, and the second antigen-binding domain comprises an scFv comprising the amino acid sequence of SEQ ID NO: 211; or (iv) the first CAR comprises the amino acid sequence of SEQ ID NO: 82 or 224, and the second CAR comprises the amino acid sequence of SEQ ID NO: 225 or 229; or (v) the isolated nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 215.

[0031] In some embodiments, provided herein is a polypeptide molecule encoded by the nucleic acid molecule disclosed herein.

[0032] In some embodiments, a CAR is provided herein, the CAR comprising: (a) a first antigen-binding domain that is an anti-BCMA binding domain, the anti-BCMA binding domain comprising a heavy-chain variable region (VH) comprising heavy-chain complementarity-determining region 1 (HC CDR1), heavy-chain complementarity-determining region 2 (HC CDR2), and heavy-chain complementarity-determining region 3 (HC CDR3), and a light-chain variable region (VL) comprising light-chain complementarity-determining region 1 (LC CDR1), light-chain complementarity-determining region 2 (LC CDR2), and light-chain complementarity-determining region 3 (LC CDR3), wherein HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 comprise (i) amino acid sequences of SEQ ID NO: 86, 130, 88, 95, 131, and 132, respectively; (ii) amino acid sequences of SEQ ID NO: 44, 45, 84, 54, 55, and 56, respectively; or (iii) amino acid sequences of SEQ ID NO: 179, 180, 181, 147, 182, and 183, respectively, the first antigen-binding domain; and (b) a second antigen-binding domain.

[0033] In some embodiments, a CAR is provided herein, the CAR comprising: (a) a first antigen-binding domain that is an anti-BCMA binding domain, the anti-BCMA binding domain comprising (i) a VH comprising HC CDR1, HC CDR2, and HC CDR3 of an anti-BCMA sequence listed in Table 20 or 26 and a VL comprising LC CDR1, LC CDR2, and LC CDR3 of an anti-BCMA sequence listed in Table 20 or 26, the VH and VL being linked by a linker comprising the amino acid sequence of SEQ ID NO: 243; (ii) a VH and a VL comprising the amino acid sequences of SEQ ID NO: 239 and 242, respectively, the VH and VL being linked by a linker comprising the amino acid sequence of SEQ ID NO: 243; or (iii) an scFv comprising the amino acid sequence of SEQ ID NO: 200, the first antigen-binding domain; and (b) a second antigen-binding domain.

[0034] In some embodiments, vectors are provided herein that comprise a nucleic acid molecule disclosed herein or a nucleic acid molecule encoding a CAR disclosed herein. In some embodiments, the vector is selected from a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, or a retroviral vector. In some embodiments, the vector comprises an EF-1 promoter comprising the nucleic acid sequence of SEQ ID NO: 11.

[0035] In some embodiments, cells are provided herein that comprise a nucleic acid molecule disclosed herein, a nucleic acid molecule encoding a CAR disclosed herein, a polypeptide disclosed herein, a CAR disclosed herein, or a vector disclosed herein. In some embodiments, the cell is a T cell or an NK cell.

[0036] In some embodiments, methods of making cells are provided herein that comprise the step of transducing the cells with a vector disclosed herein, optionally, the cell is a T cell or an NK cell. In some embodiments, methods of making RNA engineered cells are provided herein that comprise the step of introducing in vitro transcribed RNA or synthetic RNA into the cells, wherein the RNA comprises a nucleic acid molecule disclosed herein, a nucleic acid molecule encoding a CAR disclosed herein. In some embodiments, the cell is a T cell or an NK cell.

[0037] In some embodiments, provided herein is a method of generating a population of cells (e.g., T cells) that express a chimeric antigen receptor (CAR), the method comprising: (i) contacting (e.g., binding) a population of cells (e.g., T cells, such as T cells isolated from cryopreserved or fresh leukapheresis products) with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates co-stimulatory molecules on the surface of the cells; (ii) contacting the population of cells (e.g., T cells) with a nucleic acid molecule disclosed herein or a nucleic acid molecule encoding a CAR disclosed herein, thereby providing a population of cells (e.g., T cells) that contain the nucleic acid molecule, and (iii) harvesting the population of cells (e.g., T cells) for storage (e.g., re-formulating the population of cells in a cryopreservation medium) or administration, (a) Step (ii) is performed together with step (i) or within 20 hours after the start of step (i), such as within 12, 13, 14, 15, 16, 17, or 18 hours after the start of step (i), such as within 18 hours after the start of step (i), and step (iii) is performed within 30 (e.g., 26) hours after the start of step (i), such as within 22, 23, 24, 25, 26, 27, 28, 29, or 30 hours after the start of step (i), such as within 24 hours after the start of step (i), (b) Step (ii) is performed together with step (i) or within 20 hours after the start of step (i), such as within 12, 13, 14, 15, 16, 17, or 18 hours after the start of step (i), such as within 18 hours after the start of step (i), and step (iii) is performed within 30 hours after the start of step (ii), such as within 22, 23, 24, 25, 26, 27, 28, 29, or 30 hours after the start of step (ii), or (c) The population of cells from step (iii) is not expanded or is expanded by no more than 5, 10, 15, 20, 25, 30, 35, or 40%, such as no more than 10%, as evaluated by the number of viable cells, compared to the population of cells at the start of step (i), Optionally, the nucleic acid molecule of step (ii) is on a viral vector, optionally, the nucleic acid molecule of step (ii) is an RNA molecule on a viral vector, and optionally, step (ii) comprises transducing a population of cells (e.g., T cells) with a viral vector comprising a nucleic acid molecule encoding a CAR.

[0038] In some embodiments, provided herein is a method of generating a population of cells (e.g., T cells) that express a chimeric antigen receptor (CAR), the method comprising: (1) contacting a population of cells (e.g., T cells, e.g., T cells isolated from frozen or fresh leukapheresis products) with a cytokine selected from IL-2, IL-7, IL-15 (e.g., hetIL-15 (IL-15 / sIL-15Ra)), IL-21, IL-6 (e.g., IL-6 / sIL-6Ra) or combinations thereof; (2) contacting the population of cells (e.g., T cells) with a nucleic acid molecule disclosed herein or a nucleic acid molecule encoding a CAR disclosed herein, thereby providing a population of cells (e.g., T cells) that contain the nucleic acid molecule; and (3) harvesting the population of cells (e.g., T cells) for storage (e.g., reconstitution of the population of cells in a cryopreservation medium) or administration. (a) Step (2) is performed together with step (1) or within 5 hours after the start of step (1), e.g., within 1, 2, 3, 4 or 5 hours after the start of step (1), and Step (3) is performed within 26 hours after the start of step (1), e.g., within 22, 23 or 24 hours after the start of step (1), e.g., within 24 hours after the start of step (1), or (b) The population of cells from step (3) is not expanded or is expanded by no more than 5, 10, 15, 20, 25, 30, 35 or 40%, e.g., no more than 10%, as evaluated by the number of viable cells compared to the population of cells at the start of step (1). Optionally, the nucleic acid molecule of step (2) is on a viral vector, optionally, the nucleic acid molecule of step (ii) is an RNA molecule on a viral vector, and optionally, step (ii) comprises transducing a population of cells (e.g., T cells) with a viral vector comprising a nucleic acid molecule encoding a CAR.

[0039] In some embodiments, a population of cells engineered to express a CAR (a “population of CAR-expressing cells”) is disclosed herein, the population comprising: (a) naive cells, such as naive T cells, such as CD45RO−CCR7+ T cells, at approximately the same percentage as in the same population of cells prior to being engineered to express the CAR; (b) a change of within about 5% to about 10% of naive cells, such as naive T cells, such as CD45RO−CCR7+ T cells, as compared to the percentage of naive cells, such as naive T cells, such as CD45RO−CCR7+ T cells in the same population of cells prior to being engineered to express the CAR; (c) an increased percentage of naive cells, such as naive T cells, such as CD45RO−CCR7+ T cells, such as at least 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, or 3-fold increased, as compared to the percentage of naive cells, such as naive T cells, such as CD45RO−CCR7+ T cells in the same population of cells prior to being engineered to express the CAR; (d) central memory cells, such as central memory T cells, such as CCR7+CD45RO+ T cells, at approximately the same percentage as in the same population of cells prior to being engineered to express the CAR; (e) a change of within about 5% to about 10% of central memory cells, such as central memory T cells, such as CCR7+CD45RO+ T cells, as compared to the percentage of central memory cells, such as central memory T cells, such as CCR7+CD45RO+ T cells in the same population of cells prior to being engineered to express the CAR; (f) a decreased percentage of central memory cells, such as central memory T cells, such as CCR7+CD45RO+ T cells, such as at least 20, 25, 30, 35, 40, 45, or 50% decreased, as compared to the percentage of central memory cells, such as central memory T cells, such as CCR7+CD45RO+ T cells in the same population of cells prior to being engineered to express the CAR.(g)A percentage of stem memory T cells, such as CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, that is approximately the same as the percentage of stem memory T cells, such as CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in the same population of cells before being engineered to express a CAR; (h)A change within about 5% to about 10% of stem memory T cells, such as CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, compared to the percentage of stem memory T cells, such as CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in the same population of cells before being engineered to express a CAR; or (i)An increased percentage of stem memory T cells, such as CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, compared to the percentage of stem memory T cells, such as CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in the same population of cells before being engineered to express a CAR. In some embodiments, the population comprises the cells disclosed herein. In some embodiments, the population comprises cells comprising a dual CAR or diabody CAR disclosed herein. In some embodiments, the population comprises cells comprising: (a) a first antigen-binding domain that is an anti-BCMA binding domain, the anti-BCMA binding domain comprising a heavy chain variable region (VH) comprising heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3), and a light chain variable region (VL) comprising light chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2), and light chain complementarity determining region 3 (LC CDR3), wherein HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 comprise: (i) the amino acid sequences of SEQ ID NOs: 86, 130, 88, 95, 131, and 132, respectively; (ii) the amino acid sequences of SEQ ID NOs: 44, 45, 84, 54, 55, and 56, respectively; or (iii) the amino acid sequences of SEQ ID NOs: 179, 180, 181, 147, 182, and 183, respectively, and (b) a second antigen-binding domain.;

[0040] In some embodiments, provided herein is a pharmaceutical composition comprising a cell disclosed herein or a population of cells disclosed herein and a pharmaceutically acceptable carrier.

[0041] In some embodiments, the population of cells is made by the methods disclosed herein. In some embodiments, the population (a) a first cell population comprising an anti-BCMA CAR but not an anti-CD19 CAR; (b) a second cell population comprising an anti-CD19 CAR but not an anti-BCMA CAR; and (c) a third cell population comprising both an anti-BCMA CAR and an anti-CD19 CAR is included.

[0042] In some embodiments, (i) the total number of viable cells of the combined second and third populations is about 110% or less (e.g., about 105%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1% or less) of the total number of viable cells of the combined first and third populations; (ii) the total number of viable cells of the combined first and third populations is about 90% or more (e.g., about 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 750%, 1000%, 2000%, 5000, 10000% or more) of the total number of viable cells of the combined second and third populations; (iii) the total number of viable cells of the combined first and third populations is about 5% or more (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% or more) of the total number of viable cells.

[0043] In some embodiments, the population further includes a fourth cell population that does not contain a CAR.

[0044] In some embodiments, (i) the total number of viable cells in the second population is about 110% or less (e.g., about 105%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1% or less) of the total number of viable cells in the first and third populations combined; (ii) The total number of viable cells in the second population is about 45% to about 50% (e.g., about 47%) or less of the total number of viable cells in the first and third populations combined; about 50% to about 55% (e.g., about 53%) or less; about 60% to about 65% (e.g., about 63%) or less; or about 80% to about 85% (e.g., about 82%) or less.

[0045] In some embodiments, methods of providing anti-tumor immunity in a subject are disclosed herein, which include administering to the subject an effective amount of a cell, a population of cells, or a pharmaceutical composition disclosed herein. In some embodiments, methods of treating a subject having a disease associated with BCMA expression are disclosed herein, which include administering to the subject an effective amount of a cell, a population of cells, or a pharmaceutical composition disclosed herein. In some embodiments, the disease associated with BCMA expression is (i) a cancer or malignant disease or pre-cancerous condition selected from one or more of myelodysplasia, myelodysplastic syndrome or pre-leukemia, or (ii) a non-cancer related indication associated with BCMA expression. In some embodiments, the cancer is a hematological cancer or a solid cancer. In some embodiments, the disease is acute leukemia, B-cell acute lymphoblastic leukemia ("BALL"), T-cell acute lymphoblastic leukemia ("TALL"), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative disorder, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström macroglobulinemia, prostate cancer (e.g., castration-resistant or treatment-resistant prostate cancer or metastatic prostate cancer), pancreatic cancer, lung cancer, plasma cell proliferative disorders (e.g., asymptomatic myeloma (smoldering multiple myeloma or asymptomatic myeloma), monoclonal gammopathy of undetermined significance (MGUS), Waldenström macroglobulinemia, plasmacytoma (e.g., plasma cell dyscrasia, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma and multiple plasmacytoma), systemic amyloid light chain amyloidosis or POEMS syndrome (also known as Crow-Fukase syndrome, Takatsuki disease and PEP syndrome)) or combinations thereof. In some embodiments, the disease is multiple myeloma.

[0046] In some embodiments, the population of cells or pharmaceutical composition is about 1 x 10 6 ~Approx. 1×10 8 (For example, about 2 × 10 6 ~Approx. 5×10 7 , about 5×10 6 ~about 2×10 7 , about 1×10 6 ~Approx. 1×10 7 , about 1×10 7 ~Approx. 1×10 8 , about 1×10 6 ~Approx. 3×10 6 , about 2×10 6 ~Approx. 4×10 6 , about 3×10 6 ~Approx. 5×10 6 , about 4×10 6 ~about 6×10 6 , about 5×10 6 ~Approx. 7×10 6 , about 6×10 6 ~Approx. 8×10 6 , about 7×10 6 ~Approx. 9×10 6 , about 8×10 6 ~Approx. 1×10 7 , about 9×10 6 ~about 2×10 7 , about 1×10 7 ~Approx. 3×10 7 , about 2×10 7 ~Approx. 4×10 7 , about 3×10 7 ~Approx. 5×10 7 , about 4×10 7 ~about 6×10 7 , about 5×10 7 ~Approx. 7×10 7 , about 6×10 7 ~Approx. 8×10 7 , about 7×10 7 ~Approx. 9×10 7 , about 8×10 7 ~Approx. 1×10 8 , about 1×10 6 , about 2×10 6 , about 3×10 6 , about 4×10 6 , about 5×10 6 , about 6×10 6 , about 7×106 , about 8×10 6 , about 9×10 6 , about 1×10 7 , about 2×10 7 , about 3×10 7 , about 4×10 7 , about 5×10 7 , about 6×10 7 , about 7×10 7 , about 8×10 7 , about 9×10 7 or approximately 1 x 10 8 ) viable CAR-positive cells (e.g., BCMA CAR+ T cells). In some embodiments, the population of cells or pharmaceutical composition is administered to a subject at a dose of about 5 x 10 6 ~Approx. 2×10 7 A dose of 1000 CAR-positive viable cells (e.g., BCMA CAR+ T cells) is administered to the subject.

[0047] In some embodiments, the population of cells or pharmaceutical composition is administered to the subject in one or more (e.g., two, three, four, or more) doses. In some embodiments, the population of cells or pharmaceutical composition is administered to the subject in two doses. In some embodiments, the one or more doses include a first dose and a second dose, and the number of viable CAR-positive cells (e.g., BCMA CAR+ T cells) in the first dose is greater than, equal to, or less than the number of viable CAR-positive cells (e.g., BCMA CAR+ T cells) in the second dose.

[0048] In some embodiments, the one or more doses include a first dose and a second dose; (a) The first dose is approximately 1 × 10 6 ~Approx. 1×10 7 (For example, about 2 × 10 6 ~Approx. 8×10 6 , about 4×10 6 ~about 6×10 6 , about 1×10 6 ~Approx. 5×10 6 , about 5×10 6 ~Approx. 1×10 7 , about 1×10 6 ~Approx. 3×10 6 , about 2×10 6~about 4×10 6 、about 3×10 6 ~about 5×10 6 、about 4×10 6 ~about 6×10 6 、about 5×10 6 ~about 7×10 6 、about 6×10 6 ~about 8×10 6 、about 7×10 6 ~about 9×10 6 、about 8×10 6 ~about 1×10 7 、about 1×10 6 、about 2×10 6 、about 3×10 6 、about 4×10 6 、about 5×10 6 、about 6×10 6 、about 7×10 6 、about 8×10 6 、about 9×10 6 or about 1×10 7 ) contain CAR-positive viable cells (e.g., BCMA CAR+ T cells); (b) The second dose is about 1×10 7 ~about 1×10 8 (e.g., about 2×10 7 ~about 8×10 7 、about 4×10 7 ~about 6×10 7 、about 1×10 7 ~about 5×10 7 、about 5×10 7 ~about 1×10 8 、about 1×10 7 ~about 3×10 7 、about 2×10 7 ~about 4×10 7 、about 3×10 7 ~about 5×10 7 、about 4×10 7 ~about 6×10 7 、about 5×10 7 ~about 7×10 7 、about 6×10 7 ~about 8×10 7 、about 7×10 7 ~about 9×10 7 、about 8×10 7 ~about 1×10 8 、about 1×107 , about 2×10 7 , about 3×10 7 , about 4×10 7 , about 5×10 7 , about 6×10 7 , about 7×10 7 , about 8×10 7 , about 9×10 7 or about 1×10 8 ) CAR-positive viable cells (e.g., BCMA CAR+ T cells); (c) The number of CAR-positive viable cells (e.g., BCMA CAR+ T cells) in the first dose is 1 / X (where X is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100) or less than the number of CAR-positive viable cells (e.g., BCMA CAR+ T cells) in the second dose; and / or (d) The number of CAR-positive viable cells (e.g., BCMA CAR+ T cells) in the first dose is about 1% to about 100% (e.g., about 10% to about 90%, about 20% to about 80%, about 30% to about 70%, about 40% to about 60%, about 10% to about 50%, about 50% to about 90%, about 10% to about 30%, about 20% to about 40%, about 30% to about 50%, about 50% to about 70%, about 60% to about 80% or about 70% to about 90%) of the number of CAR-positive viable cells (e.g., BCMA CAR+ T cells) in the second dose.

[0049] In some embodiments, the first dose comprises about 5×10 6 CAR-positive viable cells (e.g., BCMA CAR+ T cells). In some embodiments, the second dose comprises about 1×10 7 to about 2×10 7 CAR-positive viable cells (e.g., BCMA CAR+ T cells).

[0050] In some embodiments, the method further comprises administering a second therapeutic agent to the subject. In some embodiments, the second therapeutic agent is selected from: (i) a PD-1 inhibitor, optionally a PD-1 inhibitor selected from the group consisting of PDR001, nivolumab, pembrolizumab, pidilizumab, MEDI0680, REGN2810, TSR-042, PF-06801591, and AMP-224; (ii) a PD-L1 inhibitor, optionally a PD-L1 inhibitor selected from the group consisting of FAZ053, atezolizumab, avelumab, durvalumab, and BMS-936559; (iii) a LAG-3 inhibitor, optionally a LAG-3 inhibitor selected from the group consisting of LAG525, BMS-986016, TSR-033, MK-4280, and REGN3767; (iv) a TIM-3 inhibitor, optionally a TIM-3 inhibitor selected from the group consisting of MBG453, TSR-022, and LY3321367; (v) a CTLA-4 inhibitor, optionally an ipilimumab or tremelimumab CTLA-4 inhibitor; (vi) an interleukin-15 (IL-15) polypeptide, an interleukin-15 receptor alpha (IL-15Ra) polypeptide, or a combination of both the IL-15 polypeptide and the IL-15Ra polypeptide, such as hetIL-15; (vii) an interleukin-12 (IL-12) polypeptide; or (viii) an mTOR inhibitor, optionally an RAD001 or rapamycin mTOR inhibitor.

[0051] In some embodiments, provided herein are cells comprising: (a) a first chimeric antigen receptor (CAR) comprising a first antigen-binding domain that binds to a first antigen, a first transmembrane domain, and a first intracellular signaling domain (e.g., a first primary signaling domain and / or a first co-stimulatory signaling domain), optionally comprising a first leader sequence and / or a first hinge region; and (b) a second CAR comprising a second antigen-binding domain that binds to a second antigen, a second transmembrane domain, and a second intracellular signaling domain (e.g., a second primary signaling domain and / or a second co-stimulatory signaling domain), optionally comprising a second leader sequence and / or a second hinge region, wherein: (i) the first leader sequence and the second leader sequence are encoded by different (e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100% different) nucleic acid sequences, and optionally, the first and second leader sequences comprise the same amino acid sequence; (ii) the first hinge region and the second hinge region are encoded by different (e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100% different) nucleic acid sequences, and optionally, the first and second hinge regions comprise the same amino acid sequence; (iii) the first transmembrane domain and the second transmembrane domain are encoded by different (e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100% different) nucleic acid sequences, and optionally, the first and second transmembrane domains comprise the same amino acid sequence;and / or (iv) the first intracellular signaling domain and the second intracellular signaling domain are encoded by different (e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100% different) nucleic acid sequences, optionally, the first primary signaling domain and the second primary signaling domain are encoded by different (e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100% different) nucleic acid sequences, and / or the first co-stimulatory signaling domain and the second co-stimulatory signaling domain are encoded by different (e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100% different) nucleic acid sequences.;

[0052] In some embodiments, a nucleic acid molecule comprises: (a) a first nucleic acid sequence encoding a first CAR, wherein the first CAR comprises a first antigen-binding domain that binds a first antigen, a first transmembrane domain, and a first intracellular signaling domain (e.g., a first primary signaling domain and / or a first costimulatory signaling domain), and optionally the first CAR comprises a first leader sequence and / or a first hinge region; and (b) a second nucleic acid sequence encoding a second CAR, wherein the second CAR comprises a second antigen-binding domain that binds a second antigen, a second transmembrane domain, and a first intracellular signaling domain (e.g., a first primary signaling domain and / or a first costimulatory signaling domain), and optionally and a second nucleic acid sequence comprising a first leader sequence and a second intracellular signaling domain (e.g., a second primary signaling domain and / or a second costimulatory signaling domain), and optionally the second CAR comprises a second leader sequence and / or a second hinge region, wherein (i) the first leader sequence and the second leader sequence are encoded by different nucleic acid sequences (e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% different), and optionally wherein the first and second leader sequences comprise the same amino acid sequence; (ii) the first hinge region and the second hinge region are encoded by different (e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% different) nucleic acid sequences, optionally wherein the first and second hinge regions comprise the same amino acid sequence; (iii) the first transmembrane domain and the second transmembrane domain are different (e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, or 100% different). , 90%, 95%, or 100% different) nucleic acid sequences, optionally wherein the first and second transmembrane domains comprise the same amino acid sequence; and / or (iv) the first intracellular signaling domain and the second intracellular signaling domain are encoded by different (e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% different) nucleic acid sequences, optionally wherein the first intracellular signaling domain and the second intracellular signaling domain comprise the same amino acid sequence.

[0053] In some embodiments, the first and second leader sequences comprise the same amino acid sequence. Without being bound by theory, such nucleic acid molecules exhibit lower recombination than similar nucleic acid molecules except where the first and second leader sequences are encoded by the same nucleic acid sequence.

[0054] In some embodiments, the first and second hinge regions comprise the same amino acid sequence. Without being bound by theory, such nucleic acid molecules exhibit lower recombination than similar nucleic acid molecules except where the first and second hinge regions are encoded by the same nucleic acid sequence.

[0055] In some embodiments, the first and second transmembrane domains comprise the same amino acid sequence. Without being bound by theory, such nucleic acid molecules exhibit lower recombination than similar nucleic acid molecules except where the first and second transmembrane domains are encoded by the same nucleic acid sequence.

[0056] In some embodiments, the first and second intracellular signaling domains comprise the same amino acid sequence. Without being bound by theory, such nucleic acid molecules exhibit lower recombination than similar nucleic acid molecules except where the first and second intracellular signaling domains are encoded by the same nucleic acid sequence.

[0057] In some embodiments, the first and second primary signaling domains comprise the same amino acid sequence. Without being bound by theory, such nucleic acid molecules exhibit lower recombination than similar nucleic acid molecules except where the first and second primary signaling domains are encoded by the same nucleic acid sequence.

[0058] In some embodiments, the first and second primary signaling domains comprise different amino acid sequences.

[0059] In some embodiments, the first co-stimulatory signaling domain and the second co-stimulatory signaling domain comprise the same amino acid sequence. Without being bound by theory, such nucleic acid molecules exhibit lower recombination than similar nucleic acid molecules except that the first co-stimulatory signaling domain and the second co-stimulatory signaling domain are encoded by the same nucleic acid sequence.

[0060] In some embodiments, the first co-stimulatory signaling domain and the second co-stimulatory signaling domain comprise different amino acid sequences (e.g., the first and second co-stimulatory signaling domains each comprise a 4-1BB co-stimulatory domain sequence and a CD28 co-stimulatory domain sequence; or each comprise a CD28 co-stimulatory domain sequence and a 4-1BB co-stimulatory domain sequence).

[0061] In some embodiments, the first leader sequence and the second leader sequence comprise the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto. In some embodiments, the first leader sequence and the second leader sequence are encoded by nucleic acid sequences comprising SEQ ID NOs: 199 and 210, respectively (or nucleic acid sequences having at least about 85%, 90%, 95% or 99% sequence identity thereto) or nucleic acid sequences comprising SEQ ID NOs: 210 and 199, respectively (or nucleic acid sequences having at least about 85%, 90%, 95% or 99% sequence identity thereto).

[0062] In some embodiments, the first hinge region and the second hinge region comprise the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto. In some embodiments, the first hinge region and the second hinge region are encoded by nucleic acid sequences comprising SEQ ID NOs: 337 and 13, respectively (or nucleic acid sequences having at least about 85%, 90%, 95% or 99% sequence identity thereto); or nucleic acid sequences comprising SEQ ID NOs: 13 and 337, respectively (or nucleic acid sequences having at least about 85%, 90%, 95% or 99% sequence identity thereto).

[0063] In some embodiments, the first transmembrane domain and the second transmembrane domain comprise the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto. In some embodiments, the first transmembrane domain and the second transmembrane domain are each a nucleic acid sequence comprising SEQ ID NO: 338 and 17 (or a nucleic acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto); or are each encoded by a nucleic acid sequence comprising SEQ ID NO: 17 and 338 (or a nucleic acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto).

[0064] In some embodiments, the first co-stimulatory signaling domain and the second co-stimulatory signaling domain comprise the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto. In some embodiments, the first co-stimulatory signaling domain and the second co-stimulatory signaling domain are each a nucleic acid sequence comprising SEQ ID NO: 204 and 18 (or a nucleic acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto); or are each encoded by a nucleic acid sequence comprising SEQ ID NO: 18 and 204 (or a nucleic acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto).

[0065] In some embodiments, the first primary signaling domain and the second primary signaling domain comprise the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto. In some embodiments, the first primary signaling domain and the second primary signaling domain are each a nucleic acid sequence comprising SEQ ID NO: 205 and 21 (or a nucleic acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto); or are each encoded by a nucleic acid sequence comprising SEQ ID NO: 21 and 205 (or a nucleic acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto).

[0066] In some embodiments, the first and second antigens are different. The first or second antigen may be BCMA, CD19, CD5, CD10, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD30, CD34, CD37, CD38, CD40, CD53, CD69, CD72, CD73, CD74, CD75, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD 85, CD86, CD123, CD135, CD138, CD179, CD269, Flt3, ROR1, FcRn5, FcRn2, CS-1, CXCR4, 5, 7, IL-7 / 3R, IL7 / 4 / 3R or IL4R, and optionally the B cell antigen is selected from CD19, CD20, CD22, FcRn5, FcRn2, CS-1, CD138, CD123, CD 33, CD34, CLL-1, folate receptor beta, FLT3, EGFRvIII, mesothelin, GD2, Tn antigen, sTn antigen, Tn-O-glycopeptide, sTn-O-glycopeptide, PSMA, CD97, TAG72, CD44v6, CEA, EPCAM, KIT, IL-13Ra2, legumain, GD3, CD171, IL-11Ra, PSCA, MAD-CT-1, MAD-CT-2, VEGFR2, Lewis Y, CD24, PDGFR-β, SSEA-4, folate receptor alpha, ERBB (e.g., ERBB2), Her2 / neu, MUC1, EGFR, NCAM, ephrin B2, CAIX, LMP2, sLe, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, FAP, legumain, HPV The antigen is selected from E6 or E7, ML-IAP, CLDN6, TSHR, GPRC5D, ALK, polysialic acid, Fos-related antigen, neutrophil elastase, TRP-2, CYP1B1, sperm protein 17, beta-human chorionic gonadotropin, AFP, thyroglobulin, PLAC1, globo H, RAGE1, MN-CA IX, human telomerase reverse transcriptase, intestinal carboxylesterase, mut hsp 70-2, NA-17, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, NY-ESO-1, GPR20, Ly6k, OR51E2, TARP, GFRα4, or a peptide of any of these antigens presented by MHC.In some embodiments, the first or second antigen-binding domain comprises an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity with a CDR, VH, VL or scFv disclosed herein or thereof.

[0067] In some embodiments, a CAR comprising a first VH (VH1), a first VL (VL1), a second VH (VH2), a second VL (VL2), a transmembrane domain, and an intracellular signaling domain is provided herein, where VH1 and VL1 bind to a first antigen, and VH2 and VL2 bind to a second antigen. In some embodiments, VH1, VL1, VH2, and VL2 are arranged from the N-terminus to the C-terminus in the following configuration: VH1 - optionally linker 1 ("L1") - VH2 - optionally linker 2 ("L2") - VL2 - optionally linker 3 ("L3") - VL1. In some embodiments, VH1, VL1, VH2, and VL2 are arranged from the N-terminus to the C-terminus in the following configuration: VH1 - linker 1 - VL2 - linker 2 - VH2 - linker 3 - VL1. In some embodiments, VH1, VL1, VH2, and VL2 are arranged from the N-terminus to the C-terminus in the following configuration: VL1 - linker 1 - VH2 - linker 2 - VL2 - linker 3 - VH1. In some embodiments, VH1, VL1, VH2, and VL2 are arranged from the N-terminus to the C-terminus in the following configuration: VL1 - linker 1 - VL2 - linker 2 - VH2 - linker 3 - VH1. In some embodiments, VH1, VL1, VH2, and VL2 are arranged from the N-terminus to the C-terminus in the following configuration: VH2 - optionally L1 - VH1 - optionally L2 - VL1 - optionally L3 - VL2. In some embodiments, VH1, VL1, VH2, and VL2 are arranged from the N-terminus to the C-terminus in the following configuration: VH2 - optionally L1 - VL1 - optionally L2 - VH1 - optionally L3 - VL2. In some embodiments, VH1, VL1, VH2, and VL2 are arranged from the N-terminus to the C-terminus in the following configuration: VL2 - linker 1 - VH1 - linker 2 - VL1 - linker 3 - VH2. In some embodiments, VH1, VL1, VH2, and VL2 are arranged from the N-terminus to the C-terminus in the following configuration: VL2 - linker 1 - VL1 - linker 2 - VH1 - linker 3 - VH2. In some embodiments, VH1, VL1, VH2, and VL2 are arranged from the N-terminus to the C-terminus in the following configuration: VH1 - linker 1 ("L1") - VH2 - linker 2 ("L2") - VL2 - linker 3 ("L3") - VL1.In some embodiments, VH1, VL1, VH2, and VL2 are arranged in the following configuration from the N-terminus to the C-terminus: VH1-L1-VL2-L2-VH2-L3-VL1. In some embodiments, VH1, VL1, VH2, and VL2 are arranged in the following configuration from the N-terminus to the C-terminus: VL1-L1-VH2-L2-VL2-L3-VH1. In some embodiments, VH1, VL1, VH2, and VL2 are arranged in the following configuration from the N-terminus to the C-terminus: VL1-L1-VL2-L2-VH2-L3-VH1. In some embodiments, VH1, VL1, VH2, and VL2 are arranged in the following configuration from the N-terminus to the C-terminus: VH2-L1-VH1-L2-VL1-L3-VL2. In some embodiments, VH1, VL1, VH2, and VL2 are arranged in the following configuration from the N-terminus to the C-terminus: VH2-L1-VL1-L2-VH1-L3-VL2. In some embodiments, VH1, VL1, VH2, and VL2 are arranged in the following configuration from the N-terminus to the C-terminus: VL2-L1-VH1-L2-VL1-L3-VH2. In some embodiments, VH1, VL1, VH2, and VL2 are arranged in the following configuration from the N-terminus to the C-terminus: VL2-L1-VH1-L2-VL1-L3-VH2. In some embodiments, L1 or L3 comprises the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, L2 comprises the amino acid sequence of SEQ ID NO: 63 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.In some embodiments, the CAR comprises the following configuration from the N-terminus to the C-terminus: (i) VH1 - optionally linker 1 (“L1”) - VH2 - optionally linker 2 (“L2”) - VL2 - optionally linker 3 (“L3”) - VL1 - optionally hinge region - transmembrane domain - intracellular signaling domain; (ii) VH1 - optionally L1 - VL2 - optionally L2 - VH2 - optionally L3 - VL1 - optionally hinge region - transmembrane domain - intracellular signaling domain; (iii) VL1 - optionally L1 - VH2 - optionally L2 - VL2 - optionally L3 - VH1 - optionally hinge region - transmembrane domain - intracellular signaling domain; (iv) VL1 - optionally L1 - VL2 - optionally L2 - VH2 - optionally L3 - VH1 - optionally hinge region - transmembrane domain - intracellular signaling domain; (v) VH2 - optionally L1 - VH1 - optionally L2 - VL1 - optionally L3 - VL2 - optionally hinge region - transmembrane domain - intracellular signaling domain; (vi) VH2 - optionally L1 - VL1 - optionally L2 - VH1 - optionally L3 - VL2 - optionally hinge region - transmembrane domain - intracellular signaling domain; (vii) VL2 - optionally L1 - VH1 - optionally L2 - VL1 - optionally L3 - VH2 - optionally hinge region - transmembrane domain - intracellular signaling domain; or (viii) VL2 - optionally L1 - VL1 - optionally L2 - VH1 - optionally L3 - VH2 - optionally hinge region - transmembrane domain - intracellular signaling domain. In some embodiments, the first and second antigens are different.In some embodiments, the first or second antigen is BCMA, CD19, CD5, CD10, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD30, CD34, CD37, CD38, CD40, CD53, CD69, CD72, CD73, CD74, CD75, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83 , CD84, CD85, CD86, CD123, CD135, CD138, CD179, CD269, Flt3, ROR1, FcRn5, FcRn2, CS-1, CXCR4, 5, 7, IL-7 / 3R, IL7 / 4 / 3R or IL4R, and optionally the B cell antigen is selected from CD19, CD20, CD22, FcRn5, FcRn2, CS-1, CD138, CD1 23, CD33, CD34, CLL-1, folate receptor β, FLT3, EGFRvIII, mesothelin, GD2, Tn antigen, sTn antigen, Tn-O-glycopeptide, sTn-O-glycopeptide, PSMA, CD97, TAG72, CD44v6, CEA, EPCAM, KIT, IL-13Ra2, Legman, GD3, CD171, IL-11Ra, PSCA, MAD-CT- 1, MAD-CT-2, VEGFR2, Lewis Y, CD24, PDGFR-β, SSEA-4, folate receptor α, ERBB (e.g., ERBB2), Her2 / neu, MUC1, EGFR, NCAM, ephrin B2, CAIX, LMP2, sLe, HMWMAA, o-acetyl-GD2, folate receptor β, TEM1 / CD248, TEM7R, FAP, legumain, HPV The antigen is selected from E6 or E7, ML-IAP, CLDN6, TSHR, GPRC5D, ALK, polysialic acid, Fos-related antigen, neutrophil elastase, TRP-2, CYP1B1, sperm protein 17, beta-human chorionic gonadotropin, AFP, thyroglobulin, PLAC1, globo H, RAGE1, MN-CA IX, human telomerase reverse transcriptase, intestinal carboxylesterase, mut hsp 70-2, NA-17, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, NY-ESO-1, GPR20, Ly6k, OR51E2, TARP, GFRα4, or a peptide of any of these antigens presented by MHC.In some embodiments, VH1, VL1, VH2 or VL2 comprises a CDR, VH or VL sequence disclosed herein or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto. In some embodiments, the hinge region, transmembrane domain or intracellular signaling domain (e.g., primary signaling domain and / or costimulatory signaling domain) is a hinge region sequence, transmembrane domain sequence or intracellular signaling domain sequence (e.g., primary signaling domain sequence and / or costimulatory signaling domain sequence) disclosed herein or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto.

[0068] In some embodiments, nucleic acid molecules encoding the diabody CARs disclosed herein are provided herein. In some embodiments, vectors comprising nucleic acid molecules encoding the diabody CARs disclosed herein are provided herein. In some embodiments, cells comprising a CAR disclosed herein, a diabody CAR disclosed herein or a vector comprising a nucleic acid molecule encoding a diabody CAR disclosed herein are provided herein. In some embodiments, pharmaceutical compositions comprising a diabody CAR disclosed herein and a pharmaceutically acceptable carrier are provided herein. In some embodiments, methods of making cells comprising a diabody CAR disclosed herein are disclosed herein. In some embodiments, methods of treating a subject, e.g., a subject having cancer, using cells comprising a diabody CAR disclosed herein are disclosed herein.

[0069] In some embodiments, the present disclosure relates to methods of generating immune effector cells (e.g., T cells or NK cells) engineered to express a CAR, and compositions generated using such methods. Using the methods disclosed herein (e.g., the ARM process or cytokine process disclosed herein), cells expressing the dual CAR or diabody CAR disclosed herein can be generated. Also disclosed are methods of using such compositions to treat a subject's disease, such as cancer.

[0070] In some embodiments, the present invention features a method of generating a population of cells (e.g., T cells) that express a chimeric antigen receptor (CAR). The method includes: (i) contacting a population of cells (e.g., T cells, such as T cells isolated from frozen or fresh leukapheresis products) with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates co-stimulatory molecules on the surface of the cells (e.g., binding); (ii) contacting the population of cells (e.g., T cells) with a nucleic acid molecule encoding the CAR (e.g., a DNA or RNA molecule), thereby providing a population of cells (e.g., T cells) that contain the nucleic acid molecule; and (iii) harvesting the population of cells (e.g., T cells) for storage (e.g., re-formulation of the population of cells in a cryopreservation medium) or administration. (a) Step (ii) is performed together with step (i) or within 20 hours after the start of step (i), such as within 12, 13, 14, 15, 16, 17, or 18 hours after the start of step (i), such as within 18 hours after the start of step (i), and step (iii) is performed within 26 hours after the start of step (i), such as within 22, 23, 24, or 25 hours after the start of step (i), such as within 24 hours after the start of step (i); (b) Step (ii) is performed together with step (i) or within 20 hours after the start of step (i), such as within 12, 13, 14, 15, 16, 17, or 18 hours after the start of step (i), such as within 18 hours after the start of step (i), and step (iii) is performed within 30 hours after the start of step (ii), such as within 22, 23, 24, 25, 26, 27, 28, 29, or 30 hours after the start of step (ii); or (c) The population of cells from step (iii) is not increased or is increased by no more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40%, such as no more than 10%, as evaluated by the number of viable cells, compared to the population of cells at the start of step (i). In some embodiments, the nucleic acid molecule in step (ii) is a DNA molecule. In some embodiments, the nucleic acid molecule in step (ii) is an RNA molecule.In some embodiments, the nucleic acid molecule in step (ii) is on a viral vector, e.g., a viral vector selected from a lentiviral vector, an adenoviral vector, or a retroviral vector. In some embodiments, the nucleic acid molecule in step (ii) is on a non-viral vector. In some embodiments, the nucleic acid molecule in step (ii) is on a plasmid. In some embodiments, the nucleic acid molecule in step (ii) is not on any vector. In some embodiments, step (ii) comprises transducing a population of cells (e.g., T cells) with a viral vector comprising a nucleic acid molecule encoding a CAR. In some embodiments, step (ii) is performed together with step (i). In some embodiments, step (ii) is performed within 20 hours after the initiation of step (i). In some embodiments, step (ii) is performed within 12, 13, 14, 15, 16, 17, or 18 hours after the initiation of step (i). In some embodiments, step (ii) is performed within 18 hours after the initiation of step (i). In some embodiments, step (iii) is performed within 26 hours after the initiation of step (i). In some embodiments, step (iii) is performed within 22, 23, 24, or 25 hours after the initiation of step (i). In some embodiments, step (iii) is performed within 24 hours after the initiation of step (i). In some embodiments, step (iii) is performed within 30 hours after the initiation of step (ii). In some embodiments, step (iii) is performed within 22, 23, 24, 25, 26, 27, 28, 29, or 30 hours after the initiation of step (ii). In some embodiments, the nucleic acid molecule encoding a CAR is a nucleic acid molecule disclosed herein. In some embodiments, the nucleic acid molecule comprises a first nucleic acid sequence encoding a first CAR and a second nucleic acid sequence encoding a second CAR. In some embodiments, the first and second nucleic acid sequences are located on a single nucleic acid molecule, for example, the first nucleic acid sequence and the second nucleic acid sequence are separated by a third nucleic acid sequence encoding a self-cleavage site (e.g., a P2A site, a T2A site, an E2A site, or an F2A site). In some embodiments, the first and second nucleic acid sequences are located on separate nucleic acid molecules.In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding a CAR, the CAR comprising a first VH (VH1), a first VL (VL1), a second VH (VH2), a second VL (VL2), a transmembrane domain, and an intracellular signaling domain, wherein VH1 and VL1 bind to a first antigen, and VH2 and VL2 bind to a second antigen, and VH1, VL1, VH2, and VL2 are arranged, from the N-terminus to the C-terminus, in the following configurations: VH1 - optionally linker 1 ("L1") - VH2 - optionally linker 2 ("L2") - VL2 - optionally linker 3 ("L3") - VL1, VH1 - optionally L1 - VL2 - optionally L2 - VH2 - optionally L3 - VL1, VL1 - optionally L1 - VH2 - optionally L2 - VL2 - optionally L3 - VH1, VL1 - optionally L1 - VL2 - optionally L2 - VH2 - optionally L3 - VH1, VH2 - optionally L1 - VH1 - optionally L2 - VL1 - optionally L3 - VL2, VH2 - optionally L1 - VL1 - optionally L2 - VH1 - optionally L3 - VL2, VL2 - optionally L1 - VH1 - optionally L2 - VL1 - optionally L3 - VH2; or VL2 - optionally L1 - VL1 - optionally L2 - VH1 - optionally L3 - VH2.

[0071] In some embodiments, the population of cells from step (iii) is not expanded. In some embodiments, the population of cells from step (iii) is expanded by 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40% or less, as evaluated by the number of viable cells, compared to the population of cells at the start of step (i). In some embodiments, the population of cells from step (iii) is expanded by 10% or less, as evaluated by the number of viable cells, compared to the population of cells at the start of step (i).

[0072] In some embodiments, the nucleic acid molecule comprises a first nucleic acid sequence encoding a first CAR and a second nucleic acid sequence encoding a second CAR, wherein the first and second nucleic acid sequences are disposed on separate nucleic acid molecules.

[0073] In some embodiments, the first and second nucleic acid molecules are on separate viral vectors, and step (ii) comprises transducing a population of cells (e.g., T cells) with a first viral vector comprising a nucleic acid molecule encoding a first CAR and a second viral vector comprising a second nucleic acid molecule encoding a second CAR.

[0074] In some embodiments, the first CAR comprises an anti-BCMA binding domain (e.g., an anti-BCMA CAR), and the second CAR comprises an anti-CD19 binding domain (e.g., an anti-CD19 CAR).

[0075] In some embodiments, in step (ii), the first viral vector is contacted with the cell population at a multiplicity of infection (MOI) that is higher than, equal to, or lower than the MOI when the second viral vector is contacted with the cell population. In some embodiments, in step (ii), the first viral vector is contacted with the cell population at a multiplicity of infection (MOI) that is higher than the MOI when the second viral vector is contacted with the cell population.

[0076] In some embodiments, in step (ii), the first viral vector is contacted with the cell population at a first MOI and the second viral vector is contacted with the cell population at a second MOI such that the resulting cell population comprises a first cell population that comprises an anti-BCMA CAR but not an anti-CD19 CAR, a second cell population that comprises an anti-CD19 CAR but not an anti-BCMA CAR, and a third cell population that comprises both an anti-BCMA CAR and an anti-CD19 CAR. (a) For example, as determined by the method described in Example 10, the total number of viable cells in the combined second and third populations is about 110% or less (e.g., about 105%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1% or less) of the total number of viable cells in the combined first and third populations; (b) For example, as determined by the method described in Example 10, the total number of viable cells combining the first and third populations is about 90% or more of the total number of viable cells combining the second and third populations (e.g., about 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 750%, 1000%, 2000%, 5000, 10000% or more than that); (c) For example, as determined by the method described in Example 10, the total number of viable cells combining the first and third populations is about 5% or more of the total number of viable cells of the resulting population (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% or more); (d) For example, as determined by the method described in Example 10, the total number of viable cells in the second population is about 110% or less of the total number of viable cells combining the first and third populations (e.g., about 105%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1% or less than that); or (e) For example, as determined by the method described in Example 10, the total number of viable cells combining the first and third populations is about 90% or more of the total number of viable cells in the second population (e.g., about 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 750%, 1000%, 2000%, 5000, 10000% or more than that). In some embodiments, in step (ii), the second viral vector is contacted with the cell population at a specific MOI (e.g., at an MOI that is sufficiently lower than the MOI when the first viral vector is contacted with the cell population, whereby in the resulting cell population, the following MOI is achieved: (a) For example, as determined by the method described in Example 10, the total number of viable cells combining the second and third populations is about 110% or less of the total number of viable cells combining the first and third populations (e.g., about 105%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1% or less than that); (b) For example, as determined by the method described in Example 10, the total number of viable cells combining the first and third populations is about 90% or more of the total number of viable cells combining the second and third populations (e.g., about 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 750%, 1000%, 2000%, 5000, 10000% or more than that); (c) For example, as determined by the method described in Example 10, the total number of viable cells combining the first and third populations is about 5% or more of the total number of viable cells of the resulting population (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% or more); (d) For example, as determined by the method described in Example 10, the total number of viable cells in the second population is about 110% or less of the total number of viable cells combining the first and third populations (e.g., about 105%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1% or less than that); or (e) For example, as determined by the method described in Example 10, the total number of viable cells combining the first and third populations is about 90% or more of the total number of viable cells in the second population (e.g., about 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 750%, 1000%, 2000%, 5000, 10000% or more than that).

[0077] In some embodiments, in step (ii), the cell population is contacted with the first viral vector at the first MOI and with the second viral vector at the second MOI, such that the resulting cell population comprises: (a) For example, as determined by the method described in Example 10, the total number of viable cells combining the second and third populations is about 110% or less of the total number of viable cells combining the first and third populations (e.g., about 105%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1% or less than that); (b) For example, as determined by the method described in Example 10, the total number of viable cells combining the first and third populations is about 90% or more of the total number of viable cells combining the second and third populations (e.g., about 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 750%, 1000%, 2000%, 5000, 10000% or more than that); (c) For example, as determined by the method described in Example 10, the total number of viable cells combining the first and third populations is about 5% or more of the total number of viable cells of the resulting population (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% or more); (d) For example, as determined by the method described in Example 10, the total number of viable cells in the second population is about 110% or less of the total number of viable cells combining the first and third populations (e.g., about 105%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1% or less than that); or (e) For example, as determined by the method described in Example 10, the total number of viable cells combining the first and third populations is about 90% or more of the total number of viable cells in the second population (e.g., about 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 750%, 1000%, 2000%, 50:00, 10000% or more than that).

[0078] In some embodiments, in step (ii), the cell population is (a) the first viral vector at an MOI of about 1 to about 10 (e.g., about 2 to about 9, about 3 to about 8, about 4 to about 7, about 5 to about 6, about 1 to about 8, about 1 to about 6, about 1 to about 4, about 8 to about 10, about 6 to about 10, about 4 to about 10, about 1 to 3, about 2 to about 4, about 3 to about 5, about 4 to about 6, about 5 to about 7, about 6 to about 8, about 7 to about 9, about 8 to about 10, about 2.5 to about 5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9 or about 10); (b) A second viral vector at an MOI of about 0.1 to about 5 (e.g., about 0.2 to about 4, about 0.3 to about 3, about 0.4 to about 2, about 0.5 to about 1, about 0.6 to about 0.9, about 0.7 to about 0.8, about 0.1 to about 4, about 0.1 to about 3, about 0.1 to about 2, about 0.1 to about 1, about 0.1 to about 0.5, about 4 to about 5, about 3 to about 5, about 2 to about 5, about 1 to about 5, about 0.5 to about 5, about 0.2 to about 5, about 0.1 to about 0.5, about 0.2 to about 1, about 0.5 to about 2, about 1 to about 3, about 2 to about 4, about 3 to about 5, about 0.5 to about 1, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 2, about 3, about 4 or about 5); (c) A first viral vector at an MOI that is at least 10% (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%) higher than the MOI when contacting the second viral vector with the cell population, or at an MOI that is at least about 1-fold (e.g., at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or 100-fold, e.g., about 2 to about 50-fold, about 3 to 20-fold, about 5 to about 15-fold or about 8 to about 10-fold); and / or (d) A second viral vector at an MOI that is 1 / X (where X is 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100) or less of the MOI when contacting the first viral vector with the cell population and contacting.

[0079] In some embodiments, the first viral vector is contacted with the cell population at an MOI of about 2.5 to about 5. In some embodiments, the second viral vector is contacted with the cell population at an MOI of about 0.5 to about 1.0. In some embodiments, the first viral vector is at an MOI that is about 8-fold to about 10-fold higher than the MOI when contacting the second viral vector with the cell population. In some embodiments, the second viral vector is at an MOI that is 1 / X (where X is 6, 8, 10 or 12) or less of the MOI when contacting the first viral vector with the cell population.

[0080] In some embodiments, in step (ii), the cell population is (a) the first viral vector at an MOI of about 4 to about 5 (e.g., about 4.75); and / or (b) the second viral vector at an MOI of about 0.2 to about 1 (e.g., about 0.5); and bring it into contact with.

[0081] In some embodiments, in step (ii), the cell population is about 1 x 10 8 ~Approx. 5×10 9 (For example, about 2 × 10 8 ~about 2×10 9 or about 4 x 10 8 ~Approx. 1×10 9 In some embodiments, the cells comprise about 1 x 10 total viable cells. 6 ~Approx. 1×10 7 (For example, about 2 × 10 6 ~Approx. 5×10 6 or about 3 x 10 6 ~Approx. 4×10 6 ) viable cells / mL of culture medium.

[0082] In some embodiments, the agent that stimulates the CD3 / TCR complex is an agent that stimulates CD3. In some embodiments, the agent that stimulates a co-stimulatory molecule is an agent that stimulates CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, CD2, CD226, or any combination thereof. In some embodiments, the agent that stimulates a co-stimulatory molecule is an agent that stimulates CD28. In some embodiments, the agent that stimulates the CD3 / TCR complex is selected from an antibody (e.g., a single-domain antibody (e.g., a heavy-chain variable domain antibody), a peptibody, a Fab fragment, or a scFv), a small molecule, or a ligand (e.g., a naturally occurring ligand, a recombinant ligand, or a chimeric ligand). In some embodiments, the agent that stimulates a co-stimulatory molecule is selected from an antibody (e.g., a single-domain antibody (e.g., a heavy-chain variable domain antibody), a peptibody, a Fab fragment, or a scFv), a small molecule, or a ligand (e.g., a naturally occurring ligand, a recombinant ligand, or a chimeric ligand). In some embodiments, the agent that stimulates the CD3 / TCR complex does not include beads. In some embodiments, the agent that stimulates a co-stimulatory molecule does not include beads. In some embodiments, the agent that stimulates the CD3 / TCR complex includes an anti-CD3 antibody. In some embodiments, the agent that stimulates a co-stimulatory molecule includes an anti-CD28 antibody. In some embodiments, the agent that stimulates the CD3 / TCR complex includes an anti-CD3 antibody covalently bound to a colloidal polymeric nanomatrix. In some embodiments, the agent that stimulates a co-stimulatory molecule includes an anti-CD28 antibody covalently bound to a colloidal polymeric nanomatrix. In some embodiments, the agent that stimulates the CD3 / TCR complex and the agent that stimulates a co-stimulatory molecule include T Cell TransAct™.

[0083] In some embodiments, the agent that stimulates the CD3 / TCR complex does not contain a hydrogel. In some embodiments, the agent that stimulates the costimulatory molecule does not contain a hydrogel. In some embodiments, the agent that stimulates the CD3 / TCR complex does not contain alginate. In some embodiments, the agent that stimulates the costimulatory molecule does not contain alginate.

[0084] In some embodiments, the agent that stimulates the CD3 / TCR complex contains a hydrogel. In some embodiments, the agent that stimulates the costimulatory molecule contains a hydrogel. In some embodiments, the agent that stimulates the CD3 / TCR complex contains alginate. In some embodiments, the agent that stimulates the costimulatory molecule contains alginate. In some embodiments, the agent that stimulates the CD3 / TCR complex or the agent that stimulates the costimulatory molecule contains MagCloudz™ from Quad Technologies.

[0085] In some embodiments, step (i) increases the percentage of CAR-expressing cells in the population of cells from step (iii), e.g., the population of cells from step (iii) exhibits a higher percentage of CAR-expressing cells (e.g., at least 10, 20, 30, 40, 50, or 60% higher) compared to cells made by a similar method except without step (i).

[0086] In some embodiments, the percentage of naive cells, such as naive T cells, such as CD45RA+CD45RO-CCR7+ T cells, in the cell population from step (iii) is the same as the percentage of naive cells, such as naive T cells, such as CD45RA+CD45RO-CCR7+ cells, in the cell population at the start of step (i). In some embodiments, the percentage of naive cells, such as naive T cells, such as CD45RA+CD45RO-CCR7+ T cells, in the cell population from step (iii) is different from the percentage of naive cells, such as naive T cells, such as CD45RA+CD45RO-CCR7+ cells, in the cell population at the start of step (i) by 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12% or less. In some embodiments, the percentage of naive cells, such as naive T cells, such as CD45RA+CD45RO-CCR7+ T cells, in the cell population from step (iii) is different from the percentage of naive cells, such as naive T cells, such as CD45RA+CD45RO-CCR7+ cells, in the cell population at the start of step (i) by 5 or 10% or less.

[0087] In some embodiments, the population of cells from step (iii) shows a higher percentage (e.g., at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40% higher) of naive cells, such as naive T cells, such as CD45RA+CD45RO-CCR7+ T cells, compared to cells made by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i). In some embodiments, the population of cells from step (iii) shows a higher percentage (e.g., at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40% higher) of naive cells, such as naive T cells, such as CD45RA+CD45RO-CCR7+ T cells, compared to cells made by a similar method except that it further includes a step of growing a population of cells (e.g., T cells) in vitro for more than 3 days, e.g., for 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii).

[0088] In some embodiments, the percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, in the cell population from step (iii) is the same as the percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, in the cell population at the start of step (i). In some embodiments, the percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, in the cell population from step (iii) is different from the percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, in the cell population at the start of step (i) by 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12% or less. In some embodiments, the percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, in the cell population from step (iii) is different from the percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, in the cell population at the start of step (i) by 5 or 10% or less.

[0089] In some embodiments, the population of cells from step (iii) shows a lower percentage (e.g., at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40% lower) of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, compared to cells made by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i). In some embodiments, the population of cells from step (iii) shows a lower percentage (e.g., at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40% lower) of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, compared to cells made by a similar method except that the population of cells (e.g., T cells) is further grown in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii).

[0090] In some embodiments, the percentage of stem memory T cells, such as CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in the population of cells from step (iii) is increased compared to the percentage of stem memory T cells, such as CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in the population of cells at the start of step (i). In some embodiments, the percentage of CAR-expressing stem memory T cells, such as CAR-expressing CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in the population of cells from step (iii) is increased compared to the percentage of CAR-expressing stem memory T cells, such as CAR-expressing CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in the population of cells at the start of step (i). In some embodiments, the percentage of stem memory T cells, such as CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in the population of cells from step (iii) is higher than the percentage of stem memory T cells, such as CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in a population of cells produced by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), such as more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of step (i). In some embodiments, the percentage of CAR-expressing stem memory T cells, such as CAR-expressing CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in the population of cells from step (iii) is higher than the percentage of CAR-expressing stem memory T cells, such as CAR-expressing CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in cells produced by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), such as more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of step (i).In some embodiments, the percentage of stem memory T cells, such as CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in the population of cells from step (iii) is higher than that in the population of cells produced by a similar method except that the population of cells (e.g., T cells) is grown in vitro for more than 3 days, such as 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii). In some embodiments, the percentage of CAR-expressing stem memory T cells, such as CAR-expressing CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in the population of cells from step (iii) is higher than that in the population of cells produced by a similar method except that the population of cells (e.g., T cells) is grown in vitro for more than 3 days, such as 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii).

[0091] In some embodiments, the median GeneSetScore of the cell population from step (iii) (Up TEM vs. Down TSCM) is approximately the same as or differs by no more than about 25, 50, 75, 100, or 125% (e.g., increased by no more than that) from the median GeneSetScore of the cell population at the start of step (i) (Up TEM vs. Down TSCM). In some embodiments, the median GeneSetScore of the cell population from step (iii) (Up TEM vs. Down TSCM) is lower (e.g., at least about 100, 150, 200, 250, or 300% lower) than the median GeneSetScore of the cell population generated by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i). In some embodiments, the median GeneSetScore of the cell population from step (iii) (Up TEM vs. Down TSCM) is lower (e.g., at least about 100, 150, 200, 250, or 300% lower) than the median GeneSetScore of the cell population generated by a similar method except that the method further includes a step of growing a population of cells (e.g., T cells) in vitro for more than 3 days, e.g., for 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii). In some embodiments, the median GeneSetScore of the cell population from step (iii) (Up Treg vs. Down Teff) is approximately the same as or differs by no more than about 25, 50, 100, 150, or 200% (e.g., increased by no more than that) from the median GeneSetScore of the cell population at the start of step (i) (Up Treg vs. Down Teff).In some embodiments, the median GeneSetScore (Up Treg vs. Down Teff) of the cell population from step (iii) is lower (e.g., at least about 50, 100, 125, 150, or 175% lower) than the median GeneSetScore (Up Treg vs. Down Teff) of the cell population prepared by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i). In some embodiments, the median GeneSetScore (Up Treg vs. Down Teff) of the cell population from step (iii) is lower (e.g., at least about 50, 100, 150, or 175% lower) than the median GeneSetScore (Up Treg vs. Down Teff) of the cell population prepared by a similar method except that the method further includes a step of growing a population of cells (e.g., T cells) in vitro for more than 3 days, e.g., for 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii). In some embodiments, the median GeneSetScore (Down stemness) of the cell population from step (iii) is approximately the same as or differs by no more than about 25, 50, 100, 150, 200, or 250% (e.g., is increased by no more than that) from the median GeneSetScore (Down stemness) of the cell population at the start of step (i). In some embodiments, the median GeneSetScore (Down stemness) of the cell population from step (iii) is lower (e.g., at least about 50, 100, or 125% lower) than the median GeneSetScore (Down stemness) of the cell population prepared by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i).In some embodiments, the median GeneSetScore (Down stemness) of the cell population from step (iii) is lower (e.g., at least about 50, 100, or 125% lower) than the median GeneSetScore (Down stemness) of a cell population produced by a similar method except that the cell population (e.g., T cells) is grown in vitro for more than 3 days, such as 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii). In some embodiments, the median GeneSetScore (Up hypoxia) of the cell population from step (iii) is approximately the same as or differs by no more than about 125, 150, 175, or 200% (e.g., increased by no more than that) from the median GeneSetScore (Up hypoxia) of the cell population at the start of step (i). In some embodiments, the median GeneSetScore (Up hypoxia) of the cell population from step (iii) is lower (e.g., at least about 40, 50, 60, 70, or 80% lower) than the median GeneSetScore (Up hypoxia) of a cell population produced by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), such as more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i). In some embodiments, the median GeneSetScore (Up hypoxia) of the cell population from step (iii) is lower (e.g., at least about 40, 50, 60, 70, or 80% lower) than the median GeneSetScore (Up hypoxia) of a cell population produced by a similar method except that the cell population (e.g., T cells) is grown in vitro for more than 3 days, such as 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii). In some embodiments, the median GeneSetScore (Up autophagy) of the cell population from step (iii) is approximately the same as or differs by no more than about 180, 190, 200, or 210% (e.g., increased by no more than that) from the median GeneSetScore (Up autophagy) of the cell population at the start of step (i).In some embodiments, the median GeneSetScore (Up autophagy) of the population of cells from step (iii) is lower (e.g., at least about 20, 30, or 40% lower) than the median GeneSetScore (Up autophagy) of a population of cells made by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i). In some embodiments, the median GeneSetScore (Up autophagy) of the population of cells from step (iii) is lower (e.g., at least about 20, 30, or 40% lower) than the median GeneSetScore (Up autophagy) of a population of cells made by a similar method except that the population of cells (e.g., T cells) is grown in vitro for more than 3 days, e.g., for 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii).

[0092] In some embodiments, the population of cells from step (iii) secretes IL-2 at a higher level (e.g., at least 2, 4, 6, 8, 10, 12, or 14 times higher) than cells made by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i), or cells made by a similar method except that the population of cells (e.g., T cells) is grown in vitro for more than 3 days, e.g., for 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii), after being incubated with cells expressing an antigen recognized by the CAR.

[0093] In some embodiments, the population of cells from step (iii), after being administered in vivo, persists longer or proliferates at a higher level (e.g., at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90% higher) compared to cells made by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i). In some embodiments, the population of cells from step (iii), after being administered in vivo, persists longer or proliferates at a higher level (e.g., at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90% higher) compared to cells made by a similar method except that it further includes a step of proliferating a population of cells (e.g., T cells) in vitro for more than 3 days, e.g., for 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii).

[0094] In some embodiments, the population of cells from step (iii), after being administered in vivo, has more potent antitumor activity (e.g., at a lower dose, e.g., 0.15×10 6 , 0.2×10 6 , 0.25×10 6 or 0.3×10 6 viable CAR-expressing cells) compared to cells made by a similar method except that step (iii) is performed more than 26 hours after the start of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (i), or compared to cells made by a similar method except that it further includes a step of proliferating a population of cells (e.g., T cells) in vitro for more than 3 days, e.g., for 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii).

[0095] In some embodiments, the population of cells from step (iii) does not grow as evaluated, for example, by the number of viable cells, compared to the population of cells at the start of step (i). In some embodiments, the population of cells from step (iii) decreases, as evaluated, for example, by the number of viable cells, from the number of viable cells in the population of cells at the start of step (i). In some embodiments, the population of cells from step (iii) grows by 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35 or 40% or less as evaluated, for example, by the number of viable cells, compared to the population of cells at the start of step (i). In some embodiments, the population of cells from step (iii) does not grow or grows by less than 0.5, 1, 1.5 or 2 hours, for example less than 1 or 1.5 hours, compared to the population of cells at the start of step (i).

[0096] In some embodiments, steps (i) and (ii) are performed in a cell culture medium containing IL-2, IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)), IL-6 (e.g., IL-6 / sIL-6Ra)), an LSD1 inhibitor or a MALT1 inhibitor. In some embodiments, steps (i) and (ii) are performed in a cell culture medium (e.g., serum-free medium) containing IL-7, IL-21 or a combination thereof. In some embodiments, steps (i) and (ii) are performed in a cell culture medium containing IL-2, IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)), IL-21, IL-7, IL-6 (e.g., IL-6 / sIL-6Ra), an LSD1 inhibitor, a MALT1 inhibitor or a combination thereof. In some embodiments, step (i) is performed in a cell culture medium containing IL-2, IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)), IL-6 (e.g., IL-6 / sIL-6Ra)), an LSD1 inhibitor or a MALT1 inhibitor. In some embodiments, step (ii) is performed in a cell culture medium containing IL-2, IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)), IL-6 (e.g., IL-6 / sIL-6Ra)), an LSD1 inhibitor or a MALT1 inhibitor. In some embodiments, step (i) is performed in a cell culture medium (e.g., serum-free medium) containing IL-7, IL-21 or a combination thereof. In some embodiments, step (ii) is performed in a cell culture medium (e.g., serum-free medium) containing IL-7, IL-21 or a combination thereof. In some embodiments, step (i) is performed in a cell culture medium containing IL-2, IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)), IL-21, IL-7, IL-6 (e.g., IL-6 / sIL-6Ra), an LSD1 inhibitor, a MALT1 inhibitor or a combination thereof. In some embodiments, step (ii) is performed in a cell culture medium containing IL-2, IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)), IL-21, IL-7, IL-6 (e.g., IL-6 / sIL-6Ra), an LSD1 inhibitor, a MALT1 inhibitor or a combination thereof. In some embodiments, the cell culture medium is a serum-free medium containing a serum replacement.In some embodiments, the serum replacement is CTS™ Immune Cell Serum Replacement (ICSR).

[0097] In some embodiments, the foregoing method further includes, prior to step (i), (iv) receiving, from an entity such as a laboratory, hospital, or healthcare provider, a fresh leukapheresis product (or an alternative source of hematopoietic tissue such as a fresh whole blood product, fresh bone marrow product, or fresh tumor or organ biopsy or resection (e.g., a fresh product from thymectomy)).

[0098] In some embodiments, the foregoing method further includes, prior to step (i), (v) isolating a population of cells (e.g., T cells, such as CD8+ and / or CD4+ T cells) to be contacted in step (i) from a fresh leukapheresis product (or an alternative source such as a fresh whole blood product, fresh bone marrow product, or fresh tumor or organ biopsy or resection (e.g., a fresh product from thymectomy)). In some embodiments, step (iii) is performed within 35 hours after the start of step (v), such as within 27, 28, 29, 30, 31, 32, 33, 34, or 35 hours after the start of step (v), such as within 30 hours after the start of step (v). In some embodiments, the population of cells from step (iii) is evaluated by the number of viable cells and does not proliferate or proliferates by 5, 10, 15, 20, 25, 30, 35, or 40% or less, such as 10% or less, compared to the population of cells at the end of step (v).

[0099] In some embodiments, the foregoing method further includes, prior to step (i), receiving cryopreserved T cells isolated from a leukapheresis product (or an alternative source of hematopoietic tissue such as cryopreserved T cells isolated from whole blood, bone marrow, or tumor or organ biopsy or resection (e.g., thymectomy)) from an entity such as a laboratory, hospital, or healthcare provider.

[0100] In some embodiments, prior to step (i), the method further includes the step of receiving, from an entity such as a laboratory, hospital, or healthcare provider, a cryopreserved leukapheresis product (or an alternative source of hematopoietic tissue such as a cryopreserved whole blood product, cryopreserved bone marrow product, or cryopreserved tumor or organ biopsy or resection (e.g., cryopreserved product from thymectomy)).

[0101] In some embodiments, prior to step (i), the method further includes the step of isolating, from a cryopreserved leukapheresis product (or an alternative source of hematopoietic tissue such as a cryopreserved whole blood product, cryopreserved bone marrow product, or cryopreserved tumor or organ biopsy or resection (e.g., cryopreserved product from thymectomy)), a population of cells (e.g., T cells, such as CD8+ and / or CD4+ T cells) to be contacted in step (i). In some embodiments, step (iii) is performed within 35 hours after the start of step (v), such as within 27, 28, 29, 30, 31, 32, 33, 34, or 35 hours after the start of step (v), such as within 30 hours after the start of step (v). In some embodiments, the population of cells from step (iii) is evaluated by the number of viable cells and is not proliferated or is proliferated by no more than 5, 10, 15, 20, 25, 30, 35, or 40%, such as no more than 10%, compared to the population of cells at the end of step (v).

[0102] In some embodiments, the cells from step (iii) are cultured for about 2 to about 4 days, such as about 3 days (e.g., about 72 hours after collection), prior to measuring the CAR expression level in a portion thereof (e.g., measuring the percentage of viable CAR-expressing cells in a portion thereof, such as measuring the percentage of viable anti-BCMA CAR-expressing cells in a portion thereof). In some embodiments, the measurement of CAR expression is performed about 4 days (e.g., 96 hours) after step (ii). In some embodiments, the CAR expression level is measured by flow cytometry.

[0103] In some embodiments, the present invention features a method of generating a population of cells (e.g., T cells) that express a chimeric antigen receptor (CAR), the method comprising: (1) contacting a population of cells (e.g., T cells, such as T cells isolated from a frozen leukapheresis product) with a cytokine selected from IL-2, IL-7, IL-15, IL-21, IL-6, or a combination thereof; (2) contacting the population of cells (e.g., T cells) with a nucleic acid molecule encoding the CAR (e.g., a DNA or RNA molecule), thereby providing a population of cells (e.g., T cells) that contain the nucleic acid molecule; and (3) harvesting the population of cells (e.g., T cells) for storage (e.g., cryopreservation medium reconstitution of the population of cells) or administration, wherein (a) step (2) is performed together with step (1) or within 5 hours after the start of step (1), such as within 1, 2, 3, 4, or 5 hours after the start of step (1); and step (3) is performed within 26 hours after the start of step (1), such as within 22, 23, 24, or 25 hours after the start of step (1), such as within 24 hours after the start of step (1); or (b) the population of cells from step (3) is not increased or is increased by no more than 5, 10, 15, 20, 25, 30, 35, or 40%, such as no more than 10%, as evaluated by the number of viable cells compared to the population of cells at the start of step (1). In some embodiments, the nucleic acid molecule in step (2) is a DNA molecule. In some embodiments, the nucleic acid molecule in step (2) is an RNA molecule. In some embodiments, the nucleic acid molecule in step (2) is on a viral vector selected from a viral vector, such as a lentiviral vector, an adenoviral vector, or a retroviral vector. In some embodiments, the nucleic acid molecule in step (2) is on a non-viral vector. In some embodiments, the nucleic acid molecule in step (2) is on a plasmid. In some embodiments, the nucleic acid molecule in step (2) is not on any vector. In some embodiments, step (2) comprises transducing the population of cells (e.g., T cells) with a viral vector containing the nucleic acid molecule encoding the CAR.In some embodiments, the nucleic acid molecule encoding the CAR is the nucleic acid molecule disclosed herein. In some embodiments, the nucleic acid molecule comprises a first nucleic acid sequence encoding a first CAR and a second nucleic acid sequence encoding a second CAR. In some embodiments, the first and second nucleic acid sequences are arranged on a single nucleic acid molecule, for example, the first nucleic acid sequence and the second nucleic acid sequence are separated by a third nucleic acid sequence encoding a self-cleaving site (e.g., P2A site, T2A site, E2A site or F2A site). In some embodiments, the first and second nucleic acid sequences are arranged on separate nucleic acid molecules. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding a CAR, and the CAR comprises a first VH (VH1), a first VL (VL1), a second VH (VH2), a second VL (VL2), a transmembrane domain and an intracellular signaling domain, wherein VH1 and VL1 bind to a first antigen, and VH2 and VL2 bind to a second antigen, and VH1, VL1, VH2 and VL2 are arranged from the N-terminus to the C-terminus in the following configurations: VH1 - optionally linker 1 (“L1”) - VH2 - optionally linker 2 (“L2”) - VL2 - optionally linker 3 (“L3”) - VL1, VH1 - optionally L1 - VL2 - optionally L2 - VH2 - optionally L3 - VL1, VL1 - optionally L1 - VH2 - optionally L2 - VL2 - optionally L3 - VH1, VL1 - optionally L1 - VL2 - optionally L2 - VH2 - optionally L3 - VH1, VH2 - optionally L1 - VH1 - optionally L2 - VL1 - optionally L3 - VL2, VH2 - optionally L1 - VL1 - optionally L2 - VH1 - optionally L3 - VL2, VL2 - optionally L1 - VH1 - optionally L2 - VL1 - optionally L3 - VH2; or VL2 - optionally L1 - VL1 - optionally L2 - VH1 - optionally L3 - VH2.

[0104] In some embodiments, step (2) is carried out together with step (1). In some embodiments, step (2) is carried out within 5 hours after the start of step (1). In some embodiments, step (2) is carried out within 1, 2, 3, 4 or 5 hours after the start of step (1). In some embodiments, step (3) is carried out within 26 hours after the start of step (1). In some embodiments, step (3) is carried out within 22, 23, 24 or 25 hours after the start of step (1). In some embodiments, step (3) is carried out within 24 hours after the start of step (1).

[0105] In some embodiments, the cell population from step (3) is not increased as evaluated by the number of viable cells, for example, compared to the cell population at the start of step (1). In some embodiments, the cell population from step (3) is increased by 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35 or 40% or less as evaluated by the number of viable cells, for example, compared to the cell population at the start of step (1). In some embodiments, the cell population from step (3) is increased by 10% or less as evaluated by the number of viable cells, for example, compared to the cell population at the start of step (1).

[0106] In some embodiments, step (1) comprises contacting a population of cells (e.g., T cells) with IL-2. In some embodiments, step (1) comprises contacting a population of cells (e.g., T cells) with IL-7. In some embodiments, step (1) comprises contacting a population of cells (e.g., T cells) with IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)). In some embodiments, step (1) comprises contacting a population of cells (e.g., T cells) with IL-21. In some embodiments, step (1) comprises contacting a population of cells (e.g., T cells) with IL-6 (e.g., IL-6 / sIL-6Ra). In some embodiments, step (1) comprises contacting a population of cells (e.g., T cells) with IL-2 and IL-7. In some embodiments, step (1) comprises contacting a population of cells (e.g., T cells) with IL-2 and IL15 (e.g., hetIL-15 (IL15 / sIL-15Ra)). In some embodiments, step (1) comprises contacting a population of cells (e.g., T cells) with IL-2 and IL-21. In some embodiments, step (1) comprises contacting a population of cells (e.g., T cells) with IL-2 and IL-6 (e.g., IL-6 / sIL-6Ra). In some embodiments, step (1) comprises contacting a population of cells (e.g., T cells) with IL-7 and IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)). In some embodiments, step (1) comprises contacting a population of cells (e.g., T cells) with IL-7 and IL-21. In some embodiments, step (1) comprises contacting a population of cells (e.g., T cells) with IL-7 and IL-6 (e.g., IL-6 / sIL-6Ra). In some embodiments, step (1) comprises contacting a population of cells (e.g., T cells) with IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)) and IL-21. In some embodiments, step (1) comprises contacting a population of cells (e.g., T cells) with IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)) and IL-6 (e.g., IL-6 / sIL-6Ra).In some embodiments, step (1) comprises contacting a population of cells (e.g., T cells) with IL-21 and IL-6 (e.g., IL-6 / sIL-6Ra). In some embodiments, step (1) comprises contacting a population of cells (e.g., T cells) with IL-7, IL15 (e.g., hetIL-15 (IL15 / sIL-15Ra)) and IL-21.

[0107] In some embodiments, the population of cells from step (3) exhibits a higher percentage of naive cells (e.g., at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40% higher) among CAR-expressing cells compared to cells made by a similar method except that the population of cells is further contacted with, for example, an anti-CD3 antibody.

[0108] In some embodiments, the percentage of naive cells, such as naive T cells, such as CD45RA+CD45RO-CCR7+ T cells, in the cell population from step (3) is the same as the percentage of naive cells, such as naive T cells, such as CD45RA+CD45RO-CCR7+ cells, in the cell population at the start of step (1). In some embodiments, the percentage of naive cells, such as naive T cells, such as CD45RA+CD45RO-CCR7+ T cells, in the cell population from step (3) is different from the percentage of naive cells, such as naive T cells, such as CD45RA+CD45RO-CCR7+ cells, in the cell population at the start of step (1) by 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12% or less. In some embodiments, the percentage of naive cells, such as naive T cells, such as CD45RA+CD45RO-CCR7+ T cells, in the cell population from step (3) is different from the percentage of naive cells, such as naive T cells, such as CD45RA+CD45RO-CCR7+ cells, in the cell population at the start of step (1) by 5 or 10% or less. In some embodiments, the percentage of naive cells, such as naive T cells, such as CD45RA+CD45RO-CCR7+ T cells, in the cell population from step (3) is increased compared to the percentage of naive cells, such as naive T cells, such as CD45RA+CD45RO-CCR7+ cells, in the cell population at the start of step (1). In some embodiments, the percentage of naive cells, such as naive T cells, such as CD45RA+CD45RO-CCR7+ T cells, in the cell population from step (3) is increased by at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20% compared to the percentage of naive cells, such as naive T cells, such as CD45RA+CD45RO-CCR7+ cells, in the cell population at the start of step (1).In some embodiments, the percentage of naive cells, such as naive T cells, such as CD45RA+CD45RO-CCR7+ T cells, in the cell population from step (3) is increased by at least 10 or 20% compared to the percentage of naive cells, such as naive T cells, such as CD45RA+CD45RO-CCR7+ cells, in the cell population at the start of step (1).

[0109] In some embodiments, the cell population from step (3) shows a higher percentage (e.g., at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40% higher) of naive cells, such as naive T cells, such as CD45RA+CD45RO-CCR7+ T cells, compared to cells produced by a similar method except that step (3) is performed more than 26 hours after the start of step (1), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (1). In some embodiments, the cell population from step (3) shows a higher percentage (e.g., at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40% higher) of naive cells, such as naive T cells, such as CD45RA+CD45RO-CCR7+ T cells, compared to cells produced by a similar method except that the cell population (e.g., T cells) is grown in vitro for more than 3 days, e.g., for 5, 6, 7, 8, or 9 days, after step (2) and before step (3).

[0110] In some embodiments, the percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, in the cell population from step (3) is the same as the percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, in the cell population at the start of step (i). In some embodiments, the percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, in the cell population from step (3) is different from the percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, in the cell population at the start of step (i) by 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12% or less. In some embodiments, the percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, in the cell population from step (3) is different from the percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, in the cell population at the start of step (i) by 5 or 10% or less. In some embodiments, the percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, in the cell population from step (3) is decreased compared to the percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, in the cell population at the start of step (1). In some embodiments, the percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, in the cell population from step (3) is decreased by at least 10 or 20% compared to the percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, in the cell population at the start of step (1).In some embodiments, the percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, in the cell population from step (3) is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20% less than the percentage of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, in the cell population at the start of step (1).

[0111] In some embodiments, the cell population from step (3) has a lower percentage (e.g., at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35 or 40% lower) of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, compared to cells made by a similar method except that step (3) is performed more than 26 hours after the start of step (1), e.g., more than 5, 6, 7, 8, 9, 10, 11 or 12 days after the start of step (1). In some embodiments, the cell population from step (3) has a lower percentage (e.g., at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35 or 40% lower) of central memory cells, such as central memory T cells, such as CD95+ central memory T cells, compared to cells made by a similar method except that the cell population (e.g., T cells) is grown in vitro for more than 3 days, e.g., 5, 6, 7, 8 or 9 days, after step (2) and before step (3).

[0112] In some embodiments, the cell population from step (3), after being administered in vivo, proliferates at a longer duration or at a higher (e.g., at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90% higher) level compared to cells produced by a similar method except that step (3) is performed more than 26 hours after the start of step (1), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the start of step (1). In some embodiments, the cell population from step (3), after being administered in vivo, proliferates at a longer duration or at a higher (e.g., at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90% higher) level compared to cells produced by a similar method except that it further includes a step of proliferating a cell population (e.g., T cells) in vitro for more than 3 days, e.g., for 5, 6, 7, 8, or 9 days, after step (2) and before step (3).

[0113] In some embodiments, the cell population from step (3) does not proliferate as evaluated, for example, by the number of viable cells, compared to the cell population at the start of step (1). In some embodiments, the cell population from step (3) proliferates by 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40% or less as evaluated, for example, by the number of viable cells, compared to the cell population at the start of step (1). In some embodiments, the cell population from step (3) proliferates by 10% or less as evaluated, for example, by the number of viable cells, compared to the cell population at the start of step (1). In some embodiments, the number of viable cells in the cell population from step (3) decreases as evaluated, for example, by the number of viable cells, from the number of viable cells in the cells at the start of step (1).

[0114] In some embodiments, the cell population from step (3) is not expanded as evaluated, for example, by the number of viable cells, compared to the cell population at the start of step (1). In some embodiments, the cell population from step (3) is expanded by less than 0.5, 1, 1.5, or 2 hours, such as less than 1 or 1.5 hours, compared to the cell population at the start of step (1).

[0115] In some embodiments, the cell population is not contacted in vitro with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates costimulatory molecules on the surface of those cells, or, if contacted, the contacting step is for less than 2 hours, such as 1 hour or less or 1.5 hours or less. In some embodiments, the agent that stimulates the CD3 / TCR complex is an agent that stimulates CD3 (e.g., an anti-CD3 antibody). In some embodiments, the agent that stimulates costimulatory molecules is an agent that stimulates CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, CD2, CD226, or any combination thereof. In some embodiments, the agent that stimulates costimulatory molecules is an agent that stimulates CD28. In some embodiments, the agent that stimulates the CD3 / TCR complex is selected from an antibody (e.g., a single domain antibody (e.g., a heavy chain variable domain antibody), peptibody, Fab fragment, or scFv), a small molecule, or a ligand (e.g., a naturally occurring ligand, a recombinant ligand, or a chimeric ligand).

[0116] In some embodiments, step (1) and / or (2) is performed in a cell culture medium containing 5, 4, 3, 2, 1, or 0% or less serum. In some embodiments, step (1) and / or (2) is performed in a cell culture medium containing 2% or less serum. In some embodiments, step (1) and / or (2) is performed in a cell culture medium containing approximately 2% serum. In some embodiments, step (1) and / or (2) is performed in a cell culture medium containing an LSD1 inhibitor or a MALT1 inhibitor. In some embodiments, step (1) is performed in a cell culture medium containing 5, 4, 3, 2, 1, or 0% or less serum. In some embodiments, step (1) is performed in a cell culture medium containing 2% or less serum. In some embodiments, step (1) is performed in a cell culture medium containing approximately 2% serum. In some embodiments, step (2) is performed in a cell culture medium containing 5, 4, 3, 2, 1, or 0% or less serum. In some embodiments, step (2) is performed in a cell culture medium containing 2% or less serum. In some embodiments, step (2) is performed in a cell culture medium containing approximately 2% serum. In some embodiments, step (1) is performed in a cell culture medium containing an LSD1 inhibitor or a MALT1 inhibitor. In some embodiments, step (2) is performed in a cell culture medium containing an LSD1 inhibitor or a MALT1 inhibitor.

[0117] In some embodiments, the aforementioned method further comprises, prior to step (i), the step of receiving from an entity, such as a laboratory, hospital, or healthcare provider, a fresh leukapheresis product (or an alternative source of hematopoietic tissue such as a fresh whole blood product, a fresh bone marrow product, or a fresh tumor or organ biopsy or resection (e.g., a fresh product from thymectomy)).

[0118] In some embodiments, prior to step (i), the foregoing method further includes the step of isolating, from a source of fresh leukapheresis product (or an alternative source of hematopoietic tissue such as fresh whole blood product, fresh bone marrow product, or fresh tumor or organ biopsy or resection (e.g., fresh product from thymectomy)), a population of cells (e.g., T cells, such as CD8+ and / or CD4+ T cells) to be contacted in step (i). In some embodiments, step (iii) is performed within 35 hours after the start of step (v), such as within 27, 28, 29, 30, 31, 32, 33, 34, or 35 hours after the start of step (v), such as within 30 hours after the start of step (v). In some embodiments, the population of cells from step (iii) is evaluated by the number of viable cells and is not increased or is increased by no more than 5, 10, 15, 20, 25, 30, 35, or 40%, such as no more than 10%, compared to the population of cells at the end of step (v).

[0119] In some embodiments, prior to step (i), the foregoing method further includes the step of receiving, from an entity such as a laboratory, hospital, or healthcare provider, cryopreserved T cells isolated from an alternative source such as a leukapheresis product (or cryopreserved T cells isolated from whole blood, bone marrow, or tumor or organ biopsy or resection (e.g., thymectomy)).

[0120] In some embodiments, prior to step (i), the foregoing method further includes the step of receiving, from an entity such as a laboratory, hospital, or healthcare provider, a cryopreserved leukapheresis product (or an alternative source of hematopoietic tissue such as a cryopreserved whole blood product, cryopreserved bone marrow product, or cryopreserved tumor or organ biopsy or resection (e.g., cryopreserved product from thymectomy)).

[0121] In some embodiments, prior to step (i), the foregoing method further includes isolating a population of cells (e.g., T cells, such as CD8+ and / or CD4+ T cells) to be contacted in step (i) from (v) cryopreserved leukapheresis products (or cryopreserved whole blood products, cryopreserved bone marrow products, or alternative sources of hematopoietic tissue such as cryopreserved tumor or organ biopsies or resections (e.g., cryopreserved products from thymectomy)). In some embodiments, step (iii) is performed within 35 hours after the start of step (v), such as within 27, 28, 29, 30, 31, 32, 33, 34, or 35 hours after the start of step (v), such as within 30 hours after the start of step (v). In some embodiments, the population of cells from step (iii) is evaluated by the number of viable cells and is not increased or is increased by 5, 10, 15, 20, 25, 30, 35, or 40% or less, such as 10% or less, compared to the population of cells at the end of step (v).

[0122] In some embodiments, the population of cells at the start of step (i) or step (1) is enriched for IL6R-expressing cells (e.g., cells positive for IL6Rα and / or IL6Rβ). In some embodiments, the population of cells at the start of step (i) or step (1) comprises 40, 45, 50, 55, 60, 65, or 70% or more IL6R-expressing cells (e.g., cells positive for IL6Rα and / or IL6Rβ).

[0123] In some embodiments, steps (i) and (ii) or steps (1) and (2) are performed in a cell culture medium comprising IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)). In some embodiments, IL-15 increases the ability of the population of cells to proliferate, for example, after 10, 15, 20, or 25 days. In some embodiments, IL-15 increases the percentage of IL6Rβ-expressing cells in the population of cells.

[0124] In some embodiments of the methods described above, the methods are performed in a closed system. In some embodiments, T cell separation, activation, transduction, incubation, and washing are all performed in a closed system. In some embodiments of the methods described above, the methods are performed within individual devices. In some embodiments, T cell separation, activation, and transduction, incubation, and washing are performed within individual devices.

[0125] In some embodiments of the methods described above, the method further comprises adding an adjuvant or a transduction enhancing reagent to the cell culture medium to enhance transduction efficiency. In some embodiments, the adjuvant or transduction reagent comprises a cationic polymer. In some embodiments, the adjuvant or transduction enhancing reagent is selected from LentiBOOST™ (Sirion Biotech), vectofusin-1, F108, hexadimethrine bromide (Polybrene), PEA, Pluronic F68, Pluronic F127, Synperonic, or LentiTrans™. In some embodiments, the adjuvant is LentiBOOST™ (Sirion Biotech).

[0126] In some embodiments of the methods described above, transducing a population of cells (e.g., T cells) with a viral vector comprises subjecting the population of cells and the viral vector to centrifugal force under conditions such that transduction efficiency is enhanced. In one embodiment, the cells are transduced by spinoculation.

[0127] In some embodiments of the methods described above, cells (e.g., T cells) are activated and transduced in a cell culture flask containing a gas permeable membrane at the bottom, the gas permeable membrane supporting a large volume of medium without substantially impairing gas exchange. In some embodiments, cell growth is achieved by access to nutrients by convection, e.g., uninterrupted access.

[0128] In some embodiments of the methods described above, the CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.

[0129] In some embodiments, the antigen-binding domain binds to an antigen selected from CD19, CD20, CD22, BCMA, mesothelin, EGFRvIII, GD2, Tn antigen, sTn antigen, Tn-O-glycopeptide, sTn-O-glycopeptide, PSMA, CD97, TAG72, CD44v6, CEA, EPCAM, KIT, IL-13Ra2, legumain, GD3, CD171, IL-11Ra, PSCA, MAD-CT-1, MAD-CT-2, VEGFR2, Lewis Y, CD24, PDGFR-β, SSEA-4, folate receptor α, ERBB (e.g., ERBB2), Her2 / neu, MUC1, EGFR, NCAM, Ephrin B2, CAIX, LMP2, sLe, HMWMAA, o-acetyl-GD2, folate receptor β, TEM1 / CD248, TEM7R, FAP, legumicin, HPV E6 or E7, ML-IAP, CLDN6, TSHR, GPRC5D, ALK, polysialic acid, Fos-related antigen, neutrophil elastase, TRP-2, CYP1B1, sperm protein 17, β-human chorionic gonadotropin, AFP, thyroglobulin, PLAC1, globo H, RAGE1, MN-CA IX, human telomerase reverse transcriptase, intestinal carboxylesterase, mut hsp 70-2, NA-17, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, NY-ESO-1, GPR20, Ly6k, OR51E2, TARP, GFRα4, or a peptide of any of these antigens presented by MHC. In some embodiments, the antigen-binding domain comprises a CDR, VH, VL, scFv, or CAR sequence disclosed herein. In some embodiments, the antigen-binding domain comprises VH and VL, and VH and VL are linked by a linker, and optionally, the linker comprises the amino acid sequence of SEQ ID NO: 63 or 104.

[0130] In some embodiments, the transmembrane domain comprises the transmembrane domain of a protein selected from the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In some embodiments, the transmembrane domain comprises the transmembrane domain of CD8. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding the transmembrane domain, and the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 17 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0131] In some embodiments, the antigen-binding domain is linked to the transmembrane domain by a hinge region. In some embodiments, the hinge region comprises the amino acid sequence of SEQ ID NO: 2, 3, or 4 or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding the hinge region, and the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 13, 14, or 15 or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0132] In some embodiments, the intracellular signaling domain comprises a primary signaling domain. In some embodiments, the primary signaling domain comprises a functional signaling domain derived from CD3ζ, TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (ICOS), FcεRI, DAP10, DAP12 or CD66d. In some embodiments, the primary signaling domain comprises a functional signaling domain derived from CD3ζ. In some embodiments, the primary signaling domain comprises the amino acid sequence of SEQ ID NO: 9 or 10 or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto. In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding the primary signaling domain, and the nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 20 or 21 or a nucleic acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto.

[0133] In some embodiments, the intracellular signaling domain comprises a co-stimulatory signaling domain. In some embodiments, the co-stimulatory signaling domain is an MHC class I molecule, TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, 4-1BB (CD137), B7-H3, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD28-OX40, CD28-4-1BB or a ligand that specifically binds to CD83. In some embodiments, the co-stimulatory signaling domain comprises a functional signaling domain derived from 4-1BB. In some embodiments, the co-stimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto.In some embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding a co-stimulatory signaling domain, the nucleic acid sequence comprising the nucleic acid sequence of SEQ ID NO: 18 or a nucleic acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto.

[0134] In some embodiments, the intracellular signaling domain comprises a functional signaling domain derived from 4-1BB and a functional signaling domain derived from CD3ζ. In some embodiments, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 7 (or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto) and the amino acid sequence of SEQ ID NO: 9 or 10 (or an amino acid sequence having at least about 85%, 90%, 95% or 99% sequence identity thereto). In some embodiments, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO: 9 or 10.

[0135] In some embodiments, the CAR further comprises a leader sequence comprising the amino acid sequence of SEQ ID NO: 1.

[0136] In some embodiments, the invention features a population of CAR-expressing cells (e.g., autologous or allogeneic CAR-expressing T cells or NK cells) produced by any of the methods described above or any other method disclosed herein. In some embodiments, provided herein is a pharmaceutical composition comprising a population of CAR-expressing cells disclosed herein and a pharmaceutically acceptable carrier.

[0137] In some embodiments, the population comprises (a) a first cell population that comprises an anti-BCMA CAR but does not comprise an anti-CD19 CAR; (b) a second cell population that comprises an anti-CD19 CAR but does not comprise an anti-BCMA CAR; and (c) a third cell population that comprises both an anti-BCMA CAR and an anti-CD19 CAR and.

[0138] In some embodiments, (i) The total number of viable cells combining the second and third populations is about 110% or less (e.g., about 105%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1% or less) of the total number of viable cells combining the first and third populations; (ii) The total number of viable cells combining the first and third populations is about 90% or more (e.g., about 100%, 125%, 150%, 175%, 200%, 250%, 300%, 400%, 500%, 750%, 1000%, 2000%, 5000, 10000% or more) of the total number of viable cells combining the second and third populations; and / or (iii) The total number of viable cells combining the first and third populations is about 5% or more (e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% or more) of the total number of viable cells in the population.

[0139] In some embodiments, the population further comprises a fourth population of cells that do not contain a CAR.

[0140] In some embodiments, (i) The total number of viable cells in the second population is about 110% or less (e.g., about 105%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1% or less) of the total number of viable cells combining the first and third populations; (ii) The total number of viable cells in the second population is about 45% to about 50% (e.g., about 47%) or less; about 50% to about 55% (e.g., about 53%) or less; about 60% to about 65% (e.g., about 63%) or less; or about 80% to about 85% (e.g., about 82%) or less of the total number of viable cells combining the first and third populations.

[0141] In some embodiments, in the final CAR cell product manufactured using the methods described herein, the total amount of beads (e.g., CD4 beads, CD8 beads, and / or TransACT beads) is 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, or 0.5% or less of the total amount of beads added during the manufacturing process.

[0142] In some embodiments, the invention features a population of CAR-expressing cells (e.g., autologous or allogeneic CAR-expressing T cells or NK cells) that includes one or more of the following characteristics: (a) naive cells, e.g., naive T cells, e.g., CD45RO−CCR7+ cells, at a percentage that is approximately the same as the percentage of naive cells, e.g., naive T cells, e.g., CD45RO−CCR7+ cells, in the same population of cells before being engineered to express the CAR; (b) a change in the percentage of naive cells, e.g., naive T cells, e.g., CD45RO−CCR7+ cells, that is within about 5% to about 10% of the percentage of naive cells, e.g., naive T cells, e.g., CD45RO−CCR7+ cells, in the same population of cells before being engineered to express the CAR; (c) an increased percentage of naive cells, e.g., naive T cells, e.g., CD45RO−CCR7+ cells, that is, for example, at least 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, or 3-fold increased compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RO−CCR7+ cells, in the same population of cells before being engineered to express the CAR; (d) central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells, at a percentage that is approximately the same as the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells, in the same population of cells before being engineered to express the CAR; (e) a change in the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells, that is within about 5% to about 10% of the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells, in the same population of cells before being engineered to express the CAR; (f) a decreased percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells, that is, for example, at least 20, 25, 30, 35, 40, 45, or 50% decreased compared to the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells, in the same population of cells before being engineered to express the CAR;(g) A percentage of stem memory T cells, such as CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, that is approximately the same percentage as that of the same population of cells before being engineered to express a CAR; (h) A change within about 5% to about 10% of stem memory T cells, such as CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, compared to the percentage of stem memory T cells, such as CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in the same population of cells before being engineered to express a CAR; or (i) An increased percentage of stem memory T cells, such as CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, compared to the percentage of stem memory T cells, such as CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in the same population of cells before being engineered to express a CAR.;

[0143] In some embodiments, the invention features a population of CAR-expressing cells (e.g., autologous or allogeneic CAR-expressing T cells or NK cells), where (a) the median GeneSetScore of the population of cells (Up TEM vs. Down TSCM) is approximately the same as or differs by no more than about 25, 50, 75, 100, or 125% (e.g., is increased by no more than that) from the median GeneSetScore of the same population of cells (Up TEM vs. Down TSCM) before being engineered to express the CAR; (b) the median GeneSetScore of the population of cells (Up Treg vs. Down Teff) is approximately the same as or differs by no more than about 25, 50, 100, 150, or 200% (e.g., is increased by no more than that) from the median GeneSetScore of the population of cells (Up Treg vs. Down Teff) before being engineered to express the CAR; (c) the median GeneSetScore of the population of cells (Down stemness) is approximately the same as or differs by no more than about 25, 50, 100, 150, 200, or 250% (e.g., is increased by no more than that) from the median GeneSetScore of the population of cells (Down stemness) before being engineered to express the CAR; (d) the median GeneSetScore of the population of cells (Up hypoxia) is approximately the same as or differs by no more than about 125, 150, 175, or 200% (e.g., is increased by no more than that) from the median GeneSetScore of the population of cells (Up hypoxia) before being engineered to express the CAR; or (e) the median GeneSetScore of the population of cells (Up autophagy) is approximately the same as or differs by no more than about 180, 190, 200, or 210% (e.g., is increased by no more than that) from the median GeneSetScore of the population of cells (Up autophagy) before being engineered to express the CAR.

[0144] In some embodiments, the invention features a method of increasing an immune response in a subject, which comprises administering to the subject a population of CAR-expressing cells disclosed herein or a pharmaceutical composition disclosed herein, thereby increasing the immune response in the subject.

[0145] In some embodiments, a method of treating a subject's cancer is disclosed, which includes administering to the subject a population of CAR-expressing cells disclosed herein or a pharmaceutical composition disclosed herein, thereby treating the subject's cancer. In some embodiments, the cancer is, for example, mesothelioma, malignant pleural mesothelioma, non-small cell lung cancer, small cell lung cancer, squamous cell carcinoma, large cell lung cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, esophageal adenocarcinoma, breast cancer, glioblastoma, ovarian cancer, colorectal cancer, prostate cancer, cervical cancer, skin cancer, melanoma, renal cancer, liver cancer, brain tumor, thymoma, sarcoma, carcinoma, uterine cancer, renal cancer, gastrointestinal cancer, urothelial cancer, pharyngeal cancer, head and neck cancer, rectal cancer, esophageal cancer or bladder cancer, a solid cancer selected from one or more of these or its metastasis. In some embodiments, the cancer is, for example, chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), multiple myeloma, acute lymphoblastic leukemia (ALL), Hodgkin lymphoma, B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), small lymphocyte leukemia (SLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma (DLBCL), DLBCL with chronic inflammation, chronic myeloid leukemia, myeloproliferative neoplasm, follicular lymphoma, pediatric follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphocyte proliferative disorder, MALT lymphoma (mucosa-associated lymphoid tissue type extranodal follicular marginal zone lymphoma), marginal zone lymphoma, myelodysplasia, myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström macroglobulinemia, splenic marginal zone lymphoma, splenic lymphoma / leukemia, splenic diffuse red pulp small cell B-cell lymphoma, hairy cell leukemia-variant, lymphoplasmacytic lymphoma, H-chain disease, plasma cell myeloma, solitary bone plasmacytoma, extraosseous plasmacytoma, 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 occurring in HHV8-associated multicentric Castleman disease, primary effusion lymphoma, B-cell lymphoma, acute myeloid leukemia (AML) or an unclassifiable lymphoma, a liquid cancer selected from these.

[0146] In some embodiments, the method further comprises administering a second therapeutic agent to the subject. In some embodiments, the second therapeutic agent is an anti-cancer therapeutic agent, such as a chemotherapeutic agent, radiation therapy, or immunomodulatory therapy. In some embodiments, the second therapeutic agent is IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)).

[0147] In some embodiments, an isolated cell or population of cells produced by the methods disclosed herein, (a) a first nucleic acid molecule encoding a first CAR comprising an anti-BCMA binding domain, a first transmembrane domain, and a first intracellular signaling domain, wherein the anti-BCMA binding domain comprises a heavy chain variable region (VH) comprising heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3), and a light chain variable region (VL) comprising light chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2), and light chain complementarity determining region 3 (LC CDR3), and wherein HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 each comprise the amino acid sequences of SEQ ID NOs: 86, 87, 88, 95, 96, and 97, respectively, the first nucleic acid molecule; (b) a second nucleic acid molecule encoding a second CAR comprising an anti-CD19 binding domain, a second transmembrane domain, and a second intracellular signaling domain, wherein the anti-CD19 binding domain comprises a VH comprising HC CDR1, HC CDR2, and HC CDR3, and a VL comprising LC CDR1, LC CDR2, and LC CDR3, and wherein HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 each comprise the amino acid sequences of SEQ ID NOs: 295, 304, and 297-300, respectively, the second nucleic acid molecule is provided herein.

[0148] In some embodiments, an isolated cell, (a) A first nucleic acid molecule encoding a first CAR comprising an anti-BCMA binding domain, a first transmembrane domain, and a first intracellular signaling domain, wherein the anti-BCMA binding domain comprises a heavy chain variable region (VH) comprising heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3), and a light chain variable region (VL) comprising light chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2), and light chain complementarity determining region 3 (LC CDR3), and HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 each comprise the amino acid sequences of SEQ ID NOs: 86, 87, 88, 95, 96, and 97, respectively, the first nucleic acid molecule; (b) A second nucleic acid molecule encoding a second CAR comprising an anti-CD19 binding domain, a second transmembrane domain, and a second intracellular signaling domain, wherein the anti-CD19 binding domain comprises a VH comprising HC CDR1, HC CDR2, and HC CDR3, and a VL comprising LC CDR1, LC CDR2, and LC CDR3, and HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 each comprise the amino acid sequences of SEQ ID NOs: 295, 304, and 297 - 300, respectively, the second nucleic acid molecule Isolated cells comprising the same are provided herein.

[0149] In some embodiments, the VH and VL of the anti-BCMA binding domain comprise the amino acid sequences of SEQ ID NO: 93 and 102, respectively. In some embodiments, the VH and VL of the anti-CD19 binding domain comprise the amino acid sequences of SEQ ID NO: 250 and 251, respectively. In some embodiments, the VH and VL of the anti-BCMA binding domain comprise the amino acid sequences of SEQ ID NO: 93 and 102, respectively, and the VH and VL of the anti-CD19 binding domain comprise the amino acid sequences of SEQ ID NO: 250 and 251, respectively. In some embodiments, the anti-BCMA binding domain comprises the amino acid sequence of SEQ ID NO: 105. In some embodiments, the anti-CD19 binding domain comprises the amino acid sequence of SEQ ID NO: 293. In some embodiments, the anti-BCMA binding domain comprises the amino acid sequence of SEQ ID NO: 105, and the anti-CD19 binding domain comprises the amino acid sequence of SEQ ID NO: 293. In some embodiments, the first CAR comprises the amino acid sequence of SEQ ID NO: 107. In some embodiments, the second CAR comprises the amino acid sequence of SEQ ID NO: 225. In some embodiments, the first CAR comprises the amino acid sequence of SEQ ID NO: 107; the second CAR comprises the amino acid sequence of SEQ ID NO: 225. In some embodiments, the first CAR is encoded by the nucleic acid sequence of SEQ ID NO: 259, 258 or 416. In some embodiments, the second CAR is encoded by the nucleic acid sequence of SEQ ID NO: 417, 355, 356 or 354. In some embodiments, the first CAR is encoded by the nucleic acid sequence of SEQ ID NO: 259, 258 or 416, and the second CAR is encoded by the nucleic acid sequence of SEQ ID NO: 417, 355, 356 or 354.

[0150] In some embodiments, pharmaceutical compositions comprising the cells or populations of cells described herein are provided herein.

[0151] In some embodiments, methods are provided herein for providing anti-tumor immunity in a subject or treating a subject having a disease associated with the expression of BCMA, which comprise administering to the subject an effective amount of the cells or cell populations or pharmaceutical compositions described herein.

[0152] In some embodiments, the disease associated with BCMA expression is a blood cancer or a solid cancer, such as the blood cancers or solid cancers described herein.

[0153] In some embodiments, the disease is acute leukemia, B-cell acute lymphoblastic leukemia (“BALL”), T-cell acute lymphoblastic leukemia (“TALL”), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative disorder, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström macroglobulinemia, prostate cancer (e.g., castration-resistant or treatment-resistant prostate cancer or metastatic prostate cancer), pancreatic cancer, lung cancer, plasma cell proliferative disorders (e.g., asymptomatic myeloma (smoldering multiple myeloma or asymptomatic myeloma), monoclonal gammopathy of undetermined significance (MGUS), Waldenström macroglobulinemia, plasmacytoma (e.g., plasma cell dyscrasia, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma and multiple plasmacytomas), systemic amyloid light chain amyloidosis or POEMS syndrome (also known as Crow-Fukase syndrome, Takatsuki disease and PEP syndrome) or combinations thereof.

[0154] In some embodiments, the disease is multiple myeloma.

[0155] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references (e.g., sequence database reference numbers) mentioned herein are hereby incorporated by reference in their entirety. For example, all GenBank, Unigene, and Entrez sequences mentioned herein (e.g., in any table herein) are incorporated by reference. When a gene or protein refers to multiple accession numbers, all sequence variants are included.

[0156] Furthermore, the materials, methods, and examples are illustrative only and not intended to be limiting. Headings, subheadings, or numbered or lettered elements, such as (a), (b), (i), etc., are provided merely for ease of reading. Even if headings or numbered or lettered elements are used herein, steps or elements need not be performed in alphabetical order, nor do steps or elements necessarily have to be distinct from one another. Other features, objects, and advantages of the present invention will become apparent from the description, drawings, and claims.

Brief Description of the Drawings

[0157]

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Best Mode for Carrying Out the Invention

[0158] Definitions Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0159] The terms "a" and "an" refer to one or more than one (i.e., at least one) of the grammatical referents of the article. By way of example, "an element" means one element or more than one element.

[0160] The term "about", when referring to a measurable value such as an amount, a temporal duration, etc., means a variation of ±20%, or in some instances ±10%, or in some instances ±5%, or in some instances ±1%, or in some instances ±0.1% from the specified value, such that such variation is included as being appropriate for the practice of the methods of the present disclosure.

[0161] The compositions and methods of the present invention include polypeptides and nucleic acids having a specified sequence or sequences that are substantially identical or similar thereto, e.g., sequences that are at least 85%, 90% or 95% or more identical to the specified sequence. In connection with amino acid sequences, the term "substantially identical" is used herein to refer to an amino acid sequence of a first and a second amino acid sequence that contains a sufficient or minimal number of amino acid residues that are either i) identical to, or ii) conservative substitutions of, the aligned amino acid residues within the second amino acid sequence such that the first and second amino acid sequences can have a common structural domain and / or a common functional activity, e.g., an amino acid sequence having at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence, e.g., a sequence described herein, and containing a common structural domain.

[0162] In connection with nucleotide sequences, the term "substantially identical" is used herein to refer to a first nucleotide sequence, e.g., a reference sequence, e.g., a sequence described herein, that contains a sufficient or minimum number of nucleotides that are identical to the aligned nucleotides within a second nucleic acid sequence such that the first and second nucleotide sequences encode a polypeptide having a common functional activity, or encode a common structural polypeptide domain or a common functional polypeptide activity, e.g., a nucleotide sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the reference sequence.

[0163] The term "variant" refers to a polypeptide having an amino acid sequence that is substantially identical to a reference amino acid sequence or that is encoded by a substantially identical nucleotide sequence. In some embodiments, the variant is a functional variant.

[0164] The term "functional variant" refers to a polypeptide having an amino acid sequence that is substantially identical to a reference amino acid sequence or that is encoded by a substantially identical nucleotide sequence and that may have one or more activities of the reference amino acid sequence.

[0165] The term cytokine (e.g., IL-2, IL-7, IL-15, IL-21 or IL-6) encompasses full-length naturally occurring cytokines, fragments or variants, such as functional variants (including fragments and functional variants having at least 10%, 30%, 50% or 80% of the activity of a naturally occurring cytokine, such as immunomodulatory activity). In some embodiments, the cytokine is substantially identical (e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to a naturally occurring cytokine or has an amino acid sequence encoded by a nucleotide sequence that is substantially identical (e.g., at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity) to a naturally occurring nucleotide sequence encoding the cytokine. In some embodiments, as understood from the context, the cytokine further comprises a receptor domain, such as a cytokine receptor domain (e.g., IL-15 / IL-15R).

[0166] As used herein, the term "BCMA" refers to B cell maturation antigen. BCMA (also known as TNFRSF17, BCM or CD269) is a member of the tumor necrosis factor receptor (TNFR) family and is expressed primarily on terminally differentiated B cells, such as memory B cells and plasma cells. Its ligands are called B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) of the TNF family. BCMA is involved in mediating the survival of plasma cells for maintaining long-term humoral immunity. The gene for BCMA is encoded on chromosome 16 and produces a primary mRNA transcript 994 nucleotides in length (NCBI accession number NM_001192.2), which encodes a 184 amino acid protein (NP_001183.2). A second antisense transcript derived from the BCMA locus has been described, which may play a specific role in regulating BCMA expression. (Laabi Y. et al., Nucleic Acids Res., 1994, 22:1147-1154). Another transcript variant has been described, but its significance is unclear (Smirnova AS et al. Mol Immunol., 2008, 45(4):1179-1183). A second isoform, also known as TV4, has been identified (Uniprot identifier Q02223-2). As used herein, "BCMA" encompasses proteins that include mutations of full-length wild-type BCMA, such as point mutations, fragments, insertions, deletions and splice variants.

[0167] The phrase "diseases associated with BCMA expression" includes, but is not limited to, diseases associated with cells expressing BCMA (e.g., wild-type or mutant BCMA) or conditions associated with cells expressing BCMA (e.g., wild-type or mutant BCMA), such as proliferative diseases such as cancer or malignant diseases or myelodysplasia, myelodysplastic syndrome or pre-leukemic pre-cancerous conditions such as pre-leukemia; or non-cancer-related indications associated with cells expressing BCMA (e.g., wild-type or mutant BCMA). To avoid misunderstanding, diseases associated with BCMA expression may include, for example, conditions associated with cells that do not currently express BCMA but previously expressed BCMA due to, for example, treatment with a molecule targeting BCMA (e.g., a BCMA inhibitor described herein) such that BCMA expression is downregulated. In one aspect, the cancer associated with BCMA (e.g., wild-type or mutant BCMA) expression is a blood cancer. In one aspect, the blood cancer is leukemia or lymphoma. In one aspect, the cancer associated with BCMA (e.g., wild-type or mutant BCMA) expression is a malignant disease of differentiated plasma B cells. In one aspect, the cancer associated with BCMA (e.g., wild-type or mutant BCMA) expression includes, but is not limited to, acute leukemias such as B-cell acute lymphoblastic leukemia ("BALL"), T-cell acute lymphoblastic leukemia ("TALL"), acute lymphoblastic leukemia (ALL); cancers and malignant diseases including, but not limited to, chronic leukemias such as chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), etc., but is not limited thereto.Another cancer or blood cancer associated with the expression of BCMA (e.g., wild-type or mutant BCMA) includes, but is not limited to, for example, B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative disorders, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndromes, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström macroglobulinemia, and further includes a diverse collection of hematological disorders that coincide with insufficient production (or dysplasia) of myeloid blood cells, such as "preleukemia", etc. In some embodiments, the cancer is multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, or glioblastoma. In some embodiments, the disease associated with the expression of BCMA includes plasma cell proliferative disorders, such as asymptomatic myeloma (smoldering multiple myeloma or asymptomatic myeloma), monoclonal gammopathy of undetermined significance (MGUS), Waldenström macroglobulinemia, plasmacytoma (e.g., plasma cell dysplasia, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma, and multiple plasmacytoma), systemic amyloid light chain amyloidosis, and POEMS syndrome (also known as Crow-Fukase syndrome, Takatsuki disease, and PEP syndrome). Still another disease associated with the expression of BCMA (e.g., wild-type or mutant BCMA) includes, but is not limited to, for example, atypical and / or non-classical cancers, malignant diseases, precancerous states, or proliferative diseases associated with the expression of BCMA (e.g., wild-type or mutant BCMA), such as the cancers described herein, such as prostate cancer (e.g., castration-resistant or treatment-resistant prostate cancer or metastatic prostate cancer), pancreatic cancer, or lung cancer.

[0168] Non-cancer related pathologies associated with the expression of BCMA (e.g., wild-type or mutant BCMA) include viral infections; e.g., HIV, fungal infections; e.g., C. neoformans, autoimmune diseases; e.g., rheumatoid arthritis, systemic lupus erythematosus (SLE or lupus), pemphigus vulgaris and Sjögren's syndrome; inflammatory bowel disease, ulcerative colitis; transplantation-related allo-specific immune disorders related to mucosal immunity; unwanted immune responses to biologics (e.g., factor VIII) when humoral immunity is important. In some embodiments, non-cancer related indications associated with the expression of BCMA include, but are not limited to, for example, autoimmune diseases (e.g., lupus), inflammatory diseases (allergies and asthma), and transplantation. In some embodiments, the tumor antigen-expressing cells expressed or have ever expressed the mRNA encoding the tumor antigen. In one embodiment, the tumor antigen-expressing cells produce a tumor antigen protein (e.g., wild-type or mutant), and the tumor antigen protein may be present at normal or low levels. In one embodiment, the tumor antigen-expressing cells produced a detectable level of tumor antigen protein at one point in time, but then substantially stopped producing detectable tumor antigen protein.

[0169] The term "chimeric antigen receptor" or alternatively "CAR" or "CAR molecule" refers to a recombinant polypeptide construct comprising at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as the "intracellular signaling domain") derived from a stimulatory molecule, as defined below. In some embodiments, the domains in the CAR polypeptide construct are included, for example, as a chimeric fusion protein within the same polypeptide chain. In some embodiments, the domains within the CAR polypeptide construct are not adjacent to each other and are provided, for example, within different polypeptide chains, such as in RCAR as described herein.

[0170] In some embodiments, the cytoplasmic signaling domain comprises a primary signaling domain (e.g., the primary signaling domain of CD3ζ). In some embodiments, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one co-stimulatory molecule as defined below. In some embodiments, the co-stimulatory molecule is selected from 41BB (i.e., CD137), CD27, ICOS, and / or CD28. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a co-stimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising two functional signaling domains derived from one or more co-stimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more co-stimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises an optional leader sequence at the amino terminus (N-terminus) of the CAR fusion protein. In some embodiments, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, which leader sequence is optionally cleaved from the antigen recognition domain (e.g., scFv) during cell processing and localization of the CAR to the cell membrane.

[0171] A chimeric antigen receptor (CAR) that targets a specific tumor antigen X, such as those described herein (where X can be a tumor marker as described herein), and includes an antigen-binding domain (e.g., scFv, single-domain antibody, or TCR (e.g., TCRα-binding domain or TCRβ-binding domain)) is also referred to as an XCAR. For example, a CAR that includes an antigen-binding domain targeting BCMA is referred to as a BCMA CAR. The CAR can be expressed in any cell, such as an immune effector cell (e.g., T cell or NK cell) described herein.

[0172] The term "signaling domain" refers to the functional portion of a protein that acts by transmitting information intracellularly through a specified signaling pathway to regulate cell activity by generating a second messenger or by functioning as an effector in response to such a messenger.

[0173] The term "antibody," as used herein, refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. An antibody can be polyclonal or monoclonal, multichain or single-chain, or an intact immunoglobulin, and can be derived from a natural or recombinant source. An antibody can be a tetramer of immunoglobulin molecules.

[0174] The term "antibody fragment" refers to at least a part of an intact antibody or a recombinant variant thereof, and also refers to an antigen-binding domain sufficient to confer recognition and specific binding of the antibody fragment to a target, such as an antigen, for example, a variable region of an antigen determinant of an intact antibody. Examples of antibody fragments include Fab, Fab’, F(ab’)2 and Fv fragments, scFv antibody fragments, linear antibodies, single domain antibodies (either VL or VH) such as sdAb, camelid VHH domains, and bispecific antibodies formed from antibody fragments such as two or more, for example two, Fab fragments linked by disulfide bridges in the hinge region, or two or more, for example two, isolated CDRs or other epitope fragments of linked antibodies, including but not limited to these. Antibody fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, for example, Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antibody fragments can also be grafted onto scaffolds based on polypeptides such as fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199, which describes fibronectin polypeptide minibodies). The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light chain and heavy chain variable regions are linked in proximity by a short flexible polypeptide linker and can be expressed as a single-chain polypeptide, and the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, as used herein, an scFv can have the VL and VH variable regions in either order with respect to, for example, the N-terminal and C-terminal ends of the polypeptide, and the scFv can comprise VL-linker-VH or VH-linker-VL.

[0175] In some embodiments, the scFv is NH2-V L-Linker-V H -COOH or NH2-V H -Linker-V L may contain the structure of -COOH.

[0176] As used herein, the term "complementary determining region" or "CDR" refers to the amino acid sequences within the antibody variable regions that confer antigen specificity and binding affinity. For example, generally, each heavy chain variable region (e.g., HCDR1, HCDR2, and HCDR3) has three CDRs, and each light chain variable region (LCDR1, LCDR2, and LCDR3) has three CDRs. The exact amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (the "Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 (the "Chothia" numbering scheme), or combinations thereof. In some embodiments, in the combined Kabat and Chothia numbering scheme, the CDRs correspond to amino acid residues that are part of the Kabat CDR, part of the Chothia CDR, or both.

[0177] The portion of the CAR composition of the present invention comprising an antibody or an antibody fragment thereof can exist in various forms. For example, here, the antigen-binding domain can be expressed as part of a polypeptide chain, such as a single-domain antibody fragment (sdAb), a single-chain antibody (scFv), or, for example, a human or humanized antibody (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In some embodiments, the antigen-binding domain of the CAR of the present invention comprises an antibody fragment. In some embodiments, the CAR comprises an antibody fragment comprising an scFv.

[0178] As used herein, the terms "binding domain" or "antibody molecule" (also referred to herein as "anti-target binding domain") refer to a protein comprising at least one immunoglobulin variable domain sequence, such as an immunoglobulin chain or a fragment thereof. The terms "binding domain" or "antibody molecule" encompass antibodies and antibody fragments. In some embodiments, the antibody molecule is a multispecific antibody molecule. For example, this contains a plurality of immunoglobulin variable domain sequences, where the first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and the second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In certain embodiments, the multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity for no more than two antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence having binding specificity for a first epitope and a second immunoglobulin variable domain sequence having binding specificity for a second epitope.

[0179] The terms "bispecific antibody" and "plurality of bispecific antibodies" refer to a molecule that binds the antigen-binding sites of two antibodies within a single molecule. Thus, a bispecific antibody can bind to two different antibodies simultaneously or sequentially. Methods for making bispecific antibodies are known in the art. Various formats for linking two antibodies are also known in the art. As forms of the bispecific antibodies of the present invention, diabodies, single-chain antibodies, Fab dimerization (Fab-Fab), Fab-scFv, and tandem antibodies, among others well known to those skilled in the art, may be mentioned, but are not limited thereto.

[0180] The term "antibody heavy chain" refers to the larger of the two polypeptide chains present in an antibody molecule in its naturally occurring conformation, and this usually determines the class to which the antibody belongs.

[0181] The term "antibody light chain" refers to the smaller of the two polypeptide chains present in an antibody molecule in its naturally occurring conformation. Kappa (κ) and lambda (λ) light chains refer to the two major antibody light chain isotypes.

[0182] The term "recombinant antibody" refers to an antibody made using recombinant DNA techniques, such as an antibody expressed by, for example, a bacteriophage or yeast expression system. This term should also be interpreted to mean a DNA molecule encoding an antibody, an antibody-expressing DNA molecule, or an antibody created by the synthesis of an amino acid sequence specifying the antibody, where the DNA or amino acid sequence has been obtained using well-known recombinant DNA or amino acid sequence techniques available in the art.

[0183] The term "antigen" or "Ag" refers to a molecule that elicits an immune response. This immune response may include either or both antibody production or activation of specific immunocompetent cells. One of ordinary skill in the art will understand that any macromolecule, including virtually any protein or peptide, can be an antigen. Additionally, an antigen may be derived from recombinant DNA or genomic DNA. One of ordinary skill in the art will thus understand that any DNA containing a nucleotide sequence or partial nucleotide sequence that encodes a protein that elicits an immune response encodes an "antigen" as the term is used herein. Further, one of ordinary skill in the art will understand that an antigen need not be encoded by the full-length nucleotide sequence of a gene. The present invention includes, but is not limited to, the use of partial nucleotide sequences of two or more genes, and it is readily apparent that those nucleotide sequences may be arranged in various combinations such that they encode a polypeptide that elicits a desired immune response. Additionally, one of ordinary skill in the art will understand that an antigen need not be encoded by a "gene" at all. An antigen may be synthetically created, or obtained from a biological sample, or may even be a macromolecule other than a polypeptide. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or body fluids, along with other biological components.

[0184] The terms "anti-tumor effect" and "anti-cancer effect" are used interchangeably and refer to a biological effect that can manifest by various means including, but not limited to, a decrease in tumor volume or cancer volume, a decrease in the number of tumor cells or cancer cells, a decrease in the number of metastases, an increase in mean lifespan, a reduction in tumor cell proliferation or cancer cell proliferation, a reduction in tumor cell survival or cancer cell survival, or an improvement in various physiological symptoms associated with the cancerous condition. An "anti-tumor effect" or "anti-cancer effect" may also first manifest by the ability of the peptides, polynucleotides, cells, and antibodies of the present invention to prevent the development of tumors or cancer.

[0185] The term "self" refers to any material derived from the same individual that will later be reintroduced into that individual.

[0186] The term "allogeneic" refers to any material derived from a different animal of the same species as the individual into which the material is introduced. Two or more individuals are said to be allogeneic to each other when the genes at one or more loci are not identical. In some embodiments, allogeneic materials from individuals of the same species can be genetically different enough to interact antigenically with each other.

[0187] The term "xenogeneic" refers to a graft derived from an animal of a different species.

[0188] As used herein, the term "apheresis" refers to an extracorporeal procedure approved in the art in which blood from a donor or patient is withdrawn from the donor or patient and passed through a device that separates selected specific components and then the remainder is returned to the donor or patient, for example, by reinfusion. Thus, in the context of an "apheresis sample", it refers to a sample obtained using apheresis.

[0189] The term "cancer" refers to a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein and include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and the like. In some embodiments, the cancers treated by the methods described herein include multiple myeloma, Hodgkin lymphoma, or non-Hodgkin lymphoma.

[0190] The terms "tumor" and "cancer" are used interchangeably herein and, for example, both terms encompass solid and liquid, e.g., diffuse or circulating tumors. As used herein, the terms "cancer" or "tumor" include pre-cancerous as well as malignant cancers and tumors.

[0191] "Derive" or "derived from", as used herein, refers to the relationship between a first molecule and a second molecule. This generally refers to the structural similarity between the first molecule and the second molecule, and does not imply or encompass limiting the method or source of the first molecule with respect to the second molecule from which it is derived. For example, in the case of an intracellular signaling domain derived from the CD3ζ molecule, the intracellular signaling domain retains a sufficient CD3ζ structure to have the required function, i.e., the ability to generate a signal under appropriate conditions. This does not imply or encompass limiting the particular method of making the intracellular signaling domain, e.g., it does not mean that one must start with the CD3ζ sequence to provide the intracellular signaling domain and delete unnecessary sequences or introduce mutations to arrive at the intracellular signaling domain.

[0192] The term "conservative array modification" refers to an amino acid modification in which the binding properties of an antibody or antibody fragment containing the amino acid sequence are not significantly affected or changed. Such conservative modifications include amino acid substitutions, additions, and deletions. The modifications can be introduced into the antibodies or antibody fragments of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Accordingly, one or more amino acid residues within the CAR of the present invention can be replaced with other amino acid residues from the same side chain family, and the altered CAR can be tested using the functional assays described herein.

[0193] In the context of stimulation by stimulatory and / or costimulatory molecules, the term "stimulation" refers to a response, such as a primary or secondary response, induced by the binding of a stimulatory molecule (e.g., the TCR / CD3 complex) and / or a costimulatory molecule (e.g., CD28 or 4-1BB) to its cognate ligand, thereby mediating signal transduction events such as, but not limited to, signal transduction via the TCR / CD3 complex. Stimulation can mediate changes in the expression of specific molecules and / or reorganization of the cytoskeletal structure.

[0194] The term "stimulatory molecule" refers to a molecule that is expressed by a T cell and provides a primary cytoplasmic signaling sequence that regulates the primary activation of the TCR complex for at least some aspects of the T cell signaling pathway. In some embodiments, the ITAM-containing domain within the CAR repeats the signaling of the primary TCR independently of the endogenous TCR complex. In some embodiments, the primary signal is, for example, the primary signal initiated by the binding of the TCR / CD3 complex to an MHC molecule loaded with a peptide, which results in the mediation of a T cell response including, but not limited to, proliferation, activation, differentiation, etc. The primary cytoplasmic signaling sequence that acts in a stimulatory manner (also referred to as the "primary signaling domain") may include an immunoreceptor tyrosine-based activation motif or a signaling motif known as an ITAM. Examples of ITAM-containing primary cytoplasmic signaling sequences that are particularly useful in the present invention include, but are not limited to, those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FcεRI and CD66d, DAP10 and DAP12. In a specific CAR of the present invention, the intracellular signaling domain of any one or more of the CARS of the present invention includes an intracellular signaling sequence, for example, the primary signaling sequence of CD3-ζ. The term "antigen-presenting cell" or "APC" refers to an immune system cell (e.g., B cell, dendritic cell, etc.) such as an accessory cell that presents a foreign antigen complexed with a major histocompatibility complex (MHC) on its surface. A T cell can recognize such a complex using its T cell receptor (TCR). The APC processes the antigen and presents it to the T cell.

[0195] The "intracellular signaling domain", when this term is used herein, refers to the intracellular portion of a molecule. In embodiments, the intracellular signaling domain transduces an effector function signal and directs the cell to perform a particular function. The entire intracellular signaling domain can be used, but 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 a truncated portion can be used in place of the full-length chain as long as it transduces the effector function signal. The term intracellular signaling domain is therefore intended to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

[0196] The intracellular signaling domain generates signals that promote the immune effector functions of CAR-containing cells, such as CAR T cells. Examples of immune effector functions of CAR T cells include cytolytic activity and helper activity, including cytokine secretion.

[0197] In some embodiments, the intracellular signaling domain can include a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from molecules involved in primary or antigen-dependent stimulation. In some embodiments, the intracellular signaling domain can include a co-stimulatory intracellular domain. Exemplary co-stimulatory intracellular signaling domains include those derived from molecules involved in co-stimulatory signals or antigen-independent stimulation. For example, in the case of CAR T cells, the primary intracellular signaling domain can include the cytoplasmic sequence of the T cell receptor, and the co-stimulatory intracellular signaling domain can include the cytoplasmic sequence from a co-receptor or co-stimulatory molecule.

[0198] The primary intracellular signaling domain can include a signaling motif known as an immunoreceptor activation tyrosine motif or ITAM. Examples of primary cytoplasmic signaling sequences that include ITAM include, but are not limited to, those derived from CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FcεRI, CD66d, DAP10, and DAP12.

[0199] The term "ζ" or alternatively "ζ chain", "CD3-ζ", or "TCR-ζ" refers to CD247. Swiss-Prot accession number P20963 provides an exemplary human CD3ζ amino acid sequence. The "ζ stimulation domain" or alternatively the "CD3-ζ stimulation domain" or "TCR-ζ stimulation domain" refers to the stimulation domain of CD3ζ or a variant thereof (e.g., a molecule having a mutation, e.g., a point mutation, a fragment, an insertion, or a deletion). In some embodiments, the cytoplasmic domain of ζ includes residues 52 to 164 of GenBank accession number BAG36664.1 or a variant thereof (e.g., a molecule having a mutation, e.g., a point mutation, a fragment, an insertion, or a deletion). In some embodiments, the "ζ stimulation domain" or "CD3-ζ stimulation domain" is the sequence provided as SEQ ID NO: 9 or 10 or a variant thereof (e.g., a molecule having a mutation, e.g., a point mutation, a fragment, an insertion, or a deletion).

[0200] The term "costimulatory molecule" refers to an cognate binding partner on a T cell that specifically binds to a costimulatory ligand and thereby mediates, among other things, a costimulatory response of the T cell, such as proliferation. Costimulatory molecules are cell surface molecules other than the antigen receptor or its ligand that are required for an efficient immune response. Costimulatory molecules include, but are not limited to, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecule (SLAM protein), activating NK cell receptors, BTLA, Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD28-OX40, CD28-4-1BB, and ligands that specifically bind to CD83.

[0201] The costimulatory intracellular signaling domain refers to the intracellular portion of a costimulatory molecule.

[0202] The intracellular signaling domain can include the entire intracellular portion of the molecule from which it is derived, the entire native intracellular signaling domain, or a functional fragment thereof.

[0203] The term "4-1BB" refers to CDR137 or tumor necrosis factor receptor superfamily member 9. Swiss-Prot accession number P20963 provides an exemplary human 4-1BB amino acid sequence. The "4-1BB co-stimulatory domain" refers to the co-stimulatory domain of 4-1BB or a variant thereof (e.g., a molecule having a mutation, e.g., a point mutation, a fragment, an insertion, or a deletion). In some embodiments, the "4-1BB co-stimulatory domain" is the sequence provided as SEQ ID NO: 7 or a variant thereof (e.g., a molecule having a mutation, e.g., a point mutation, a fragment, an insertion, or a deletion).

[0204] As used herein, the term "immune effector cell" refers to a cell involved in promoting an immune response, e.g., an immune effector response. Examples of immune effector cells include T cells, e.g., α / β T cells and γ / δ T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes.

[0205] As used herein, the term "immune effector function or immune effector response" refers to the function or response of an immune effector cell, e.g., that enhances or promotes an immune attack on a target cell. For example, an immune effector function or response refers to the property of a T cell or NK cell that promotes the death or growth or proliferation inhibition of a target cell. In the case of T cells, primary stimulation and co-stimulation are examples of immune effector functions or responses.

[0206] The term "effector function" refers to a specialized function of a cell. The effector function of a T cell can be, for example, cytolytic activity or helper activity including the secretion of cytokines.

[0207] The term "encode" refers to the unique property of a specific nucleotide sequence within a polynucleotide, such as a gene, cDNA, or mRNA, which serves as a template for the synthesis of other polymers and macromolecules in biological processes having either a defined nucleotide sequence (e.g., rRNA, tRNA, and mRNA) or a defined amino acid sequence, and the resulting biological properties. Thus, a gene, cDNA, or RNA encodes a protein when the protein is produced in a cell or other biological system by transcription and translation of the mRNA corresponding to that gene. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is typically provided in the sequence listing, and the non-coding strand used as the transcription template for the gene or cDNA are said to encode the protein or other product of that cDNA of the gene.

[0208] Unless otherwise specified, the term "nucleotide sequence encoding an amino acid sequence" refers to degenerate versions of each other and includes all nucleotide sequences encoding the same amino acid sequence. The phrase "nucleotide sequence encoding a protein or RNA" may also include introns to the extent that the nucleotide sequence encoding the protein may contain one or more introns in any version thereof.

[0209] The terms "effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to the amount of a compound, formulation, material, or composition described herein that is effective in achieving a particular biological result.

[0210] The term "endogenous" refers to any substance produced from or within an organism, cell, tissue, or system.

[0211] The term "exogenous" refers to any material introduced from outside or produced outside of an organism, cell, tissue, or system.

[0212] The term "expression" refers to the transcription and / or translation of a specific nucleotide sequence. In some embodiments, expression includes the translation of mRNA introduced into a cell.

[0213] The term "transfer vector" refers to a composition comprising an isolated nucleic acid, which can be used for delivering the isolated nucleic acid into a cell. In the art, many vectors are known, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "transfer vector" includes self-replicating plasmids or viruses. This term should also be interpreted to further include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral transfer vectors include but are not limited to adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, etc.

[0214] The term "expression vector" refers to a vector comprising a recombinant polynucleotide that contains an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains cis-acting elements sufficient for expression, and other expression elements can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., those contained in naked or liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0215] The term "lentivirus" refers to a genus of the family Retroviridae. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells, and lentiviruses can deliver a large amount of genetic information into the DNA of host cells, and thus are one of the most efficient methods of gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses.

[0216] The term "lentiviral vector" refers to a vector derived from at least a portion of the lentiviral genome, including, in particular, a self-inactivating lentiviral vector as provided in Milone et al., Mol. Ther. 17(8):1453-1464 (2009). Other examples of lentiviral vectors that can be used clinically include, but are not limited to, for example, the LENTIVECTOR® gene delivery technology from Oxford BioMedica, the LENTIMAX™ vector system from Lentigen, and the like. Non-clinical types of lentiviral vectors are also available and would be known to those of skill in the art.

[0217] The terms "homologous" or "identity" refer to subunit sequence identity between two polymer molecules, e.g., between two nucleic acid molecules such as two DNA molecules or two RNA molecules, or between two polypeptide molecules. When the subunit positions in both of the two molecules are occupied by the same monomer subunit, e.g., when each position in two DNA molecules is occupied by adenine, they are homologous or identical at that position. The homology between two sequences is a direct function of the number of matching or homologous positions. For example, if half of the positions in two sequences (e.g., 5 positions in a 10-subunit-long polymer) are homologous, the two sequences are 50% homologous. If 90% of the positions (e.g., 9 out of 10) match or are homologous, the two sequences are 90% homologous.

[0218] A "humanized" form of a non-human (e.g., mouse) antibody is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (such as the Fv, Fab, Fab’, F(ab’)2 of an antibody or other antigen-binding portion sequence) that includes minimal sequences derived from a non-human immunoglobulin. In most cases, a humanized antibody and its antibody fragment are those in which residues from the recipient's complementarity-determining regions (CDRs) in a human immunoglobulin (recipient antibody or antibody fragment) have been replaced with residues from the CDRs of a non-human species (donor antibody), such as a mouse, rat, or rabbit, that have the desired specificity, affinity, and capacity. In some examples, residues in the Fv framework region (FR) of the human immunoglobulin are replaced with the corresponding non-human residues. Additionally, a humanized antibody / antibody fragment can include residues that are not found in either the recipient antibody or the transferred CDR or framework sequences. These modifications can further refine and optimize the performance of the antibody or antibody fragment. Generally, a humanized antibody or its antibody fragment will include substantially all of at least one and typically two variable domains, with all or substantially all of the CDR regions corresponding to those of a non-human immunoglobulin and all or most of the FR regions being of human immunoglobulin sequence. A humanized antibody or antibody fragment can also include the immunoglobulin constant region (Fc), typically at least a portion of the Fc of a human immunoglobulin. For further details, see Jones et al., Nature, 321:522-525, 1986; Reichmann et al., Nature, 332:323-329, 1988; Presta, Curr. Op. Struct. Biol., 2:593-596, 1992.

[0219] "Fully human" refers to an immunoglobulin such as an antibody or antibody fragment that is of entirely human origin or consists of an amino acid sequence identical to that of an antibody or immunoglobulin in human form.

[0220] The term "isolated" means modified or removed from its natural state. For example, a nucleic acid or peptide that naturally exists in a living animal is not "isolated", but the same nucleic acid or peptide that is partially or completely separated from its coexisting materials in its natural state is "isolated". An isolated nucleic acid or protein can exist in a substantially purified form or can exist in a non-natural environment such as, for example, a host cell.

[0221] In the context of the present invention, the following abbreviations are used with respect to commonly occurring nucleobases. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0222] The term "operably linked" or "transcriptional control" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence that results in the expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functionally related state with respect to the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. DNA sequences that are operably linked may be contiguous with each other and, for example, when it is necessary to join two protein coding regions, are within the same reading frame.

[0223] The term "parenteral" administration of an immunogenic composition includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, intratumoral, or infusion techniques.

[0224] The terms "nucleic acid", "nucleic acid molecule", "polypeptide" or "polynucleotide molecule" refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in either single-stranded or double-stranded form and polymers thereof. Unless specifically limited, this term encompasses nucleic acids including known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in the same manner as naturally occurring nucleotides. In some embodiments, "nucleic acid", "nucleic acid molecule", "polypeptide" or "polynucleotide molecule" includes nucleotide / nucleoside derivatives or analogs. Unless otherwise indicated, a particular nucleic acid sequence implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions, e.g., conservative substitutions), alleles, orthologs, SNPs and complementary sequences as well as the explicitly indicated sequences. Specifically, degenerate codon substitutions, e.g., conservative substitutions, can be achieved by creating sequences in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0225] The terms "peptide," "polypeptide," and "protein" are used synonymously and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can be included, including the sequence of the protein or the sequence of the peptide. A polypeptide includes any peptide or protein that contains two or more amino acids joined to each other by peptide bonds. As used herein, this term refers to both short chains, which are generally also referred to in the art as, for example, peptides, oligopeptides, and oligomers, and the many types of longer chains, which are generally referred to in the art as proteins. "Polypeptide" specifically includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins. Polypeptides include natural peptides, recombinant peptides, or combinations thereof.

[0226] The term "promoter" refers to a DNA sequence recognized by the synthetic machinery of a cell or an introduced synthetic machinery that is necessary to initiate specific transcription of a polynucleotide sequence.

[0227] The term "promoter / regulatory sequence" refers to a nucleic acid sequence necessary for the expression of a gene product operably linked to that promoter / regulatory sequence. In some examples, this sequence can be a core promoter sequence, and in other examples, this sequence can also include enhancer sequences and other regulatory elements necessary for the expression of the gene product. A promoter / regulatory sequence can, for example, be one that expresses a gene product in a tissue-specific manner.

[0228] The term "constitutive" promoter refers to a nucleotide sequence that causes the production of a gene product in a cell under most or all physiological conditions of the cell when operably linked to a polynucleotide encoding or specifying that gene product.

[0229] The term "inducible" promoter refers to a nucleotide sequence that causes production of a gene product in a cell only when an inducer substantially corresponding to the promoter is present in the cell, when operably linked to a polynucleotide encoding or specifying the gene product.

[0230] The term "tissue-specific" promoter refers to a nucleotide sequence that causes production of a gene product in a cell only when the cell is substantially of the tissue type corresponding to the promoter, when operably linked to a polynucleotide encoding or specified by the gene.

[0231] The terms "cancer-associated antigen", "tumor antigen", "hyperproliferative disorder antigen", and "antigen associated with a hyperproliferative disorder" are used interchangeably to refer to antigens common to a particular hyperproliferative disorder. In some embodiments, these terms refer to molecules (typically proteins, carbohydrates, or lipids) that are expressed on the surface of cancer cells, either fully or as fragments (e.g., MHC / peptide), and that are useful for the preferential targeting of pharmacological agents to cancer cells. In some embodiments, a tumor antigen is a marker expressed by both normal and cancer cells, such as a lineage marker, such as CD19 on B cells. In some embodiments, a tumor antigen is a cell surface molecule that is overexpressed in cancer cells compared to normal cells (e.g., 1-fold overexpression, 2-fold overexpression, 3-fold or greater overexpression compared to normal cells). In some embodiments, a tumor antigen is a cell surface molecule that is inappropriately synthesized in cancer cells, such as a molecule that contains deletions, additions, or mutations compared to the molecule expressed in normal cells. In some embodiments, a tumor antigen will be expressed only on the cell surface of cancer cells, either fully or as a fragment (e.g., MHC / peptide), and will not be synthesized or expressed on the surface of normal cells. In some embodiments, the hyperproliferative disorder antigen of the invention is derived from primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, kidney cancer, and breast cancer, prostate cancer (e.g., castration-resistant or treatment-resistant prostate cancer or metastatic prostate cancer), ovarian cancer, pancreatic cancer, etc., or a plasma cell proliferative disorder, such as asymptomatic myeloma (smoldering multiple myeloma or asymptomatic myeloma), monoclonal gammopathy of undetermined significance (MGUS), Waldenström macroglobulinemia, plasmacytoma (e.g., plasma cell dyscrasia, solitary myeloma, solitary plasmacytoma, extramedullary plasmacytoma, and multiple plasmacytoma), systemic light chain amyloidosis, and POEMS syndrome (also known as Crow-Fukase syndrome, Takatsuki disease, and PEP syndrome). In some embodiments, the CAR of the invention comprises a CAR that includes an antigen-binding domain (e.g., an antibody or antibody fragment) that binds to an MHC-presented peptide.Typically, peptides derived from endogenous proteins fit into the pockets of major histocompatibility complex (MHC) class I molecules and are recognized by the T cell receptor (TCR) on CD8+ T lymphocytes. The MHC class I complex is constitutively expressed by all nucleated cells. In cancer, virus-specific and / or tumor-specific peptide / MHC complexes serve as a unique class of cell surface targets for immunotherapy. TCR-like antibodies targeting peptides derived from viral or tumor antigens in the context of human leukocyte antigen (HLA)-A1 or HLA-A2 have been described (see, for example, Sastry et al., J Virol. 2011 85(5):1935-1942; Sergeeva et al., Blood, 2011 117(16):4262-4272; Verma et al., J Immunol 2010 184(4):2156-2165; Willemsen et al., Gene Ther 2001 8(21):1601-1608; Dao et al., Sci Transl Med 2013 5(176):176ra33; Tassev et al., Cancer Gene Ther 2012 19(2):84-100). For example, TCR-like antibodies can be identified by screening libraries such as human scFv phage display libraries.

[0232] The term "tumor-supporting antigen" or "cancer-supporting antigen" refers, interchangeably, to a molecule (typically a protein, carbohydrate, or lipid) that is expressed on the surface of cells that are not themselves cancerous but that support cancer cells, for example, by promoting their growth or survival, such as resistance to immune cells. Exemplary cells of this type include stromal cells and myeloid-derived suppressor cells (MDSC). The tumor-supporting antigen itself need not play a role in supporting tumor cells as long as the antigen is present on the cells that support the cancer cells.

[0233] The term "flexible polypeptide linker" or "linker", when used in connection with an scFv, refers to a peptide linker composed of amino acids such as glycine and / or serine residues used alone or in combination to link together both the variable heavy chain region and the variable light chain region. In some embodiments, the flexible polypeptide linker is a Gly / Ser linker and includes the amino acid sequence (Gly-Gly-Gly-Ser)n, where n is a positive integer of 1 or more, for example n = 1, n = 2, n = 3, n = 4, n = 5 and n = 6, n = 7, n = 8, n = 9 and n = 10 (SEQ ID NO: 41). In some embodiments, the flexible polypeptide linker includes, but is not limited to, (Gly4Ser)4 (SEQ ID NO: 27) or (Gly4Ser)3 (SEQ ID NO: 28). In some embodiments, the linker includes multiple repeats of (Gly2Ser), (GlySer) or (Gly3Ser) (SEQ ID NO: 29). Also included within the scope of the present invention are the linkers described in WO 2012 / 138475 pamphlet (incorporated herein by reference).

[0234] As used herein, a 5' cap (RNA cap, RNA 7-methylguanosine cap or RNA m 7 G cap, also referred to) is a modified guanine nucleotide added to the "front" or 5' end of a eukaryotic messenger RNA immediately after transcription initiation. The 5' cap consists of a terminal group linked to the first transcribed nucleotide. Its presence is important for recognition by ribosomes and protection from RNases. Capping is linked to transcription and occurs cotranscriptionally such that each affects the other. Immediately after transcription initiation, a cap synthesis complex associated with RNA polymerase binds to the 5' end of the mRNA being synthesized. This enzyme complex catalyzes the chemical reactions necessary for mRNA capping. The synthesis proceeds as a multi-step biochemical reaction. The capping moiety can be modified to modulate functions such as the stability or translational efficiency of the mRNA.

[0235] As used herein, "in vitro transcribed RNA" refers to RNA synthesized in vitro. In some embodiments, the RNA is mRNA. Generally, in vitro transcribed RNA is generated from an in vitro transcription vector. The in vitro transcription vector contains a template used for generating the in vitro transcribed RNA.

[0236] As used herein, "poly(A)" is a series of adenosines that are attached to mRNA by polyadenylation. In some embodiments of constructs for transient expression, the poly(A) is from 50 to 5000 (SEQ ID NO: 30). In some embodiments, the poly(A) is more than 64. In some embodiments, the poly(A) is more than 100. In some embodiments, the poly(A) is more than 300. In some embodiments, the poly(A) is more than 400. The poly(A) sequence can be chemically or enzymatically modified to modulate mRNA functions such as localization, stability, or translation efficiency.

[0237] As used herein, "polyadenylation" refers to the covalent attachment of a polyadenylate moiety or a modified variant thereof to a messenger RNA molecule. In eukaryotes, most messenger RNA (mRNA) molecules are polyadenylated at their 3' ends. The 3' poly(A) tail is a long sequence of adenine nucleotides (often several hundred) that is added to the mRNA precursor by the action of an enzyme, polyadenylate polymerase. In higher eukaryotes, the poly(A) tail is added to transcripts containing a specific sequence, the polyadenylation signal. The poly(A) tail and the proteins bound to it help protect the mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, nuclear export of mRNA, and translation. Polyadenylation occurs in the nucleus immediately after transcription from DNA to RNA, but can also occur later in the cytoplasm. After transcription has terminated, the mRNA strand is cleaved by the action of an endonuclease complex associated with RNA polymerase. The cleavage site is usually characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA has been cleaved, adenosine residues are added to the free 3' end of the cleavage site.

[0238] As used herein, "transient" refers to the expression of a transgene that is not integrated over a period of hours, days or weeks, which expression period is shorter than the expression period of a gene when integrated into the genome or contained in a stable plasmid replicon in a host cell.

[0239] As used herein, the terms "treating", "treatment" and "being treated" refer to a reduction or improvement in the progression, severity and / or duration of a proliferative disorder or an improvement in one or more symptoms (preferably one or more recognizable symptoms) of a proliferative disorder brought about by the administration of one or more therapies (e.g., one or more therapeutic agents such as a CAR of the present invention). In a specific embodiment, the terms "treating", "treatment" and "being treated" refer to an improvement in at least one measurable physical parameter of a proliferative disorder, such as tumor growth, that may not necessarily be recognizable to the patient. In other embodiments, the terms "treating", "treatment" and "being treated" refer to either or both of the inhibition of the progression of a proliferative disorder, e.g., by stabilization of a recognizable symptom, and physiological inhibition, e.g., by stabilization of a physical parameter. In other embodiments, the terms "treating", "treatment" and "being treated" refer to a reduction or stabilization of tumor size or the number of cancerous cells.

[0240] The term "signaling pathway" refers to the biochemical relationships among various signaling molecules that play a role in the transmission of signals from one part of a cell to another part of the cell. The phrase "cell surface receptor" includes molecules and molecular complexes that have the ability to receive a signal and transmit the signal across the cell's membrane.

[0241] The term "subject" is intended to include living organisms (e.g., mammals, such as humans) capable of mounting an immune response.

[0242] The term "substantially purified" cells refers to cells that are essentially free of other cell types. Substantially purified cells also refer to cells that are separated from other cell types with which they are normally associated in their natural state. In some examples, a substantially purified cell population refers to a homogeneous cell population. In other examples, the term simply refers to cells that are separated from the cells with which they are naturally associated in their natural state. In some embodiments, these cells are cultured in vitro. In some embodiments, these cells are not cultured in vitro.

[0243] As used herein, the term "therapeutic" means treatment. A therapeutic effect is achieved by reduction, suppression, remission, or eradication of a disease state.

[0244] As used herein, the term "prevention" means the prevention or prophylactic treatment of a disease or condition.

[0245] The terms "transfected", or "transformed", or "transduced" refer to the process of introducing or transferring exogenous nucleic acid into a host cell. A "transfected", or "transformed", or "transduced" cell has been transfected, transformed, or transduced with exogenous nucleic acid. Cells include primary subject cells and their progeny.

[0246] The term "specifically binds" refers to an antibody or ligand that recognizes and binds to a cognate binding partner (e.g., a stimulatory and / or co-stimulatory molecule present on a T cell) protein present in a sample, but does not substantially recognize or bind to other molecules in the sample.

[0247] As used herein, the term "regulatable chimeric antigen receptor (RCAR)" refers to a series of polypeptides, typically two polypeptides in the simplest embodiments, that confer specificity for and intracellular signaling in target cells, typically cancer cells, when present in immune effector cells. In some embodiments, the RCAR comprises at least one extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") that includes a functional signaling domain derived from a stimulatory molecule and / or a co-stimulatory molecule as defined herein in relation to CAR molecules. In some embodiments, these series of polypeptides in the RCAR are not adjacent to each other and are, for example, within different polypeptide chains. In some embodiments, the RCAR comprises a dimerization switch that can bind the polypeptides to each other in the presence of a dimerization molecule, for example, can bind an antigen-binding domain to an intracellular signaling domain. In some embodiments, the RCAR is expressed in a cell described herein (e.g., an immune effector cell), e.g., an RCAR-expressing cell (also referred to as an "RCARX cell"). In some embodiments, the RCARX cell is a T cell and is referred to as an RCART cell. In some embodiments, the RCARX is an NK cell and is referred to as an RCARN cell. The RCAR can confer specificity for and regulatable intracellular signaling or proliferation in RCAR-expressing cells to target cells, typically cancer cells, which can optimize the immune effector properties of the RCAR-expressing cells. In multiple embodiments, the RCAR cell acquires specificity for a target cell comprising an antigen bound to the antigen-binding domain at least in part depending on the antigen-binding domain.

[0248] "Membrane anchor" or "membrane tethering domain," when the term is used herein, refers to a polypeptide or moiety sufficient to bind an extracellular or intracellular domain to the plasma membrane, such as a myristoyl group.

[0249] When the term "switch domain" is used herein to refer to, for example, RCAR, it refers to an entity that binds to another switch domain in the presence of a dimerization molecule, typically a polypeptide-based entity. This binding results in a functional coupling between a first entity linked, for example, fused to the first switch domain and a second entity linked, for example, fused to the second switch domain. The first and second switch domains are collectively referred to as a dimerization switch. In a plurality of embodiments, the first and second switch domains are the same as each other. For example, both are polypeptides having the same primary amino acid sequence and are collectively referred to as a homodimerization switch. In a plurality of embodiments, the first and second switch domains are different from each other. For example, they are polypeptides having different primary amino acid sequences and are collectively referred to as a heterodimerization switch. In a plurality of embodiments, the switch is intracellular. In a plurality of embodiments, the switch is extracellular. In a plurality of embodiments, the switch domain is a polypeptide-based entity, such as FKBP or FRB-based, and the dimerization molecule is a small molecule, such as a rapalog. In a plurality of embodiments, the switch domain is an scFv that binds to a polypeptide-based entity, such as a myc peptide, and the dimerization molecule is a polypeptide, a fragment thereof, or a multimer of a polypeptide, such as a myc ligand or a multimer of a myc ligand that binds to one or more myc scFvs. In a plurality of embodiments, the switch domain is a polypeptide-based entity, such as a myc receptor, and the dimerization molecule is an antibody or a fragment thereof, such as a myc antibody.

[0250] When the term "dimerization molecule" is used herein to refer to, for example, RCAR, it refers to a molecule that promotes the binding of a first switch domain and a second switch domain. In a plurality of embodiments, the dimerization molecule does not naturally exist in the subject or does not exist at a concentration that causes significant dimerization. In a plurality of embodiments, the dimerization molecule is a small molecule, such as rapamycin or a rapalog, such as RAD001.

[0251] Dosage “Low immunosuppressive dosage” refers to the dosage of an mTOR inhibitor that, when used in combination with an mTOR inhibitor, such as an allosteric mTOR inhibitor, such as RAD001 or rapamycin, or a catalytic mTOR inhibitor, partially but not completely inhibits mTOR activity, as measured, for example, by inhibition of P70 S6 kinase activity. For example, the method of assessing mTOR activity by inhibition of P70 S6 kinase activity is detailed herein. This dosage is insufficient to bring about complete immunosuppression but is sufficient to enhance the immune response. In some embodiments, a low immunosuppressive dosage of an mTOR inhibitor results in a decrease in the number of PD-1 positive T cells and / or an increase in the number of PD-1 negative T cells or an increase in the ratio of PD-1 negative T cells / PD-1 positive T cells. In some embodiments, a low immunosuppressive dosage of an mTOR inhibitor results in an increase in the number of naive T cells. In some embodiments, a low immunosuppressive dosage of an mTOR inhibitor results in for example, an increase in the expression of one or more of the following markers: CD62L in memory T cells, such as memory T cell precursors high , CD127 high , CD27 + and BCL2; for example, a decrease in the expression of KLRG1 in memory T cells, such as memory T cell precursors; and for example, the following characteristics: an increase in CD62L high , an increase in CD127 high , an increase in CD27 + , a decrease in KLRG1 and an increase in BCL2, or any one or a combination of them, resulting in an increase in the number of memory T cell precursors having one or more of the above, where any of the aforementioned changes occur, at least temporarily, for example, compared to untreated subjects.

[0252] “Refractory” as used herein refers to a disease, such as cancer, that does not respond to treatment. In multiple embodiments, a refractory cancer may be resistant to treatment before or at the start of treatment. In other embodiments, a refractory cancer may become resistant during treatment. A refractory cancer is also referred to as a resistant cancer.

[0253] As used herein, "recurred" or "recurrence" refers to the recurrence or reappearance of a disease (e.g., cancer) or the signs and symptoms of a disease such as cancer after an improvement or responsive period, for example, after a previous treatment (e.g., cancer treatment). The initial responsive period can include a decrease in the level of cancer cells to less than a certain threshold, e.g., less than 20%, 1%, 10%, 5%, 4%, 3%, 2% or 1%. The reappearance can include an increase in the level of cancer cells beyond a certain threshold, e.g., beyond 20%, 1%, 10%, 5%, 4%, 3%, 2% or 1%. For example, the reappearance can include, e.g., in relation to B-ALL, the reappearance of blast cells in, e.g., blood, bone marrow (>5%) or any extramedullary site after a complete response. In this regard, a complete response can include <5% BM blast cells. More generally, in some embodiments, a response (e.g., a complete response or a partial response) can include the absence of detectable MRD (minimal residual disease). In one embodiment, the initial period of responsiveness lasts at least 1, 2, 3, 4, 5 or 6 days; at least 1, 2, 3 or 4 weeks; at least 1, 2, 3, 4, 6, 8, 10 or 12 months; or at least 1, 2, 3, 4 or 5 years.

[0254] Ranges: Throughout this disclosure, various embodiments of the invention may be presented in range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the invention. Accordingly, a range description should be considered to have specifically disclosed all the possible subranges as well as the individual numerical values within that range. For example, a range description such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc. as well as the individual numerical values within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3 and 6. As another example, a range such as 95-99% identity should be considered to include those having 95%, 96%, 97%, 98% or 99% identity and subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This applies regardless of the width of the range.

[0255] As used herein, a "gene editing system" refers to a system, e.g., one or more molecules, that directs and performs the modification, e.g., deletion, of one or more nucleic acids at or near the site of genomic DNA targeted by the system. Gene editing systems are known in the art and are described in more detail below.

[0256] Administered "in combination" means, as used herein, that two or more different treatments are delivered to a subject during the course of a disease's continued affliction of the subject, e.g., after the subject has been diagnosed with the disease and before the disease has been cured or eliminated, or the treatment has been discontinued for some other reason. In some embodiments, the delivery of one treatment continues even when the delivery of a second treatment begins, such that there is overlap with respect to administration. This may be referred to herein as "simultaneous" or "co-delivery." In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatments are more effective by combination administration. For example, the second treatment is more effective, e.g., the same effect is seen with a lesser amount of the second treatment, or the second treatment reduces symptoms to a greater extent than seen when the second treatment is administered without the first treatment, or a similar situation as the first treatment. In some embodiments, the delivery is such that a reduction in symptoms or other parameter related to the disorder is higher than seen when either is delivered without the other. The effects of the two treatments may be partially additive, fully additive, or supra-additive. The delivery can be such that the effect of the first treatment delivered is still detectable when the second treatment is delivered.

[0257] As used interchangeably herein, the terms "depletion (deletion)" or "to deplete (delete)" refer to a decrease or reduction in the level or amount of cells, proteins, or macromolecules in a sample after a process, such as a selection step, such as a negative selection, has been performed. Depletion can be a complete or partial depletion of cells, proteins, or macromolecules. In some embodiments, depletion is at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% decrease or reduction in the level or amount of cells, proteins, or macromolecules compared to the level or amount of cells, proteins, or macromolecules in the sample before the process is performed.

[0258] As used herein, "naïve T cells" refer to antigen-inexperienced T cells. In some embodiments, antigen-inexperienced T cells have encountered their cognate antigen in the thymus but not in the periphery. In some embodiments, naïve T cells are precursors of memory T cells. In some embodiments, naïve T cells express both CD45RA and CCR7 but do not express CD45RO. In some embodiments, naïve T cells can be characterized by the expression of CD62L, CD27, CCR7, CD45RA, CD28, and CD127 and the absence of CD95 or CD45RO isoforms. In some embodiments, naïve T cells express CD62L, IL-7 receptor α, IL-6 receptor, and CD132 but do not express CD25, CD44, CD69, or CD45RO. In some embodiments, naïve T cells express CD45RA, CCR7, and CD62L but do not express CD95 or the IL-2 receptor β. In some embodiments, the surface expression level of the marker is evaluated using flow cytometry.

[0259] The term "central memory T cell" refers to a subset of T cells that are CD45RO positive and express CCR7 in humans. In some embodiments, central memory T cells express CD95. In some embodiments, central memory T cells express IL-2R, IL-7R, and / or IL-15R. In some embodiments, central memory T cells express CD45RO, CD95, IL-2 receptor β, CCR7, and CD62L. In some embodiments, the surface expression level of the marker is evaluated using flow cytometry.

[0260] The terms "stem memory T cell", "stem cell memory T cell", "stem cell-like memory T cell", "memory stem T cell", "T memory stem cell", "T stem cell memory cell", or "TSCM cell" refer to a subset of memory T cells having stem cell-like capabilities, such as the ability to self-renew and / or the pluripotency to reconstitute memory and / or effector T cell subsets. In some embodiments, stem memory T cells express CD45RA, CD95, IL-2 receptor β, CCR7, and CD62L. In some embodiments, the surface expression level of the marker is evaluated using flow cytometry. In some embodiments, exemplary stem memory T cells are disclosed in Gattinoni et al., Nat Med. 2017 January 06;23(1):18-27, the entire content of which is incorporated herein by reference.

[0261] For the purposes of clarification, unless otherwise noted, classifying a cell or population of cells as "not expressing" or "absent" or "negative" for a particular marker does not necessarily mean the absence of the marker. One of ordinary skill in the art can readily compare the cells to positive and / or negative controls, and / or set a predetermined threshold, and using conventional detection methods, e.g., flow cytometry, as described in the examples herein, classify a cell or population of cells as not expressing a marker or negative for a marker when the cell has an expression level below a predetermined threshold or the population of cells has an overexpression level below a predetermined threshold.

[0262] As used herein, the term "GeneSetScore (Up TEM vs. Down TSCM)" of a cell refers to a score representing the degree to which the cell exhibits an effector memory T cell (TEM) phenotype relative to the stem cell memory T cell (TSCM) phenotype. A higher GeneSetScore (Up TEM vs. Down TSCM) indicates an increase in the TEM phenotype, while a lower GeneSetScore (Up TEM vs. Down TSCM) indicates an increase in the TSCM phenotype. In some embodiments, GeneSetScore (Up TEM vs. Down TSCM) is determined by measuring the expression of one or more genes that are upregulated in TEM cells and / or downregulated in TSCM, such as MXRA7, CLIC1, NAT13, TBC1D2B, GLCCI1, DUSP10, APOBEC3D, CACNB3, ANXA2P2, TPRG1, EOMES, MATK, ARHGAP10, ADAM8, MAN1A1, SLFN12L, SH2D2A, EIF2C4, CD58, MYO1F, RAB27B, ERN1, NPC1, NBEAL2, APOBEC3G, SYTL2, SLC4A4, PIK3AP1, PTGDR, MAF, PLEKHA5, ADRB2, PLXND1, GNAO1, THBS1, PPP2R2B, CYTH3, KLRF1, FLJ16686, AUTS2, PTPRM, GNLY, and GFPT2. In some embodiments, GeneSetScore (Up TEM vs. Down TSCM) is determined for each cell using RNA-seq, such as single cell RNA-seq (scRNA-seq), as exemplified in Example 7 with reference to FIG. 25A. In some embodiments, GeneSetScore (Up TEM vs. Down TSCM) is calculated by obtaining the average log-normalized gene expression value of all genes in the gene set.

[0263] As used herein, the term "GeneSetScore(Up Treg vs.Down Teff)" of a cell refers to a score that represents the degree to which the cell exhibits regulatory T cells (Tregs) relative to the effector T cell (Teff) phenotype. A higher GeneSetScore(Up Treg vs.Down Teff) indicates an increase in the Treg phenotype, while a lower GeneSetScore(Up Treg vs.Down Teff) indicates an increase in the Teff phenotype. In some embodiments, GeneSetScore(Up Treg vs.Down Teff) is one or more genes that are upregulated in Treg cells and / or downregulated in Teff cells, such as C12orf75, SELPLG, SWAP70, RGS1, PRR11, SPATS2L, SPATS2L, TSHR, C14orf145, CASP8, SYT11, ACTN4, ANXA5, GLRX, HLA-DMB, PMCH, RAB11FIP1, IL32, FAM160B1, SHMT2, FRMD4B, CCR3, TNFRSF13B, NTNG2, CLDND1, BARD1, FCER1G, TYMS, ATP1B1, GJB6, FGL2, TK1, SLC2A8, CDKN2A, SKAP2, GPR55, CDCA7, S100A4, GDPD5, PMAIP1, ACOT9, CEP55, SGMS1, ADPRH, AKAP2, HDAC9, IKZF4, CARD17, VAV3, OBFC2A, ITGB1, CIITA, SETD7, HLA-DMA, CCR10, KIAA0101, SLC14A1, PTTG3P, DUSP10, FAM164A, PYHIN1, MYO1F, SLC1A4, MYBL2, PTTG1, RRM2, TP53INP1, CCR5, ST8SIA6, TOX, BFSP2, ITPRIPL1, NCAPH, HLA-DPB2, SYT4, NINJ2, FAM46C, CCR4, GBP5, C15orf53, LMCD1, MKI67, NUSAP1, PDE4A, E2F2, CD58, ARHGEF12, LOC100188949, FAS, HLA-DPB1, SELP, WEE1, HLA-DPA1, FCRL1, ICA1, CNTNAP1, OAS1, METTL7A, CCR6, HLA-DRB4, ANXA2P3, STAM, HLA-DQB2,It is determined by measuring the expression of one or more genes selected from the group consisting of LGALS1, ANXA2, PI16, DUSP4, LAYN, ANXA2P2, PTPLA, ANXA2P1, ZNF365, LAIR2, LOC541471, RASGRP4, BCAS1, UTS2, MIAT, PRDM1, SEMA3G, FAM129A, HPGD, NCF4, LGALS3, CEACAM4, JAKMIP1, TIGIT, HLA-DRA, IKZF2, HLA-DRB1, FANK1, RTKN2, TRIB1, FCRL3, and FOXP3. In some embodiments, GeneSetScore (Up Treg vs. Down Teff) is determined using RNA-seq, such as single-cell RNA-seq (scRNA-seq), as illustrated in Example 7 with reference to FIG. 25B. In some embodiments, GeneSetScore (Up Treg vs. Down Teff) is calculated by obtaining the average log-normalized gene expression values of all genes in the gene set.,

[0264] As used herein, the term "GeneSetScore (Down stemness)" of a cell refers to a score representing the degree to which the cell exhibits a stem cell phenotype. A lower GeneSetScore (Down stemness) indicates an increase in the stem cell phenotype. In some embodiments, GeneSetScore (Down stemness) is measured by measuring the expression of one or more genes that are downregulated in hematopoietic stem cells and upregulated in differentiated stem cells, such as one or more genes selected from the group consisting of ACE, BATF, CDK6, CHD2, ERCC2, HOXB4, MEOX1, SFRP1, SP7, SRF, TAL1, and XRCC5. In some embodiments, GeneSetScore (Down stemness) is determined using RNA-seq, such as single-cell RNA-seq (scRNA-seq), as exemplified in Example 7 with reference to FIG. 25C. In some embodiments, GeneSetScore (Down stemness) is calculated by obtaining the average log-normalized gene expression values of all genes in the gene set.

[0265] As used herein, the term "GeneSetScore(UP hypoxia)" of a cell refers to a score indicating the degree to which the cell exhibits a hypoxic phenotype. A higher GeneSetScore(UP hypoxia) indicates an increase in the hypoxic phenotype. In some embodiments, GeneSetScore(UP hypoxia) is one or more genes that are upregulated in cells under hypoxia, such as ABCB1, ACAT1, ADM, ADORA2B, AK2, AK3, ALDH1A1, ALDH1A3, ALDOA, ALDOC, ANGPT2, ANGPTL4, ANXA1, ANXA2, ANXA5, ARHGAP5, ARSE, ART1, BACE2, BATF3, BCL2L1, BCL2L2, BHLHE40, BHLHE41, BIK, BIRC2, BNIP3, BNIP3L, BPI, BTG1, C11orf2, C7orf68, CA12, CA9, CALD1, CCNG2, CCT6A, CD99, CDK1, CDKN1A, CDKN1B, CITED2, CLK1, CNOT7, COL4A5, COL5A1, COL5A2, COL5A3, CP, CTSD, CXCR4, D4S234E, DDIT3, DDIT4, 1-Dec, DKC1, DR1, EDN1, EDN2, EFNA1, EGF, EGR1, EIF4A3, ELF3, ELL2, ENG, ENO1, ENO3, ENPEP, EPO, ERRFI1, ETS1, F3, FABP5, FGF3, FKBP4, FLT1, FN1, FOS, FTL, GAPDH, GBE1, GLRX, GPI, GPRC5A, HAP1, HBP1, HDAC1, HDAC9, HERC3, HERPUD1, HGF, HIF1A, HK1, HK2, HLA-DQB1, HMOX1, HMOX2, HSPA5, HSPD1, HSPH1, HYOU1, ICAM1, ID2, IFI27, IGF2, IGFBP1, IGFBP2, IGFBP3, IGFBP5, IL6, IL8, INSIG1, IRF6, ITGA5, JUN, KDR, KRT14, KRT18, KRT19, LDHA, LDHB, LEP, LGALS1, LONP1, LOX, LRP1, MAP4, MET, MIF, MMP13, MMP2, MMP7, MPI, MT1L, MTL3P, MUC1, MXI1, NDRG1, NFIL3, NFKB1, NFKB2, NOS1, NOS2,It is determined by measuring the expression of one or more genes selected from the group consisting of NOS2P1, NOS2P2, NOS3, NR3C1, NR4A1, NT5E, ODC1, P4HA1, P4HA2, PAICS, PDGFB, PDK3, PFKFB1, PFKFB3, PFKFB4, PFKL, PGAM1, PGF, PGK1, PGK2, PGM1, PIM1, PIM2, PKM2, PLAU, PLAUR, PLIN2, PLOD2, PNN, PNP, POLM, PPARA, PPAT, PROK1, PSMA3, PSMD9, PTGS1, PTGS2, QSOX1, RBPJ, RELA, RIOK3, RNASEL, RPL36A, RRP9, SAT1, SERPINB2, SERPINE1, SGSM2, SIAH2, SIN3A, SIRPA, SLC16A1, SLC16A2, SLC20A1, SLC2A1, SLC2A3, SLC3A2, SLC6A10P, SLC6A16, SLC6A6, SLC6A8, SORL1, SPP1, SRSF6, SSSCA1, STC2, STRA13, SYT7, TBPL1, TCEAL1, TEK, TF, TFF3, TFRC, TGFA, TGFB1, TGFB3, TGFBI, TGM2, TH, THBS1, THBS2, TIMM17A, TNFAIP3, TP53, TPBG, TPD52, TPI1, TXN, TXNIP, UMPS, VEGFA, VEGFB, VEGFC, VIM, VPS11 and XRCC6. In some embodiments, GeneSetScore(UP hypoxia) is determined using RNA-seq, such as single cell RNA-seq (scRNA-seq), as exemplified in Example 7 with reference to FIG. 25D. In some embodiments, GeneSetScore(UP hypoxia) is calculated by obtaining the average log-normalized gene expression values of all genes in the gene set.,

[0266] As used herein, the term "GeneSetScore(UP autophagy)" of a cell refers to a score indicating the degree to which the cell exhibits an autophagy phenotype. A higher GeneSetScore(UP autophagy) indicates an increase in the autophagy phenotype. In some embodiments, GeneSetScore(UP autophagy) is one or more genes that are upregulated in cells undergoing autophagy, such as ABL1, ACBD5, ACIN1, ACTRT1, ADAMTS7, AKR1E2, ALKBH5, ALPK1, AMBRA1, ANXA5, ANXA7, ARSB, ASB2, ATG10, ATG12, ATG13, ATG14, ATG16L1, ATG16L2, ATG2A, ATG2B, ATG3, ATG4A, ATG4B, ATG4C, ATG4D, ATG5, ATG7, ATG9A, ATG9B, ATP13A2, ATP1B1, ATPAF1-AS1, ATPIF1, BECN1, BECN1P1, BLOC1S1, BMP2KL, BNIP1, BNIP3, BOC, C11orf2, C11orf41, C12orf44, C12orf5, C14orf133, C1orf210, C5, C6orf106, C7orf59, C7orf68, C8orf59, C9orf72, CA7, CALCB, CALCOCO2, CAPS, CCDC36, CD163L1, CD93, CDC37, CDKN2A, CHAF1B, CHMP2A, CHMP2B, CHMP3, CHMP4A, CHMP4B, CHMP4C, CHMP6, CHST3, CISD2, CLDN7, CLEC16A, CLN3, CLVS1, COX8A, CPA3, CRNKL1, CSPG5, CTSA, CTSB, CTSD, CXCR7, DAP, DKKL1, DNAAF2, DPF3, DRAM1, DRAM2, DYNLL1, DYNLL2, DZANK1, EI24, EIF2S1, EPG5, EPM2A, FABP1, FAM125A, FAM131B, FAM134B, FAM13B, FAM176A, FAM176B, FAM48A, FANCC, FANCF, FANCL, FBXO7, FCGR3B, FGF14, FGF7, FGFBP1, FIS1, FNBP1L, FOXO1, FUNDC1, FUNDC2, FXR2, GABARAP, GABARAPL1,GABARAPL2, GABARAPL3, GABRA5, GDF5, GMIP, HAP1, HAPLN1, HBXIP, HCAR1, HDAC6, HGS, HIST1H3A, HIST1H3B, HIST1H3C, HIST1H3D, HIST1H3E, HIST1H3F, HIST1H3G, HIST1H3H, HIST1H3I, HIST1H3J, HK2, HMGB1, HPR, HSF2BP, HSP90AA1, HSPA8, IFI16, IPPK, IRGM, IST1, ITGB4, ITPKC, KCNK3, KCNQ1, KIAA0226, KIAA1324, KRCC1, KRT15, KRT73, LAMP1, LAMP2, LAMTOR1, LAMTOR2, LAMTOR3, LARP1B, LENG9, LGALS8, LIX1, LIX1L, LMCD1, LRRK2, LRSAM1, LSM4, MAP1A, MAP1LC3A, MAP1LC3B, MAP1LC3B2, MAP1LC3C, MAP1S, MAP2K1, MAP3K12, MARK2, MBD5, MDH1, MEX3C, MFN1, MFN2, MLST8, MRPS10, MRPS2, MSTN, MTERFD1, MTMR14, MTMR3, MTOR, MTSS1, MYH11, MYLK, MYOM1, NBR1, NDUFB9, NEFM, NHLRC1, NME2, NPC1, NR2C2, NRBF2, NTHL1, NUP93, OBSCN, OPTN, P2RX5, PACS2, PARK2, PARK7, PDK1, PDK4, PEX13, PEX3, PFKP, PGK2, PHF23, PHYHIP, PI4K2A, PIK3C3, PIK3CA, PIK3CB, PIK3R4, PINK1, PLEKHM1, PLOD2, PNPO, PPARGC1A, PPY, PRKAA1, PRKAA2, PRKAB1, PRKAB2, PRKAG1, PRKAG2, PRKAG3, PRKD2, PRKG1, PSEN1, PTPN22, RAB12, RAB1A, RAB1B, RAB23, RAB24, RAB33B, RAB39, RAB7A, RB1CC1, RBM18, REEP2, REP15, RFWD3, RGS19, RHEB, RIMS3, RNF185, RNF41, RPS27A, RPTOR, RRAGA, RRAGB, RRAGC, RRAGD, S100A8, S100A9, SCN1A, SERPINB10Determined by measuring the expression of one or more genes selected from the group consisting of SESN2, SFRP4, SH3GLB1, SIRT2, SLC1A3, SLC1A4, SLC22A3, SLC25A19, SLC35B3, SLC35C1, SLC37A4, SLC6A1, SLCO1A2, SMURF1, SNAP29, SNAPIN, SNF8, SNRPB, SNRPB2, SNRPD1, SNRPF, SNTG1, SNX14, SPATA18, SQSTM1, SRPX, STAM, STAM2, STAT2, STBD1, STK11, STK32A, STOM, STX12, STX17, SUPT3H, TBC1D17, TBC1D25, TBC1D5, TCIRG1, TEAD4, TECPR1, TECPR2, TFEB, TM9SF1, TMBIM6, TMEM203, TMEM208, TMEM39A, TMEM39B, TMEM59, TMEM74, TMEM93, TNIK, TOLLIP, TOMM20, TOMM22, TOMM40, TOMM5, TOMM6, TOMM7, TOMM70A, TP53INP1, TP53INP2, TRAPPC8, TREM1, TRIM17, TRIM5, TSG101, TXLNA, UBA52, UBB, UBC, UBQLN1, UBQLN2, UBQLN4, ULK1, ULK2, ULK3, USP10, USP13, USP30, UVRAG, VAMP7, VAMP8, VDAC1, VMP1, VPS11, VPS16, VPS18, VPS25, VPS28, VPS33A, VPS33B, VPS36, VPS37A, VPS37B, VPS37C, VPS37D, VPS39, VPS41, VPS4A, VPS4B, VTA1, VTI1A, VTI1B, WDFY3, WDR45, WDR45L, WIPI1, WIPI2, XBP1, YIPF1, ZCCHC17, ZFYVE1, ZKSCAN3, ZNF189, ZNF593 and ZNF681. In some embodiments, GeneSetScore(UP autophagy) is determined using RNA-seq, such as single-cell RNA-seq (scRNA-seq), as exemplified in Example 7 with reference to FIG. 25E. In some embodiments, GeneSetScore(UP autophagy) isIt is calculated by obtaining the average log-normalized gene expression value of all genes in the gene set.

[0267] As used herein, the term "GeneSetScore (Up resting vs. Down activated)" of a cell refers to a score that represents the extent to which the cell exhibits a resting T cell phenotype relative to an activated T cell phenotype. A higher GeneSetScore (Up resting vs. Down activated) indicates an increase in the resting T cell phenotype, while a lower GeneSetScore (Up resting vs. Down activated) indicates an increase in the activated T cell phenotype. In some embodiments, GeneSetScore (Up resting vs. Down activated) is one or more genes that are upregulated in resting T cells and / or downregulated in activated T cells, such as ABCA7, ABCF3, ACAP2, AMT, ANKH, ATF7IP2, ATG14, ATP1A1, ATXN7, ATXN7L3B, BCL7A, BEX4, BSDC1, BTG1, BTG2, BTN3A1, C11orf21, C19orf22, C21orf2, CAMK2G, CARS2, CCNL2, CD248, CD5, CD55, CEP164, CHKB, CLK1, CLK4, CTSL1, DBP, DCUN1D2, DENND1C, DGKD, DLG1, DUSP1, EAPP, ECE1, ECHDC2, ERBB2IP, FAM117A, FAM134B, FAM134C, FAM169A, FAM190B, FAU, FLJ10038, FOXJ2, FOXJ3, FOXL1, FOXO1, FXYD5, FYB, HLA-E, HSPA1L, HYAL2, ICAM2, IFIT5, IFITM1, IKBKB, IQSEC1, IRS4, KIAA0664L3, KIAA0748, KLF3, KLF9, KRT18, LEF1, LINC00342, LIPA, LIPT1, LLGL2, LMBR1L, LPAR2, LTBP3, LYPD3, LZTFL1, MANBA, MAP2K6, MAP3K1, MARCH8, MAU2, MGEA5, MMP8, MPO, MSL1, MSL3, MYH3, MYLIP, NAGPA, NDST2, NISCH, NKTR, NLRP1, NOSIP, NPIP, NUMA1, PAIP2B, PAPD7, PBXIP1, PCIF1, PI4KA, PLCL2, PLEKHA1, PLEKHF2, PNISR,It is determined by measuring the expression of one or more genes selected from the group consisting of PPFIBP2, PRKCA, PRKCZ, PRKD3, PRMT2, PTP4A3, PXN, RASA2, RASA3, RASGRP2, RBM38, REPIN1, RNF38, RNF44, ROR1, RPL30, RPL32, RPLP1, RPS20, RPS24, RPS27, RPS6, RPS9, RXRA, RYK, SCAND2, SEMA4C, SETD1B, SETD6, SETX, SF3B1, SH2B1, SLC2A4RG, SLC35E2B, SLC46A3, SMAGP, SMARCE1, SMPD1, SNPH, SP140L, SPATA6, SPG7, SREK1IP1, SRSF5, STAT5B, SVIL, SYF2, SYNJ2BP, TAF1C, TBC1D4, TCF20, TECTA, TES, TMEM127, TMEM159, TMEM30B, TMEM66, TMEM8B, TP53TG1, TPCN1, TRIM22, TRIM44, TSC1, TSC22D1, TSC22D3, TSPYL2, TTC9, TTN, UBE2G2, USP33, USP34, VAMP1, VILL, VIPR1, VPS13C, ZBED5, ZBTB25, ZBTB40, ZC3H3, ZFP161, ZFP36L1, ZFP36L2, ZHX2, ZMYM5, ZNF136, ZNF148, ZNF318, ZNF350, ZNF512B, ZNF609, ZNF652, ZNF83, ZNF862, and ZNF91. In some embodiments, GeneSetScore (Up resting vs. Down activated) is determined using RNA-seq, such as single-cell RNA-seq (scRNA-seq), as illustrated in Example 7 with reference to FIG. 24D. In some embodiments, GeneSetScore (Up resting vs. Down activated) is calculated by obtaining the mean log-normalized gene expression values of all genes in the gene set.,

[0268] As used herein, the "GeneSetScore (Progressively up in memory differentiation)" of a cell refers to a score representing the stage of the cell in memory differentiation. A higher GeneSetScore (Progressively up in memory differentiation) indicates an increase in the late memory T cell phenotype, while a lower GeneSetScore (Progressively up in memory differentiation) indicates an increase in the early memory T cell phenotype. In some embodiments, GeneSetScore (UP autophagy) is one or more genes that are upregulated during memory differentiation, such as MTCH2, RAB6C, KIAA0195, SETD2, C2orf24, NRD1, GNA13, COPA, SELT, TNIP1, CBFA2T2, LRP10, PRKCI, BRE, ANKS1A, PNPLA6, ARL6IP1, WDFY1, MAPK1, GPR153, SHKBP1, MAP1LC3B2, PIP4K2A, HCN3, GTPBP1, TLN1, C4orf34, KIF3B, TCIRG1, PPP3CA, ATG4D, TYMP, TRAF6, C17orf76, WIPF1, FAM108A1, MYL6, NRM, SPCS2, GGT3P, GALK1, CLIP4, ARL4C, YWHAQ, LPCAT4, ATG2A, IDS, TBC1D5, DMPK, ST6GALNAC6, REEP5, ABHD6, KIAA0247, EMB, TSEN54, SPIRE2, PIWIL4, ZSCAN22, ICAM1, CHD9, LPIN2, SETD8, ZC3H12A, ULBP3, IL15RA, HLA-DQB2, LCP1, CHP, RUNX3, TMEM43, REEP4, MEF2D, ABL1, TMEM39A, PCBP4, PLCD1, CHST12, RASGRP1, C1orf58, C11orf63, C6orf129, FHOD1, DKFZp434F142, PIK3CG, ITPR3, BTG3, C4orf50, CNNM3, IFI16, AK1, CDK2AP1, REL, BCL2L1, MVD, TTC39C, PLEKHA2, FKBP11, EML4, FANCA, CDCA4, FUCA2, MFSD10,TBCD, CAPN2, IQGAP1, CHST11, PIK3R1, MYO5A, KIR2DL3, DLG3, MXD4, RALGDS, S1PR5, WSB2, CCR3, TIPARP, SP140, CD151, SOX13, KRTAP5-2, NF1, PEA15, PARP8, RNF166, UEVLD, LIMK1, CACNB1, TMX4, SLC6A6, LBA1, SV2A, LLGL2, IRF1, PPP2R5C, CD99, RAPGEF1, PPP4R1, OSBPL7, FOXP4, SLA2, TBC1D2B, ST7, JAZF1, GGA2, PI4K2A, CD68, LPGAT1, STX11, ZAK, FAM160B1, RORA, C8orf80, APOBEC3F, TGFBI, DNAJC1, GPR114, LRP8, CD69, CMIP, NAT13, TGFB1, FLJ00049, ANTXR2, NR4A3, IL12RB1, NTNG2, RDX, MLLT4, GPRIN3, ADCY9, CD300A, SCD5, ABI3, PTPN22, LGALS1, SYTL3, BMPR1A, TBK1, PMAIP1, RASGEF1A, GCNT1, GABARAPL1, STOM, CALHM2, ABCA2, PPP1R16B, SYNE2, PAM, C12orf75, CLCF1, MXRA7, APOBEC3C, CLSTN3, ACOT9, HIP1, LAG3, TNFAIP3, DCBLD1, KLF6, CACNB3, RNF19A, RAB27A, FADS3, DLG5, APOBEC3D, TNFRSF1B, ACTN4, TBKBP1, ATXN1, ARAP2, ARHGEF12, FAM53B, MAN1A1, FAM38A, PLXNC1, GRLF1, SRGN, HLA-DRB5, B4GALT5, WIPI1, PTPRJ, SLFN11, DUSP2, ANXA5, AHNAK, NEO1, CLIC1, EIF2C4, MAP3K5, IL2RB, PLEKHG1, MYO6, GTDC1, EDARADD, GALM, TARP, ADAM8, MSC, HNRPLL, SYT11, ATP2B4, NHSL2, MATK, ARHGAP18, SLFN12L, SPATS2L, RAB27B, PIK3R3, TP53INP1, MBOAT1, GYG1, KATNAL1, FAM46C, ZC3HAV1L, ANXA2P2, CTNNA1,It is determined by measuring the expression of one or more genes selected from the group consisting of NPC1, C3AR1, CRIM1, SH2D2A, ERN1, YPEL1, TBX21, SLC1A4, FASLG, PHACTR2, GALNT3, ADRB2, PIK3AP1, TLR3, PLEKHA5, DUSP10, GNAO1, PTGDR, FRMD4B, ANXA2, EOMES, CADM1, MAF, TPRG1, NBEAL2, PPP2R2B, PELO, SLC4A4, KLRF1, FOSL2, RGS2, TGFBR3, PRF1, MYO1F, GAB3, C17orf66, MICAL2, CYTH3, TOX, HLA-DRA, SYNE1, WEE1, PYHIN1, F2R, PLD1, THBS1, CD58, FAS, NETO2, CXCR6, ST6GALNAC2, DUSP4, AUTS2, C1orf21, KLRG1, TNIP3, GZMA, PRR5L, PRDM1, ST8SIA6, PLXND1, PTPRM, GFPT2, MYBL1, SLAMF7, FLJ16686, GNLY, ZEB2, CST7, IL18RAP, CCL5, KLRD1 and KLRB1. In some embodiments, GeneSetScore (Progressively up in memory differentiation) is determined using RNA-seq, such as single-cell RNA-seq (scRNA-seq), as exemplified in Example 7 with reference to FIG. 26B. In some embodiments, GeneSetScore (Progressively up in memory differentiation) is calculated by obtaining the average log-normalized gene expression value of all genes in the gene set.,

[0269] As used herein, the term "GeneSetScore(Up TEM vs.Down TN)" of a cell refers to a score that represents the degree to which the cell exhibits an effector memory T cell (TEM) phenotype relative to a naive T cell (TN) phenotype. A higher GeneSetScore(Up TEM vs.Down TN) indicates an increase in the TEM phenotype, while a lower GeneSetScore(Up TEM vs.Down TN) indicates an increase in the TN phenotype.In some embodiments, GeneSetScore (Up TEM vs. Down TN) is determined by measuring the expression of one or more genes that are upregulated in TEM cells and / or downregulated in TN cells, such as MYO5A, MXD4, STK3, S1PR5, GLCCI1, CCR3, SOX13, KRTAP5-2, PEA15, PARP8, RNF166, UEVLD, LIMK1, SLC6A6, SV2A, KPNA2, OSBPL7, ST7, GGA2, PI4K2A, CD68, ZAK, RORA, TGFBI, DNAJC1, JOSD1, ZFYVE28, LRP8, OSBPL3, CMIP, NAT13, TGFB1, ANTXR2, NR4A3, RDX, ADCY9, CHN1, CD300A, SCD5, PTPN22, LGALS1, RASGEF1A, GCNT1, GLUL, ABCA2, CLDND1, PAM, CLCF1, MXRA7, CLSTN3, ACOT9, METRNL, BMPR1A, LRIG1, APOBEC3G, CACNB3, RNF19A, RAB27A, FADS3, ACTN4, TBKBP1, FAM53B, MAN1A1, FAM38A, GRLF1, B4GALT5, WIPI1, DUSP2, ANXA5, AHNAK, CLIC1, MAP3K5, ST8SIA1, TARP, ADAM8, MATK, SLFN12L, PIK3R3, FAM46C, ANXA2P2, CTNNA1, NPC1, SH2D2A, ERN1, YPEL1, TBX21, STOM, PHACTR2, GBP5, ADRB2, PIK3AP1, DUSP10, PTGDR, EOMES, MAF, TPRG1, NBEAL2, NCAPH, SLC4A4, FOSL2, RGS2, TGFBR3, MYO1F, C17orf66, CYTH3, WEE1, PYHIN1, F2R, THBS1, CD58, AUTS2, FAM129A, TNIP3, GZMA, PRR5L, PRDM1, PLXND1, PTPRM, GFPT2, MYBL1, SLAMF7, ZEB2, CST7, CCL5, GZMK, and KLRB1, selected from the group consisting of one or more genes.In some embodiments, GeneSetScore (Up TEM vs. Down TN) is determined using RNA-seq, such as single-cell RNA-seq (scRNA-seq), as exemplified in Example 7 with reference to FIG. 26C. In some embodiments, GeneSetScore (Up TEM vs. Down TN) is calculated by obtaining the average log-normalized gene expression values of all genes in the gene set.

[0270] In relation to the GeneSetScore value (e.g., GeneSetScore median), when the positive GeneSetScore decreases by 100%, this value becomes 0. When the negative GeneSetScore increases by 100%, this value becomes 0. For example, in FIG. 25A, the GeneSetScore median of the Day 1 sample is -0.084; the GeneSetScore median of the Day 9 sample is 0.035; the GeneSetScore median of the input sample is -0.1. In FIG. 25A, a 100% increase in the GeneSetScore median of the input sample results in a GeneSetScore value of 0; a 200% increase in the GeneSetScore median of the input sample results in a GeneSetScore value of 0.1. In FIG. 25A, a 100% decrease in the GeneSetScore median of the Day 9 sample results in a GeneSetScore value of 0; a 200% decrease in the GeneSetScore median of the Day 9 sample results in a GeneSetScore value of -0.035.

[0271] As used herein, the term "beads" refers to individual particles having a solid surface and a size of from about 0.1 μm to several millimeters in diameter. The beads can be spherical (e.g., microspheres) or can have an irregular shape. The beads can include a variety of materials including, but not limited to, paramagnetic materials, ceramics, plastics, glass, polystyrene, methylstyrene, acrylic polymers, titanium, latex, Sepharose™, cellulose, nylon, and the like. In some embodiments, the beads are relatively uniform, spherical, superparamagnetic polystyrene beads having a diameter of about 4.5 μm and are coated with, e.g., bound to, a mixture of antibodies to CD3 (e.g., CD3ε) and CD28. In some embodiments, the beads are Dynabeads®. In some embodiments, both the anti-CD3 and anti-CD28 antibodies are bound to the same bead to mimic the stimulation of T cells by antigen-presenting cells. The properties of Dynabeads® and the use of Dynabeads® for cell isolation and expansion are known in the art and reference is made, for example, to Neurauter et al., Cell isolation and expansion using Dynabeads, Adv Biochem Eng Biotechnol 2007;106:41-73, the entire contents of which are incorporated herein by reference.

[0272] As used herein, the term "nanomatrix" refers to a nanostructure that includes a matrix of mobile polymer chains. The nanomatrix has a size of 1 to 500 nm, such as 10 to 200 nm. In some embodiments, the matrix of mobile polymer chains is bound to one or more agonists that provide an activating signal to T cells, such as agonist anti-CD3 and / or anti-CD28 antibodies. In some embodiments, the nanomatrix includes a colloidal polymer nanomatrix that is bound, such as covalently bound, to an agonist of one or more stimulatory molecules and / or an agonist of one or more costimulatory molecules. In some embodiments, the agonist of one or more stimulatory molecules is a CD3 agonist (e.g., an anti-CD3 agonist antibody). In some embodiments, the agonist of one or more stimulatory molecules is a CD28 agonist (e.g., an anti-CD28 agonist antibody). In some embodiments, the nanomatrix is characterized by the absence of a solid surface as a binding site for agonists such as, for example, anti-CD3 and / or anti-CD28 antibodies. In some embodiments, the nanomatrix is a nanomatrix as disclosed in International Publication No. WO 2014 / 048920 A1 or as provided in the MACS® GMP T Cell TransAct™ kit from Miltenyi Biotec GmbH, the entire content of which is incorporated herein by reference. MACS® GMP T Cell TransAct™ is composed of a colloidal polymer nanomatrix covalently bound to humanized recombinant agonist antibodies against human CD3 and CD28.

[0273] Various embodiments of the compositions and methods described herein are further described in detail below. Additional definitions are set forth throughout this specification.

[0274] This specification provides compositions of matter and methods of use for treating diseases such as cancer using cells that express one or more chimeric antigen receptors (CARs). In some embodiments, the invention provides cells engineered to express one or more CARs (e.g., immune effector cells such as T cells or NK cells), where the CAR T cells ("CART") or CAR NK cells exhibit anti-tumor properties.

[0275] In some embodiments, the cells express at least two CARs. In some embodiments, the cells express a first CAR that binds to a first antigen and a second CAR that binds to a second antigen. In some embodiments, the first antigen and the second antigen are different. In some embodiments, the first antigen is BCMA. In some embodiments, the first CAR is an anti-BCMA CAR comprising a CDR, VH, VL, scFv, or CAR sequence disclosed herein, such as the sequences disclosed in Tables 3-15, 19, 20, 22, and 26, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto, e.g., an anti-BCMA CAR disclosed herein. In some embodiments, the second antigen is CD19. In some embodiments, the second antigen is an anti-CD19 CAR comprising a CDR, VH, VL, scFv, or CAR sequence disclosed herein, such as the sequences disclosed in Tables 2, 19, and 22, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto, e.g., an anti-CD19 CAR disclosed herein. In some embodiments, the first CAR and the second CAR are expressed by nucleic acid sequences disposed in a single nucleic acid molecule. In some embodiments, the nucleic acid sequence encoding the first CAR and the nucleic acid sequence encoding the second CAR are separated by a nucleic acid encoding a self-cleaving site, such as a P2A site, T2A site, E2A site, or F2A site. In some embodiments, the cells are cells that express a dual CAR disclosed herein. In some embodiments, the first CAR and the second CAR are expressed by nucleic acid sequences disposed in separate nucleic acid molecules. In some embodiments, the cells are engineered using a co-transduction system disclosed herein.

[0276] In some embodiments, the cell expresses a CAR that binds to a first antigen and a second antigen. In some embodiments, the CAR is a diabody CAR disclosed herein. In some embodiments, the CAR comprises a binding domain having a first VH (VH1), a first VL (VL1), a second VH (VH2), and a second VL (VL2). In some embodiments, VH1 and VL1 bind to the first antigen, and VH2 and VL2 bind to the second antigen. In some embodiments, VH1, VL1, VH2, and VL2 are configured, from N-terminus to C-terminus, as follows: VH1 - optionally linker 1 ("L1") - VH2 - optionally linker 2 ("L2") - VL2 - optionally linker 3 ("L3") - VL1, VH1 - optionally L1 - VL2 - optionally L2 - VH2 - optionally L3 - VL1, VL1 - optionally L1 - VH2 - optionally L2 - VL2 - optionally L3 - VH1, VL1 - optionally L1 - VL2 - optionally L2 - VH2 - optionally L3 - VH1, VH2 - optionally L1 - VH1 - optionally L2 - VL1 - optionally L3 - VL2, VH2 - optionally L1 - VL1 - optionally L2 - VH1 - optionally L3 - VL2, VL2 - optionally L1 - VH1 - optionally L2 - VL1 - optionally L3 - VH2; or VL2 - optionally L1 - VL1 - optionally L2 - VH1 - optionally L3 - VH2.

[0277] In some embodiments, the CAR of the present invention binds an antigen-binding domain to an intracellular signaling molecule. For example, in some embodiments, intracellular signaling molecules include, but are not limited to, the CD3ζ chain, 4-1BB, and CD28 signaling modules and combinations thereof.

[0278] Furthermore, the present invention also provides CAR compositions and their use or methods in drugs for treating diseases, including cancer or any malignant or autoimmune disease.

[0279] Chimeric antigen receptor (CAR) The present invention provides immune effector cells (e.g., T cells or NK cells) engineered to contain one or more chimeric antigen receptors (CARs) that direct the immune effector cells to cancer. This is achieved through antigen-binding domains on the CARs that are specific for cancer-associated antigens. There are two classes of cancer-associated antigens (tumor antigens) that can be targeted by the CARs described herein: (1) cancer-associated antigens expressed on the surface of cancer cells; and (2) cancer-associated antigens that are themselves intracellular, but fragments (peptides) of such antigens are presented on the surface of cancer cells by the major histocompatibility complex (MHC).

[0280] Accordingly, immune effector cells (e.g., those obtained by the methods described herein) can be engineered to contain a CAR that targets one of the following cancer-associated antigens (tumor antigens): CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, VEGFR2, Lewis Y, CD24, PDGFR-β, PRSS21, SSEA-4, CD20, folate receptor α, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosine kinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor β, TEM1 / CD248, TEM7R, CLDN6, TSHR, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, legumain, HPV E6, E7, MAGE-A1, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 variants, prostain, survivin and telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras variants, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase and mut hsp70-2.

[0281] Table 1 lists the sequences of non-limiting examples of various components that can be part of the CAR molecules described herein, where "aa" represents an amino acid and "na" represents a nucleic acid encoding the corresponding peptide.

[0282] [T...

Claims

1. An isolated T cell or NK cell, or a population of T cells or NK cells, comprising a chimeric antigen receptor (CAR), wherein the CAR is as follows: (a) A first chimeric antigen receptor (BCMA CAR) comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), a first antigen-binding domain that binds to BCMA, a first transmembrane domain, and a first intracellular signaling domain comprising a first co-stimulatory signaling domain and a first primary signaling domain; and (b) A second chimeric antigen receptor (CD19 CAR) comprising a VH and a VL, a second antigen-binding domain that binds to CD19, a second transmembrane domain, and a second intracellular signaling domain comprising a second co-stimulatory signaling domain and a second primary signaling domain; wherein the first CAR and the second CAR each comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as set forth in one of SEQ ID NOs: 214, 216, 218, 220, or 222, and wherein the CAR comprises the amino acid sequence of SEQ ID NOs: 214, 216, 218, 220, 222, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity thereto, a cell or population of cells.

2. The isolated T cell or NK cell, or population of T cells or NK cells according to claim 1, wherein the first CAR is encoded by a first nucleic acid sequence and the second CAR is encoded by a second nucleic acid sequence, wherein the first and second nucleic acid sequences are arranged on a single nucleic acid molecule.

3. (a) The single nucleic acid molecule has the following configuration in the 5' to 3' direction: (i) A nucleic acid sequence encoding the first antigen-binding domain, a nucleic acid sequence encoding the first transmembrane domain, a nucleic acid sequence encoding the first intracellular signaling domain, a nucleic acid sequence encoding a linker comprising a P2A site, a nucleic acid sequence encoding the second antigen-binding domain, a nucleic acid sequence encoding the second transmembrane domain, a nucleic acid sequence encoding the second intracellular signaling domain, or (ii) comprising a nucleic acid sequence encoding the second antigen-binding domain, a nucleic acid sequence encoding the second transmembrane domain, a nucleic acid sequence encoding the second intracellular signaling domain, a nucleic acid sequence encoding a linker comprising a P2A site, a nucleic acid sequence encoding the first antigen-binding domain, a nucleic acid sequence encoding the first transmembrane domain, and a nucleic acid sequence encoding the first intracellular signaling domain; (b) the single nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 215, 217, 219, 221 or 223, or a nucleic acid sequence having at least 90%, 95% or 99% sequence identity thereto; or (c) the single nucleic acid molecule encodes the amino acid sequence of SEQ ID NO: 214, 216, 218, 220 or 222, or an amino acid sequence having at least 95% or 99% sequence identity thereto, the isolated T cell or NK cell, or population of T cells or NK cells according to claim 2.

4. The linker comprising the P2A site is (I) encoded by the nucleic acid sequence of SEQ ID NO: 209 or a nucleic acid sequence having at least 90%, 95% or 99% sequence identity thereto, or (II) comprises the amino acid sequence of SEQ ID NO: 208 or an amino acid sequence having at least 90%, 95% or 99% sequence identity thereto, the isolated T cell or NK cell, or population of T cells or NK cells according to claim 3.

5. The first antigen-binding domain or the second antigen-binding domain comprises VH and VL, and the VH and VL are linked by a linker, and the linker comprises the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having at least 90%, 95% or 99% sequence identity thereto, the isolated T cell or NK cell, or population of T cells or NK cells according to claim 1.

6. (i) the first transmembrane domain or the second transmembrane domain comprises the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having at least 90%, 95% or 99% sequence identity thereto; (ii) the first transmembrane domain or the second transmembrane domain is encoded by the nucleic acid sequence of SEQ ID NO: 17 or a nucleic acid sequence having at least 90%, 95% or 99% sequence identity thereto; (iii) The first antigen-binding domain or the second antigen-binding domain is linked to the first transmembrane domain or the second transmembrane domain, respectively, by a hinge region; (iv) The primary signal transduction domain includes a functional signal transduction domain derived from CD3ζ, (a) The primary signal transduction domain includes the amino acid sequence of SEQ ID NO: 9 or 10 or an amino acid sequence having at least 90%, 95% or 99% sequence identity therewith, or (b) The primary signal transduction domain is encoded by the nucleic acid sequence of SEQ ID NO: 20, 21 or 205 or a nucleic acid sequence having at least 90%, 95% or 99% sequence identity therewith; (v) The co-stimulatory signal transduction domain includes a functional signal transduction domain derived from 4-1BB (CD137), (a) The co-stimulatory signal transduction domain includes the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having at least 90%, 95% or 99% sequence identity therewith, or (b) The co-stimulatory signal transduction domain is encoded by the nucleic acid sequence of SEQ ID NO: 18 or 204 or a nucleic acid sequence having at least 90%, 95% or 99% sequence identity therewith; (vi) The first intracellular signal transduction domain or the second intracellular signal transduction domain includes a functional signal transduction domain derived from 4-1BB and a functional signal transduction domain derived from CD3ζ, (a) The first intracellular signal transduction domain or the second intracellular signal transduction domain includes the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having at least 90%, 95% or 99% sequence identity therewith and the amino acid sequence of SEQ ID NO: 9 or 10 or an amino acid sequence having at least 90%, 95% or 99% sequence identity therewith, or (b) The first intracellular signal transduction domain or the second intracellular signal transduction domain includes the amino acid sequence of SEQ ID NO: 7 and the amino acid sequence of SEQ ID NO: 9 or 10; or (vii) The first CAR further includes a first leader sequence, or the second CAR further includes a second leader sequence, (a) The first or second leader sequence includes the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 90%, 95% or 99% sequence identity therewith, or (b) The isolated T cell or NK cell, or population of T cells or NK cells according to claim 2, wherein the first or second leader sequence is encoded by a nucleic acid sequence of SEQ ID NO: 199 or 210, or a nucleic acid sequence having at least 90%, 95% or 99% sequence identity thereto.

7. (a) The hinge region comprises an amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 90%, 95% or 99% sequence identity thereto, (b) the hinge region is encoded by a nucleic acid sequence of SEQ ID NO: 13 or a nucleic acid sequence having at least 90%, 95% or 99% sequence identity thereto, (c) the hinge region and the transmembrane domain comprise an amino acid sequence of SEQ ID NO: 202 or an amino acid sequence having at least 90%, 95% or 99% sequence identity thereto, or (d) the hinge region and the transmembrane domain are encoded by a nucleic acid sequence of SEQ ID NO: 203 or 213, or a nucleic acid sequence having at least 90%, 95% or 99% sequence identity thereto, the isolated T cell or NK cell, or population of T cells or NK cells according to claim 6.

8. (a) (i) The first leader sequence and the second leader sequence are encoded by different nucleic acid sequences, (ii) the first hinge region and the second hinge region are encoded by different nucleic acid sequences, (iii) the first transmembrane domain and the second transmembrane domain are encoded by different nucleic acid sequences, and / or (iv) the first intracellular signaling domain and the second intracellular signaling domain are encoded by different nucleic acid sequences; (b) (i) The first leader sequence and the second leader sequence comprise the same amino acid sequence or different amino acid sequences, (ii) the first hinge region and the second hinge region comprise the same amino acid sequence or different amino acid sequences, (iii) the first transmembrane domain and the second transmembrane domain comprise the same amino acid sequence or different amino acid sequences, and / or (iv) the first intracellular signaling domain and the second intracellular signaling domain comprise the same amino acid sequence or different amino acid sequences; and / or (c)(i) The first leader sequence and the second leader sequence are each encoded by a nucleic acid sequence comprising SEQ ID NO: 199 and 210, respectively, or a nucleic acid sequence having at least 90%, 95% or 99% sequence identity thereto, or a nucleic acid sequence comprising SEQ ID NO: 210 and 199, respectively, or a nucleic acid sequence having at least 90%, 95% or 99% sequence identity thereto, (ii) The first hinge region and the second hinge region are each encoded by a nucleic acid sequence comprising SEQ ID NO: 337 and 13, respectively, or a nucleic acid sequence having at least 90%, 95% or 99% sequence identity thereto, or a nucleic acid sequence comprising SEQ ID NO: 13 and 337, respectively, or a nucleic acid sequence having at least 90%, 95% or 99% sequence identity thereto, (iii) The first transmembrane domain and the second transmembrane domain are each encoded by a nucleic acid sequence comprising SEQ ID NO: 338 and 17, respectively, or a nucleic acid sequence having at least 90%, 95% or 99% sequence identity thereto; or a nucleic acid sequence comprising SEQ ID NO: 17 and 338, respectively, or a nucleic acid sequence having at least 90%, 95% or 99% sequence identity thereto, (iv) The first co-stimulatory signaling domain and the second co-stimulatory signaling domain are each encoded by a nucleic acid sequence comprising SEQ ID NO: 204 and 18, respectively, or a nucleic acid sequence having at least 90%, 95% or 99% sequence identity thereto; or a nucleic acid sequence comprising SEQ ID NO: 18 and 204, respectively, or a nucleic acid sequence having at least 90%, 95% or 99% sequence identity thereto, and / or (v) The first primary signaling domain and the second primary signaling domain are each encoded by a nucleic acid sequence comprising SEQ ID NO: 205 and 21, respectively, or a nucleic acid sequence having at least 90%, 95% or 99% sequence identity thereto; or a nucleic acid sequence comprising SEQ ID NO: 21 and 205, respectively, or a nucleic acid sequence having at least 90%, 95% or 99% sequence identity thereto; or (d) The first CAR or the second CAR is encoded by a nucleic acid molecule comprising a woodchuck hepatitis post-transcriptional regulatory element (WPRE), the isolated T cell or NK cell according to claim 6, or a population of T cells or NK cells. Claim 9. (a) The first primary signal transduction domain and the second primary signal transduction domain are encoded by different nucleic acid sequences; (b) The first co-stimulatory signal transduction and the second co-stimulatory signal transduction domain are encoded by different nucleic acid sequences; (c) The first leader sequence and the second leader sequence include the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 90%, 95% or 99% sequence identity therewith; (d) The first hinge region and the second hinge region include the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 90%, 95% or 99% sequence identity therewith; (e) The first transmembrane domain and the second transmembrane domain include the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having at least 90%, 95% or 99% sequence identity therewith; (f) The first primary signal transduction domain and the second primary signal transduction domain include the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence having at least 90%, 95% or 99% sequence identity therewith, or (g) The first co-stimulatory signal transduction domain and the second co-stimulatory signal transduction domain include the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having at least 90%, 95% or 99% sequence identity therewith, an isolated T cell or NK cell according to claim 8, or a population of T cells or NK cells. Claim 10 An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR), the nucleic acid molecule comprising: (a) a first nucleic acid sequence encoding a first CAR (BCMA CAR) comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), a first antigen-binding domain that binds to BCMA, a first transmembrane domain, and a first intracellular signal transduction domain comprising a first co-stimulatory signal transduction domain and a first primary signal transduction domain; and (b) a second nucleic acid sequence encoding a second CAR (CD19 CAR) comprising a second antigen-binding domain that binds to CD19, a second transmembrane domain, and a second intracellular signal transduction domain comprising a second co-stimulatory signal transduction domain and a second primary signal transduction domain, Here, the first CAR and the second CAR each contain HCDR1, HCHR2, HCDR3, LCDR1, LCHR2, and LCDR3 described in one of SEQ ID NOs: 214, 216, 218, 220, or 222, wherein the encoded CAR contains the amino acid sequence of SEQ ID NOs: 214, 216, 218, 220, 222, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and wherein the first nucleic acid sequence and the second nucleic acid sequence are isolated nucleic acid molecules arranged on a single nucleic acid molecule. **Claim 11** a) a first chimeric antigen receptor (BCMA CAR) comprising a first antigen-binding domain that binds to BCMA and that contains a heavy chain variable domain (VH) and a light chain variable domain (VL), a first transmembrane domain, and a first intracellular signaling domain that contains a first co-stimulatory signaling domain and a first primary signaling domain; and b) a second chimeric antigen receptor (CD19 CAR) comprising a second antigen-binding domain that binds to CD19 and that contains VH and VL, a second transmembrane domain, and a second intracellular signaling domain that contains a second co-stimulatory signaling domain and a second primary signaling domain, wherein the first CAR and the second CAR each contain HCDR1, HCHR2, HCDR3, LCDR1, LCHR2, and LCDR3 described in one of SEQ ID NOs: 214, 216, 218, 220, or 222, wherein the CAR contains the amino acid sequence of SEQ ID NOs: 214, 216, 218, 220, 222, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity thereto, an isolated CAR. **Claim 12** A vector comprising the nucleic acid molecule according to claim 10. **Claim 13** The vector according to claim 12, wherein the vector is a vector selected from a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, or a retroviral vector. **Claim 14** An isolated T cell or NK cell, or a population of T cells or NK cells, comprising the nucleic acid molecule according to claim 10. **Claim 15** A method of producing a T cell or NK cell, the method comprising the step of transducing a cell with the vector according to claim 12 or 13. **Claim 16** A method for producing RNA-operated cells, comprising the step of introducing in vitro-transcribed RNA or synthetic RNA into cells, wherein the RNA contains the nucleic acid molecule according to claim 10, and the cells are T cells or NK cells.

17. A pharmaceutical composition comprising a T cell or NK cell according to any one of claims 1 to 9 or 14, or a population of T cells or NK cells, and a pharmaceutically acceptable carrier.

18. An effective amount of (a)(i)The nucleic acid molecule according to claim 10 (ii)The CAR molecule according to claim 11, or (iii )A vector according to claim 12 or 13, comprising a T cell or NK cell, or a population of T cells or NK cells; (b)A T cell or NK cell according to any one of claims 1 to 9 or 14, or a population of T cells or NK cells; or (c)The pharmaceutical composition according to claim 17, A pharmaceutical composition for use in providing anti-tumor immunity in a subject or for use in treating a disease associated with the expression of BCMA.

19. The disease associated with the expression of BCMA is (i)A cancer or malignant disease or pre-cancerous state selected from one or more of myelodysplasia, myelodysplastic syndrome or pre-leukemia, (ii)A non-cancer-related indication associated with the expression of BCMA, (iii)A blood cancer or solid cancer, (iv)Acute leukemia, B cell acute lymphoblastic leukemia (“BALL”), T cell acute lymphoblastic leukemia (“TALL”), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphocyte proliferative disorder, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström macroglobulinemia, prostate cancer, pancreatic cancer, lung cancer, plasma cell proliferative disorder, monoclonal gammopathy of undetermined significance (MGUS), plasmacytoma, systemic amyloid light chain amyloidosis or POEMS syndrome (also known as Crow-Fukase syndrome, Takatsuki disease and PEP syndrome) or a combination thereof, or (v) Multiple myeloma The pharmaceutical composition according to claim 18, which is as described above.

20. The pharmaceutical composition according to claim 18 or 19, wherein the isolated T cell or NK cell, or a population of T cells or NK cells, is formulated for administration in combination with a second therapeutic agent.

21. The second therapeutic agent is (i) a PD-1 inhibitor; (ii) a PD-L1 inhibitor; (iii) a LAG-3 inhibitor; (iv) a TIM-3 inhibitor; (v) a CTLA-4 inhibitor; (vi) an interleukin-15 (IL-15) polypeptide, an interleukin-15 receptor α (IL-15Ra) polypeptide, or a combination of both the IL-15 polypeptide and the IL-15Ra polypeptide; (vii) an interleukin-12 (IL-12) polypeptide; or (viii) an mTOR inhibitor, and is selected from the group consisting of the above, and is the pharmaceutical composition according to claim 20.

22. (i) The PD-1 inhibitor is selected from the group consisting of PDR001, nivolumab, pembrolizumab, pidilizumab, MEDI0680, REGN2810, TSR-042, PF-06801591, and AMP-224; (ii) The PD-L1 inhibitor is selected from the group consisting of FAZ053, atezolizumab, avelumab, durvalumab, and BMS-936559; (iii) The LAG-3 inhibitor is selected from the group consisting of LAG525, BMS-986016, TSR-033, MK-4280, and REGN3767; (iv) The TIM-3 inhibitor is selected from the group consisting of MBG453, TSR-022, and LY3321367; (v) The CTLA-4 inhibitor is ipilimumab or tremelimumab; (vi) The mTOR inhibitor is RAD001 or rapamycin, and is the pharmaceutical composition according to claim 21.

23. In the manufacture of a medicament for providing anti-tumor immunity in a subject or for treating a disease associated with the expression of BCMA, an effective amount of (a) (i) the nucleic acid molecule according to claim 10, (ii) the CAR molecule according to claim 11, or (iii) the vector according to claim 12 or 13, which comprises a T cell or NK cell, or a population of T cells or NK cells; (b) the isolated T cell or NK cell, or a population of T cells or NK cells, according to any one of claims 1 to 9 or 14; or (c) the pharmaceutical composition according to claim 17, for use.

24. wherein the disease associated with the expression of BCMA is (i) a cancer or malignant disease or pre-cancerous condition selected from one or more of myelodysplasia, myelodysplastic syndrome or pre-leukemia, (ii) a non-cancer-related indication associated with the expression of BCMA, (iii) a blood cancer or solid cancer, (iv) acute leukemia, B-cell acute lymphoblastic leukemia (“BALL”), T-cell acute lymphoblastic leukemia (“TALL”), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphocyte proliferative disorders, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenström macroglobulinemia, prostate cancer, pancreatic cancer, lung cancer, plasma cell proliferative disorders, monoclonal gammopathy of undetermined significance (MGUS), plasmacytoma, systemic amyloid light chain amyloidosis or POEMS syndrome (also known as Crow-Fukase syndrome, Takatsuki disease and PEP syndrome) or a combination thereof, or (v) multiple myeloma The use according to claim 23, wherein the use is as described above.

25. The use according to claim 23 or 24, wherein the T cells or NK cells, or a population of T cells or NK cells, are formulated for administration in combination with a second therapeutic agent.

26. The second therapeutic agent is (i) a PD-1 inhibitor; (ii) a PD-L1 inhibitor; (iii) a LAG-3 inhibitor; (iv) a TIM-3 inhibitor; (v) a CTLA-4 inhibitor; (vi) an interleukin-15 (IL-15) polypeptide, an interleukin-15 receptor α (IL-15Ra) polypeptide or a combination of both the IL-15 polypeptide and the IL-15Ra polypeptide; (vii) an interleukin-12 (IL-12) polypeptide; or (viii) an mTOR inhibitor The use according to claim 25, wherein the use is selected from the above. Claim 27. (i) The PD-1 inhibitor is selected from the group consisting of PDR001, nivolumab, pembrolizumab, pidilizumab, MEDI0680, REGN2810, TSR-042, PF-06801591, and AMP-224; (ii) The PD-L1 inhibitor is selected from the group consisting of FAZ053, atezolizumab, avelumab, durvalumab, and BMS-936559; (iii) The LAG-3 inhibitor is selected from the group consisting of LAG525, BMS-986016, TSR-033, MK-4280, and REGN3767; (iv) The TIM-3 inhibitor is selected from the group consisting of MBG453, TSR-022, and LY3321367; (v) The CTLA-4 inhibitor is ipilimumab or tremelimumab; (vi) The mTOR inhibitor is RAD001 or rapamycin, The use according to claim 26.

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