Chimeric antigen receptors and uses thereof

Abstract

To provide immune effector cells (for example, T cells and NK cells) that express a chimeric antigen receptor (CAR), and compositions and methods thereof.SOLUTION: Provided is an isolated cell comprising: (a) a first antigen-binding domain that is an anti-BCMA binding domain; and (b) a second antigen-binding domain, each disposed in a chimeric antigen receptor (CAR).SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Patent Application No. 62 / 940,509, filed November 26, 2019, the entire contents of which are incorporated herein by reference.

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

[0003] The present invention relates generally to immune effector cells (e.g., T cells or NK cells) engineered to express chimeric antigen receptors (CARs), and compositions and uses thereof. [Background technology]

[0004] Adoptive cell transfer (ACT) therapy with T cells, particularly T cells transduced with chimeric antigen receptors (CARs), has shown promise in trials for several hematologic cancers. Methods and processes are needed to improve the production of CAR-expressing cell therapy products, enhance product quality, and maximize their therapeutic efficacy. Summary of the Invention [Means for solving the problem]

[0005] In one aspect, the invention features a cell, e.g., an immune cell, e.g., a T cell or an NK cell, comprising a first antigen-binding domain and a second antigen-binding domain. 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, e.g., a CDR, a VH, a VL, or an 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, e.g., a CDR, a VH, a VL, or an scFv sequence disclosed in Tables 2, 19, 22, and 31.

[0006] In some embodiments, the invention provides (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), wherein HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 The present invention provides cells comprising: (i) a first antigen-binding domain; and (b) a second antigen-binding domain, wherein the CDR3 comprises the amino acid sequence of 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. In some embodiments, the first antigen-binding domain and the second antigen-binding domain are arranged in two chimeric antigen receptors (CARs). In some embodiments, the first antigen-binding domain and the second antigen-binding domain are arranged in one CAR.

[0007] In some embodiments, the HC CDRl, HC CDR2, HC CDR3, LC CDRl, LC CDR2, and LC CDR3 comprise the amino acid sequences of SEQ ID NOs: 86, 130, 88, 95, 131, and 132, respectively. In some embodiments, the HC CDRl, HC CDR2, HC CDR3, LC CDRl, LC CDR2, and LC CDR3 comprise the amino acid sequences of SEQ ID NOs: 86, 87, 88, 95, 96, and 97, respectively. In some embodiments, the HC CDRl, HC CDR2, HC CDR3, LC CDRl, LC CDR2, and LC CDR3 comprise the amino acid sequences of SEQ ID NOs: 86, 109, 88, 95, 114, and 115, respectively. In some embodiments, the HC CDRl, HC CDR2, HC CDR3, LC CDRl, LC CDR2, and LC CDR3 comprise the amino acid sequences of SEQ ID NOs: 86, 109, 88, 95, 114, and 97, respectively. In some embodiments, the 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 thereof. In some embodiments, the 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 thereof. In some embodiments, the 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 thereof. In some embodiments, the 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 thereof. In some embodiments, the VH and VL comprise the amino acid sequences of SEQ ID NOs: 93 and 102, respectively. In some embodiments, the VH and VL comprise the amino acid sequences of SEQ ID NOs: 112 and 118, respectively. In some embodiments, the VH and VL comprise the amino acid sequences of SEQ ID NOs: 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 a 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, the HC CDRl, HC CDR2, HC CDR3, LC CDRl, LC CDR2 and LC CDR3 comprise the amino acid sequences of SEQ ID NOs: 44, 45, 84, 54, 55 and 56, respectively. In some embodiments, the HC CDRl, HC CDR2, HC CDR3, LC CDRl, LC CDR2 and LC CDR3 comprise the amino acid sequences of SEQ ID NOs: 44, 45, 76, 54, 55 and 56, respectively. In some embodiments, the HC CDRl, HC CDR2, HC CDR3, LC CDRl, LC CDR2 and LC CDR3 comprise the amino acid sequences of SEQ ID NOs: 44, 45, 46, 54, 55 and 56, respectively. In some embodiments, the HC CDRl, HC CDR2, HC CDR3, LC CDRl, LC CDR2 and LC CDR3 comprise the amino acid sequences of SEQ ID NOs: 44, 45, 68, 54, 55 and 56, respectively. In some embodiments, the 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, the 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, the 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, the 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, the VH and VL comprise the amino acid sequences of SEQ ID NOs: 78 and 61, respectively. In some embodiments, the VH and VL comprise the amino acid sequences of SEQ ID NOs: 52 and 61, respectively. In some embodiments, VH and VL comprise the amino acid sequences of SEQ ID NOs: 70 and 61, respectively. In some embodiments, the first antigen-binding domain comprises a single-chain variable 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 on a 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, the HC CDRl, HC CDR2, HC CDR3, LC CDRl, LC CDR2, and LC CDR3 comprise the amino acid sequences of SEQ ID NOs: 179, 180, 181, 147, 182, and 183, respectively. In some embodiments, the HC CDRl, HC CDR2, HC CDR3, LC CDRl, LC CDR2, and LC CDR3 comprise the amino acid sequences of SEQ ID NOs: 137, 138, 139, 147, 148, and 149, respectively. In some embodiments, the HC CDRl, HC CDR2, HC CDR3, LC CDRl, LC CDR2, and LC CDR3 comprise the amino acid sequences of SEQ ID NOs: 160, 161, 162, 147, 170, and 171, respectively. In some embodiments, the 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, the 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 thereof. In some embodiments, the 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 thereof. In some embodiments, the 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 thereof. In some embodiments, the VH and VL comprise the amino acid sequences of SEQ ID NOs: 145 and 154, respectively. In some embodiments, the VH and VL comprise the amino acid sequences of SEQ ID NOs: 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 cells comprising: (a) a first antigen binding domain that is an anti-BCMA binding domain, the anti-BCMA binding domain comprising: (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 antigen binding domain. In some embodiments, the first antigen binding domain and the second antigen binding domain are arranged in two chimeric antigen receptors (CARs). In some embodiments, the first antigen-binding domain and the second antigen-binding domain are located on one CAR. In some embodiments, the second antigen-binding domain is selected from the group consisting of 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, CD1 In some embodiments, the second antigen-binding domain binds to an antigen selected from CD19, CD20, CD22, FcRn5, FcRn2, CS-1, CD138, CD123, CD33, CD34, CLL-1, folate receptor beta, or FLT3.In some embodiments, the second antigen binding domain is 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 The antibody binds to an antigen 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 on MHC.

[0011] In some embodiments, the second antigen-binding domain binds to CD19. In some embodiments, the second antigen-binding domain comprises an HC CDR1, an HC CDR2, an HC CDR3, an LC CDR1, an LC CDR2, and / or an LC CDR3 of an anti-CD19 sequence listed in Table 19 or Table 22, e.g., an HC CDR1, an HC CDR2, an HC CDR3, an LC CDR1, an LC CDR2, and an LC CDR3 comprising the sequences of SEQ ID NOs: 295 and 245-249, respectively. In some embodiments, the second antigen-binding domain comprises a VH and / or VL of an anti-CD19 sequence listed in Table 19 or Table 22, e.g., a 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 on a second CAR, wherein 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 (a) HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 comprising the amino acid sequences of 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 comprising 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 comprising 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 a VH and a VL comprising the amino acid sequence of SEQ ID NOs: 250 and 251, respectively. In some embodiments, the first antigen-binding domain comprises an scFv comprising the amino acid sequence of SEQ ID NO: 105, 80, or 64. In some embodiments, the second antigen-binding domain comprises an scFv comprising 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 a first CAR and the second antigen-binding domain is disposed in a 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 located 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 arranged on a single nucleic acid molecule. In some embodiments, the single nucleic acid molecule comprises, from 5' to 3', the following: 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, and a nucleic acid sequence encoding a second intracellular signaling domain. In some embodiments, the single nucleic acid molecule comprises, from 5' to 3', the following: 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 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. 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 arranged in a single CAR, and the CAR further comprises a transmembrane domain and an intracellular signaling domain. In some embodiments, the first antigen-binding domain comprises a first VH (VH1) and a first VL (VL1), and the second antigen-binding domain comprises a second VH (VH2) and a second VL (VL2). In some embodiments, VH1, VL1, VH2, and VL2 are arranged from N-terminus to C-terminus in the following configuration: VH2-optional linker 1 ("L1")-VL1-optional linker 2 ("L2")-VH1-optional linker 3 ("L3")-VL2. In some embodiments, VH1, VL1, VH2, and VL2 are arranged from N-terminus to C-terminus in the following configuration: VH1-optionally L1-VH2-optionally L2-VL2-optionally L3-VL1. In some embodiments, VH1, VL1, VH2, and VL2 are arranged from N-terminus to C-terminus in the following configuration: VL2-optionally L1-VL1-optionally L2-VH1-optionally L3-VH2. In some embodiments, VH1, VL1, VH2, and VL2 are arranged from N-terminus to C-terminus in the following configuration: VL2-optionally L1-VH1-optionally L2-VL1-optionally L3-VH2. In some embodiments, VH1, VL1, VH2, and VL2 are arranged from N-terminus to 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 N-terminus to C-terminus in the following configuration: VL1-optionally L1-VH2-optionally L2-VL2-optionally L3-VH1. In some embodiments, VH1, VL1, VH2, and VL2 are arranged from N-terminus to C-terminus in the following configuration: VL1-optionally L1-VL2-optionally L2-VH2-optionally L3-VH1. In some embodiments, VH1, VL1, VH2 and VL2 are arranged from N-terminus to C-terminus in the following configuration: VH1-optionally L1-VL2-optionally L2-VH2-optionally L3-VL1.In some embodiments, VH1 and VL1 comprise the amino acid sequence of SEQ ID NOs: 93 and 102, respectively (or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, VH1 and VL1 comprise the amino acid sequence of SEQ ID NOs: 333 and 334, respectively (or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, VH1 and VL1 comprise the amino acid sequence of SEQ ID NOs: 78 and 61, respectively (or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, VH1 and VL1 comprise the amino acid sequence of SEQ ID NOs: 335 and 336, respectively (or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, VH2 and VL2 comprise the amino acid sequence of SEQ ID NOs: 250 and 251, respectively (or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, VH2 and VL2 comprise the amino acid sequence of SEQ ID NOs: 331 and 332, respectively (or an amino acid sequence 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, a CAR is encoded by a nucleic acid molecule comprising, in 5' to 3' direction, 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, a CAR is encoded by a nucleic acid molecule comprising, in 5' to 3' direction, 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 the 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, a CAR comprises the following configuration, from N to C: first antigen binding domain-optional linker-second antigen binding domain-transmembrane domain-intracellular signaling domain. In some embodiments, a CAR comprises the following configuration, from N to C: second antigen binding domain-optional linker-first antigen binding domain-transmembrane domain-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 VL are connected 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 a 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., a 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 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, first intracellular signaling domain, or second intracellular signaling domain comprises a primary signaling domain (e.g., a 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 costimulatory signaling domain (e.g., a first or second costimulatory signaling domain).In some embodiments, the costimulatory signaling domain comprises a functional signaling domain derived from an MHC class I molecule, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocyte activation molecule (SLAM protein), an activating NK cell receptor, BTLA, a 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, C D49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB 7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, L A ligand that specifically binds to y9 (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 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 thereof. 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 thereof.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, first CAR, or second CAR further comprises a leader sequence (e.g., a 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 and second leader sequences 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). In some embodiments, the first and second hinge regions 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). In some embodiments, the first and second transmembrane domains 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). In some embodiments, the first intracellular signaling domain and the second intracellular signaling domain 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). In some embodiments, the first primary signaling domain and the second primary signaling domain 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). In some embodiments, the first costimulatory signaling domain and the second costimulatory signaling domain 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). 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 the second hinge region sequences comprise different amino acid sequences. In some embodiments, the first transmembrane domain and the second transmembrane domain comprise the same amino acid sequence (e.g., 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 comprise different amino acid sequences. In some embodiments, the first intracellular signaling domain and the second intracellular signaling domain comprise the same amino acid sequence. In some embodiments, the first intracellular signaling domain and the second intracellular signaling domain comprise different amino acid sequences. In some embodiments, the first primary signaling domain and the second primary signaling domain comprise the same amino acid sequence (e.g., 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 comprise different amino acid sequences. In some embodiments, the first costimulatory signaling domain and the second costimulatory signaling domain comprise the same amino acid sequence (e.g., the first costimulatory signaling domain and the second costimulatory 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 costimulatory signaling domain and the second costimulatory signaling domain comprise different amino acid sequences (e.g., the first and second costimulatory signaling domains comprise a 4-1BB costimulatory domain sequence and a CD28 costimulatory domain sequence, respectively; or a CD28 costimulatory domain sequence and a 4-1BB costimulatory domain sequence, respectively). In some embodiments, the first leader sequence and the second leader sequence are encoded by a nucleic acid sequence comprising SEQ ID NOs: 199 and 210, respectively (or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto).In some embodiments, the first and second leader sequences are encoded by a nucleic acid sequence comprising SEQ ID NOs: 210 and 199, respectively (or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the first and second hinge regions are encoded by a nucleic acid sequence comprising SEQ ID NOs: 337 and 13, respectively (or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the first and second hinge region sequences are encoded by a nucleic acid sequence comprising SEQ ID NOs: 13 and 337, respectively (or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto). In some embodiments, the first and second transmembrane domains are encoded by a nucleic acid sequence comprising SEQ ID NOs: 338 and 17, respectively (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 sequence are encoded by a nucleic acid sequence comprising SEQ ID NOs: 17 and 338 (or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto), respectively. In some embodiments, the first costimulatory signaling domain and the second costimulatory signaling domain are encoded by a nucleic acid sequence comprising SEQ ID NOs: 204 and 18 (or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto), respectively. In some embodiments, the first costimulatory signaling domain and the second costimulatory sequence are encoded by a nucleic acid sequence comprising SEQ ID NOs: 18 and 204 (or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto), respectively. In some embodiments, the first primary signaling domain and the second primary signaling domain are encoded by a nucleic acid sequence comprising SEQ ID NOs: 205 and 21 (or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto), respectively.In some embodiments, the first costimulatory signaling domain and the second costimulatory sequence are encoded by a nucleic acid sequence comprising SEQ ID NOs: 21 and 205, respectively (or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto).

[0025] In some embodiments, the CAR, first CAR, or second CAR is encoded by a nucleic acid sequence that includes a woodchuck hepatitis post-transcriptional regulatory element (WPRE).

[0026] In some embodiments, a nucleic acid molecule comprises: (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 Provided herein are nucleic acid molecules comprising: (i) a first nucleic acid sequence, wherein the CDR3 comprises the amino acid sequence of 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, wherein the first nucleic acid sequence is located on the first nucleic acid molecule and the second nucleic acid sequence is located 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 are nucleic acid molecules 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, wherein (i) the first antigen binding domain comprises HC CDRl, HC CDR2, HC CDR3, LC CDRl, LC CDR2, and LC CDRs 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 CDRl, HC CDR2, HC CDR3, LC CDRl, LC CDR2, and LC CDRs comprising the amino acid sequences of SEQ ID NOs: 295 and 245-249, respectively. (ii) the first antigen-binding domain comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 93 and 102, respectively, and the second antigen-binding domain comprises a VH and a 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 are nucleic acid molecules 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, wherein (i) the first antigen binding domain comprises HC CDRl, HC CDR2, HC CDR3, LC CDRl, LC CDR2, and LC CDRs 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 CDRl, HC CDR2, HC CDR3, LC CDRl, LC CDR2, and LC CDRs comprising the amino acid sequences of SEQ ID NOs: 295 and 245-249, respectively. (ii) the first antigen-binding domain comprises a VH and a VL comprising the amino acid sequences of SEQ ID NOs: 78 and 61, respectively, and the second antigen-binding domain comprises a VH and a 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: 80, 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: 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 are polypeptide molecules encoded by the nucleic acid molecules 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, 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), wherein the HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC The CDR3 comprises (i) a first antigen-binding domain comprising the amino acid sequence of 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 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 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 antigen-binding domain.

[0034] In some embodiments, provided herein is a vector comprising 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, provided herein is a cell comprising 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, provided herein are methods of making a cell, the method comprising transducing a cell with a vector disclosed herein, optionally wherein the cell is a T cell or an NK cell. In some embodiments, provided herein are methods of making an RNA-engineered cell, the method comprising introducing in vitro transcribed RNA or synthetic RNA into a cell, 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 are methods of producing 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, e.g., T cells isolated from a frozen or fresh leukapheresis product) with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule 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) comprising the nucleic acid molecule; and (iii) harvesting the population of cells (e.g., T cells) for storage (e.g., reformulating the population of cells in cryopreservation medium) or administration; (a) step (ii) is carried out 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 carried out 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), for example within 24 hours after the start of step (i); (b) step (ii) is carried out 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 carried out 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%, e.g., no more than 10%, relative to the population of cells at the start of step (i), e.g., as assessed by the number of viable cells; 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 the viral vector, and optionally step (ii) comprises transducing a population of cells (e.g., T cells) with the viral vector comprising the nucleic acid molecule encoding the CAR.

[0038] In some embodiments, provided herein are methods of producing a population of cells (e.g., T cells) that express a chimeric antigen receptor (CAR), the methods comprising: (1) contacting a population of cells (e.g., T cells, e.g., T cells isolated from a frozen or fresh leukapheresis product) 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 a combination 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) comprising the nucleic acid molecule; and (3) harvesting the population of cells (e.g., T cells) for storage (e.g., reformulating the population of cells in cryopreservation medium) or administration; (a) step (2) is carried out simultaneously with step (1) or within 5 hours after the initiation of step (1), e.g., within 1, 2, 3, 4, or 5 hours after the initiation of step (1); and Step (3) is carried out within 26 hours after the initiation of step (1), e.g., within 22, 23, or 24 hours after the initiation of step (1), e.g., within 24 hours after the initiation 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%, relative to the population of cells at the start of step (1), e.g., as assessed by the number of viable cells; Optionally, the nucleic acid molecule of step (ii) is on a viral vector, and optionally the nucleic acid molecule of step (ii) is an RNA molecule on the viral vector, and optionally step (ii) comprises transducing a population of cells (e.g., T cells) with the viral vector comprising the nucleic acid molecule encoding the CAR.

[0039] In some embodiments, disclosed herein are populations of cells engineered to express a CAR ("populations of CAR-expressing cells"), said populations comprising: (a) about the same percentage of naive cells, e.g., naive T cells, e.g., CD45RO-CCR7+ T cells, as compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RO-CCR7+ T cells, in the same population of cells before they were engineered to express a CAR; (b) about the same percentage of naive cells, e.g., naive T cells, e.g., CD45RO-CCR7+ T cells, as compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RO-CCR7+ T cells, in the same population of cells before they were engineered to express a CAR. (c) an increased percentage of naive cells, e.g., naive T cells, e.g., CD45RO-CCR7+ T cells, compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RO-CCR7+ T cells, in the same population of cells before being engineered to express a CAR, e.g., by at least 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, or 3-fold increase in naive cells, e.g., naive T cells, e.g., CD4 (d) about the same percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells, 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 they were engineered to express a CAR; (e) about the same percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells, 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 they were engineered to express a CAR. (f) a decreased percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells, 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 they were engineered to express a CAR, e.g., a decrease of at least 20, 25, 30, 35, 40, 45, or 50%;(g) Approximately the same percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, as compared to the percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in the same population of cells before they were engineered to express a CAR; (h) approximately the same percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in the same population of cells before they were engineered to express a CAR. or (i) an increased percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, compared to the percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in the same population of cells prior to being engineered to express a CAR. In some embodiments, the population comprises 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 (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), wherein HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC The CDR3 comprises a first antigen-binding domain and (b) a second antigen-binding domain comprising the amino acid sequence 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;

[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 produced by a method disclosed herein. In some embodiments, the population is (a) First cell population containing anti-BCMA CAR but no 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 containing both anti-BCMA and anti-CD19 CARs. Includes:

[0042] In some embodiments, (i) the total number of viable cells in the second and third populations combined 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 first and third populations combined 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 in the second and third populations combined; (iii) the total number of viable cells in the first and third populations combined 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 comprises a fourth cell population that does not comprise 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, disclosed herein are methods of providing anti-tumor immunity in a subject, comprising administering to the subject an effective amount of a cell disclosed herein, a population of cells disclosed herein, or a pharmaceutical composition disclosed herein. In some embodiments, disclosed herein are methods of treating a subject having a disease associated with BCMA expression, comprising administering to the subject an effective amount of a cell disclosed herein, a population of cells disclosed herein, or a pharmaceutical composition disclosed herein. In some embodiments, the disease associated with BCMA expression is (i) a cancer or malignant disease or precancerous condition selected from one or more of myelodysplasia, myelodysplastic syndrome, or preleukemia, 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 myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndromes, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell The disease is a neoplasm, Waldenstrom's macroglobulinemia, prostate cancer (e.g., castration-resistant or treatment-resistant prostate cancer or metastatic prostate cancer), pancreatic cancer, lung cancer, plasma cell proliferative disorder (e.g., asymptomatic myeloma (smoldering multiple myeloma or asymptomatic myeloma), monoclonal gammopathy of undetermined significance (MGUS), Waldenstrom's macroglobulinemia, plasmacytoma (e.g., dysplasmocytoma, 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 a combination 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 ~about 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~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 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 x 10 7 ) viable CAR-positive cells (e.g., BCMA CAR+ T cells); (b) The second dose is approximately 1 × 10 7 ~Approx. 1×10 8 (For example, about 2 × 10 7 ~Approx. 8×10 7 , about 4×10 7 ~about 6×10 7 , about 1×10 7 ~Approx. 5×10 7 , about 5×10 7 ~Approx. 1×10 8 , 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 1×107 , 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 x 10 8 ) viable CAR-positive cells (e.g., BCMA CAR+ T cells); (c) the number of viable CAR-positive cells (e.g., BCMA CAR+ T cells) in the first dose is less than or equal to 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) of the number of viable CAR-positive cells (e.g., BCMA CAR+ T cells) in the second dose; and / or (d) The number of viable CAR-positive 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 viable CAR-positive cells (e.g., BCMA CAR+ T cells) in the second dose.

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

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

[0051] In some embodiments, provided herein are cells comprising: (a) a first CAR comprising 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), 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 a second antigen, a second transmembrane domain, and a second intracellular signaling domain (e.g., a second primary signaling domain and / or a second costimulatory 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 different (e.g., by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, (ii) the first and second leader sequences 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; and (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. (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, and 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 costimulatory signaling domain and the second costimulatory 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 intending to be bound by theory, such nucleic acid molecules exhibit lower recombination than otherwise similar nucleic acid molecules in which 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 intending to be bound by theory, such nucleic acid molecules exhibit lower recombination than otherwise similar nucleic acid molecules in which 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 intending to be bound by theory, such nucleic acid molecules exhibit lower recombination than otherwise similar nucleic acid molecules in which the first and second transmembrane domains are encoded by the same nucleic acid sequence.

[0056] In some embodiments, the first intracellular signaling domain and the second intracellular signaling domain comprise the same amino acid sequence. Without intending to be bound by theory, such nucleic acid molecules exhibit lower recombination than otherwise similar nucleic acid molecules in which the first intracellular signaling domain and the second intracellular signaling domain are encoded by the same nucleic acid sequence.

[0057] In some embodiments, the first primary signaling domain and the second primary signaling domain comprise the same amino acid sequence. Without intending to be bound by theory, such nucleic acid molecules exhibit lower recombination than otherwise similar nucleic acid molecules in which the first primary signaling domain and the second primary signaling domain are encoded by the same nucleic acid sequence.

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

[0059] In some embodiments, the first costimulatory signaling domain and the second costimulatory signaling domain comprise the same amino acid sequence. Without intending to be bound by theory, such nucleic acid molecules exhibit lower recombination than otherwise similar nucleic acid molecules in which the first costimulatory signaling domain and the second costimulatory signaling domain are encoded by the same nucleic acid sequence.

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

[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 a nucleic acid sequence comprising SEQ ID NOs: 199 and 210, respectively (or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto), or a nucleic acid sequence comprising SEQ ID NOs: 210 and 199, respectively (or a nucleic acid sequence 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 a nucleic acid sequence comprising SEQ ID NOs: 337 and 13, respectively (or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto); or a nucleic acid sequence comprising SEQ ID NOs: 13 and 337, respectively (or a nucleic acid sequence 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 encoded by a nucleic acid sequence comprising SEQ ID NOs: 338 and 17, respectively (or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto); or a nucleic acid sequence comprising SEQ ID NOs: 17 and 338, respectively (or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto).

[0064] In some embodiments, the first costimulatory signaling domain and the second costimulatory 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 costimulatory signaling domain and the second costimulatory signaling domain are encoded by a nucleic acid sequence comprising SEQ ID NOs: 204 and 18, respectively (or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto); or a nucleic acid sequence comprising SEQ ID NOs: 18 and 204, respectively (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 encoded by a nucleic acid sequence comprising SEQ ID NOs: 205 and 21, respectively (or a nucleic acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto); or a nucleic acid sequence comprising SEQ ID NOs: 21 and 205, respectively (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 a CDR, VH, VL, or scFv disclosed herein, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto.

[0067] In some embodiments, provided herein is 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, wherein 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 N-terminus to C-terminus in the following configuration: VH1-optional linker 1 ("L1")-VH2-optional linker 2 ("L2")-VL2-optional linker 3 ("L3")-VL1. In some embodiments, VH1, VL1, VH2, and VL2 are arranged from N-terminus to 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 N-terminus to C-terminus in the following configuration: VL1-linker1-VH2-linker2-VL2-linker3-VH1. In some embodiments, VH1, VL1, VH2, and VL2 are arranged from N-terminus to C-terminus in the following configuration: VL1-linker1-VL2-linker2-VH2-linker3-VH1. In some embodiments, VH1, VL1, VH2, and VL2 are arranged from N-terminus to C-terminus in the following configuration: VH2-optional L1-VH1-optional L2-VL1-optional L3-VL2. In some embodiments, VH1, VL1, VH2, and VL2 are arranged from N-terminus to C-terminus in the following configuration: VH2-optional L1-VL1-optional L2-VH1-optional L3-VL2. In some embodiments, VH1, VL1, VH2, and VL2 are arranged from N-terminus to C-terminus in the following configuration: VL2-linker1-VH1-linker2-VL1-linker3-VH2. In some embodiments, VH1, VL1, VH2, and VL2 are arranged from N-terminus to C-terminus in the following configuration: VL2-linker1-VL1-linker2-VH1-linker3-VH2. In some embodiments, VH1, VL1, VH2, and VL2 are arranged from N-terminus to 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 from N-terminus to C-terminus in the following configuration: VH1-L1-VL2-L2-VH2-L3-VL1. In some embodiments, VH1, VL1, VH2, and VL2 are arranged from N-terminus to C-terminus in the following configuration: VL1-L1-VH2-L2-VL2-L3-VH1. In some embodiments, VH1, VL1, VH2, and VL2 are arranged from N-terminus to C-terminus in the following configuration: VL1-L1-VL2-L2-VH2-L3-VH1. In some embodiments, VH1, VL1, VH2, and VL2 are arranged from N-terminus to C-terminus in the following configuration: VH2-L1-VH1-L2-VL1-L3-VL2. In some embodiments, VH1, VL1, VH2, and VL2 are arranged from N-terminus to C-terminus in the following configuration: VH2-L1-VL1-L2-VH1-L3-VL2. In some embodiments, VH1, VL1, VH2, and VL2 are arranged from N-terminus to C-terminus in the following configuration: VL2-L1-VH1-L2-VL1-L3-VH2. In some embodiments, VH1, VL1, VH2, and VL2 are arranged from N-terminus to C-terminus in the following configuration: 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, a CAR comprises the following configuration from N-terminus to C-terminus: (i) VH1-optional linker 1 ("L1")-VH2-optional linker 2 ("L2")-VL2-optional linker 3 ("L3")-VL1-optional hinge region-transmembrane domain-intracellular signaling domain; (ii) VH1-optional L1-VL2-optional L2-VH2-optional L3-VL1-optional hinge region-transmembrane domain-intracellular signaling domain; (iii) VL1-optional L1-VH2-optional L2-VL2-optional L3-VH1-optional hinge region-transmembrane domain-intracellular signaling domain; (iv) VL1-optional L1-VL2-optional L2-VH2-optional L3-VH1-optional (v) VH2-optionally L1-VH1-optionally L2-VL1-optionally L3-VL2-optional hinge region-transmembrane domain-intracellular signaling domain; (vi) VH2-optionally L1-VL1-optionally L2-VH1-optionally L3-VL2-optional hinge region-transmembrane domain-intracellular signaling domain; (vii) VL2-optionally L1-VH1-optionally L2-VL1-optionally L3-VH2-optional hinge region-transmembrane domain-intracellular signaling domain; or (viii) VL2-optionally L1-VL1-optionally L2-VH1-optionally L3-VH2-optional 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, the 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., a primary signaling domain and / or a costimulatory signaling domain) comprises a hinge region sequence, transmembrane domain sequence, or intracellular signaling domain sequence (e.g., a primary signaling domain sequence and / or a 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, provided herein are nucleic acid molecules encoding a diabody CAR disclosed herein. In some embodiments, provided herein are vectors comprising a nucleic acid molecule encoding a diabody CAR disclosed herein. In some embodiments, provided herein are 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. In some embodiments, provided herein are pharmaceutical compositions comprising a diabody CAR disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, disclosed herein are methods of making cells comprising a diabody CAR disclosed herein. In some embodiments, disclosed herein are methods of treating a subject, e.g., a subject with cancer, using cells comprising a diabody CAR disclosed herein.

[0069] In some embodiments, the present disclosure relates to methods of producing immune effector cells (e.g., T cells or NK cells) engineered to express a CAR, and compositions produced using such methods. The methods disclosed herein (e.g., the ARM process or cytokine process disclosed herein) can be used to produce cells that express the dual CARs or diabody CARs disclosed herein. Also disclosed are methods of using such compositions to treat a disease, e.g., cancer, in a subject.

[0070] In some embodiments, the invention features a method of producing a population of cells (e.g., T cells) that express a chimeric antigen receptor (CAR), the method including the steps of: (i) contacting (e.g., binding) a population of cells (e.g., T cells, e.g., T cells isolated from a frozen or fresh leukapheresis product) with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells; (ii) contacting the population of cells (e.g., T cells) with a nucleic acid molecule (e.g., DNA or RNA) encoding a CAR; (iii) harvesting the population of cells (e.g., T cells) for storage (e.g., reformulating the population of cells in cryopreservation medium) or administration, wherein (a) step (ii) is performed together with step (i) or within 20 hours after initiation of step (i), e.g., within 12, 13, 14, 15, 16, 17, or 18 hours after initiation of step (i), e.g., within 18 hours after initiation of step (i). and step (iii) is carried out within 26 hours after the start of step (i), for example within 22, 23, 24 or 25 hours after the start of step (i), for example within 24 hours after the start of step (i); (b) step (ii) is carried out together with step (i) or within 20 hours after the start of step (i), for example within 12, 13, 14, 15, 16, 17 or 18 hours after the start of step (i), for example within 18 hours after the start of step (i), and step (iii) is carried out together with step (i), or (c) the population of cells from step (iii) is not expanded or is expanded 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%, e.g., no more than 10%, as assessed by the number of viable cells, relative 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, wherein 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 a first antigen, and VH2 and VL2 bind a second antigen, and wherein VH1, VL1, VH2, and VL2 are arranged from N-terminus to C-terminus as follows: VH1-optional linker 1 ("L1")-VH2-optional linker 2 ("L2")-VL2-optional linker 3 ("L3")-VL1; VH1-optional L1-VL2-optional 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 no more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40%, e.g., as assessed 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 no more than 10%, e.g., as assessed 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 located 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 the first CAR and a second viral vector comprising a second nucleic acid molecule encoding the 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 cell population is contacted with the first viral vector at a multiplicity of infection (MOI) that is higher than, equal to, or lower than the MOI at which the cell population is contacted with the second viral vector. In some embodiments, in step (ii), the cell population is contacted with the first viral vector at a multiplicity of infection (MOI) that is higher than the MOI at which the cell population is contacted with the second viral vector.

[0076] In some embodiments, in step (ii), the cell populations are contacted with a first viral vector at a first MOI and a second viral vector at a second MOI, such that the resulting cell populations include 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) the total number of viable cells in the second and third populations combined 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, e.g., as determined by the method described in Example 10; (b) the total number of viable cells in the first and third populations combined 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 in the second and third populations combined, e.g., as determined by the method described in Example 10; (c) the total number of viable cells in the first and third populations combined 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 resulting populations, e.g., as determined by the method described in Example 10; (d) 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, e.g., as determined by the method described in Example 10; or (e) the total number of viable cells in the first and third populations combined 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 in the second population, e.g., as determined by the method described in Example 10. In some embodiments, in step (ii), the second viral vector is contacted with the population of cells at a particular MOI (e.g., an MOI sufficiently lower than the MOI at which the first viral vector is contacted with the cell population, such that the following MOI is achieved in the resulting cell population: (a) the total number of viable cells in the second and third populations combined 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, e.g., as determined by the method described in Example 10; (b) the total number of viable cells in the first and third populations combined 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 in the second and third populations combined, e.g., as determined by the method described in Example 10; (c) the total number of viable cells in the first and third populations combined 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 resulting populations, e.g., as determined by the method described in Example 10; (d) 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, e.g., as determined by the method described in Example 10; or (e) the total number of viable cells in the first and third populations combined 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 in the second population, e.g., as determined by the method described in Example 10.

[0077] In some embodiments, in step (ii), the cell population is contacted with a first viral vector at a first MOI and the cell population is contacted with a second viral vector at a second MOI, such that the resulting cell population comprises: (a) the total number of viable cells in the second and third populations combined 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, e.g., as determined by the method described in Example 10; (b) the total number of viable cells in the first and third populations combined 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 in the second and third populations combined, e.g., as determined by the method described in Example 10; (c) the total number of viable cells in the first and third populations combined 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 resulting populations, e.g., as determined by the method described in Example 10; (d) 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, e.g., as determined by the method described in Example 10; or (e) the total number of viable cells in the first and third populations combined 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 in the second population, e.g., as determined by the method described in Example 10.

[0078] In some embodiments, in step (ii), the cell population is (a) a 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) 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 the second viral vector at an MOI of about 0.5, 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) the 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 at which the cell population is contacted with the second viral vector, or 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) higher than the MOI at which the cell population is contacted with the second viral vector; and / or (d) contacting the cell population with the first viral vector at an MOI of 1 / X or less (where X is 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100) of the first viral vector; and bring it into contact with.

[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 has an MOI that is about 8 to about 10 times higher than the MOI at which the second viral vector is contacted with the cell population. In some embodiments, the second viral vector has an MOI that is 1 / X (where X is 6, 8, 10, or 12) or less of the MOI at which the first viral vector is contacted 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 costimulatory 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 costimulatory 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 an 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 costimulatory 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 an 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 comprise a bead. In some embodiments, the agent that stimulates a costimulatory molecule does not comprise a bead. In some embodiments, the agent that stimulates the CD3 / TCR complex comprises an anti-CD3 antibody. In some embodiments, the agent that stimulates a costimulatory molecule comprises an anti-CD28 antibody. In some embodiments, the agent that stimulates the CD3 / TCR complex comprises an anti-CD3 antibody covalently attached to a colloidal polymer nanomatrix. In some embodiments, the agent that stimulates a costimulatory molecule comprises an anti-CD28 antibody covalently attached to a colloidal polymer nanomatrix. In some embodiments, the agent that stimulates the CD3 / TCR complex and the agent that stimulates a costimulatory molecule comprise T Cell TransAct™.

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

[0084] In some embodiments, the agent that stimulates the CD3 / TCR complex comprises a hydrogel. In some embodiments, the agent that stimulates a costimulatory molecule comprises a hydrogel. In some embodiments, the agent that stimulates the CD3 / TCR complex comprises alginate. In some embodiments, the agent that stimulates a costimulatory molecule comprises alginate. In some embodiments, the agent that stimulates the CD3 / TCR complex or the agent that stimulates a costimulatory molecule comprises MagCloudz™ manufactured by 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) than cells produced by an otherwise similar method without step (i).

[0086] In some embodiments, the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, in the population of cells from step (iii) is the same as the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ cells, in the population of cells at the start of step (i). In some embodiments, the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, in the population of cells from step (iii) differs by no more than 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12% from the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ cells, in the population of cells at the start of step (i). In some embodiments, the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, in the population of cells from step (iii) differs by no more than 5 or 10% from the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ cells, in the population of cells at the start of step (i).

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

[0088] In some embodiments, the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the population of cells from step (iii) is the same as the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the population of cells at the start of step (i). In some embodiments, the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the population of cells from step (iii) differs by no more than 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12% from the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the population of cells at the start of step (i). In some embodiments, the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the population of cells from step (iii) differs by no more than 5 or 10% from the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the population of cells at the start of step (i).

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

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

[0091] In some embodiments, the median GeneSetScore (Up TEM vs. Down TSCM) of the population of cells from step (iii) is about the same as or differs from (e.g., is increased by) no more than about 25, 50, 75, 100, or 125% of (i.e., is increased by) the median GeneSetScore (Up TEM vs. Down TSCM) of the population of cells at the start of step (i). In some embodiments, the median GeneSetScore (Up TEM vs. Down TSCM) of the population of cells from step (iii) is lower (e.g., at least about 100, 150, 200, 250, or 300% lower) than the median GeneSetScore (Up TEM vs. Down TSCM) of a population of cells 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 (Up TEM vs. Down TSCM) of the population of cells from step (iii) is lower (e.g., at least about 100, 150, 200, 250, or 300% lower) than the median GeneSetScore (Up TEM vs. Down TSCM) of a population of cells made by a similar method but further comprising expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii). In some embodiments, the median GeneSetScore (Up Treg vs. Down Teff) of the population of cells from step (iii) is about the same as or differs from (e.g., is increased by) no more than about 25, 50, 100, 150, or 200% of the median GeneSetScore (Up Treg vs. Down Teff) of the population of cells at the start of step (i).In some embodiments, the median GeneSetScore (Up Treg vs. Down Teff) of the population of cells 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 a population of cells generated by a similar method except that step (iii) is performed more than 26 hours after the initiation of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the initiation of step (i). In some embodiments, the median GeneSetScore (Up Treg vs. Down Teff) of the population of cells 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 a population of cells made by a similar method but further comprising expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii). In some embodiments, the median GeneSetScore (Down stemness) of the population of cells from step (iii) is about the same as or differs from (e.g., is increased by) no more than about 25, 50, 100, 150, 200, or 250% of the median GeneSetScore (Down stemness) of the population of cells at the start of step (i). In some embodiments, the median GeneSetScore (Down stemness) of the population of cells from step (iii) is lower (e.g., at least about 50, 100, or 125% lower) than the median GeneSetScore (Down stemness) of a population of cells generated by a similar method, except that step (iii) is performed more than 26 hours after the initiation of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the initiation of step (i).In some embodiments, the median GeneSetScore (Down stemness) of the population of cells from step (iii) is lower (e.g., at least about 50, 100, or 125% lower) than the median GeneSetScore (Down stemness) of a population of cells made by a similar method but further comprising expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii). In some embodiments, the median GeneSetScore (Up hypoxia) of the population of cells from step (iii) is about the same as or differs from (e.g., is increased by) no more than about 125, 150, 175, or 200% of the median GeneSetScore (Up hypoxia) of the population of cells at the start of step (i). In some embodiments, the median GeneSetScore (Up hypoxia) of the population of cells 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 population of cells made by a similar method except that step (iii) is performed more than 26 hours after the initiation of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the initiation of step (i). In some embodiments, the median GeneSetScore (Up hypoxia) of the population of cells 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 population of cells made by a similar method except that step (iii) further includes expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii). In some embodiments, the median GeneSetScore (Up autophagy) of the population of cells from step (iii) is about the same as or differs from (e.g., is increased by) no more than about 180, 190, 200, or 210% of the median GeneSetScore (Up autophagy) of the population of cells 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 initiation of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the initiation 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 method further includes expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 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), after being incubated with cells expressing the antigen recognized by the CAR, secrete IL-2 at a higher level (e.g., at least 2, 4, 6, 8, 10, 12, or 14 fold higher) than cells made by a similar method except that step (iii) is performed more than 26 hours after the initiation of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the initiation of step (i), or than cells made by a similar method except that the method further comprises expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days after step (ii) and before step (iii).

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

[0094] In some embodiments, the population of cells from step (iii), after administration in vivo, exhibits stronger anti-tumor activity (e.g., at a lower dose, e.g., 0.15×10) than cells produced by a similar method except that step (iii) is performed more than 26 hours after the initiation of step (i), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the initiation of step (i), or cells produced by a similar method except that the method further comprises expanding the population of cells (e.g., T cells) in vitro for more than 3 days, e.g., 5, 6, 7, 8, or 9 days, after step (ii) and before step (iii). 6 , 0.2 × 10 6 , 0.25×10 6 or 0.3 x 10 6 (more potent antitumor activity in surviving CAR-expressing cells).

[0095] In some embodiments, the population of cells from step (iii) is not expanded relative to the population of cells at the start of step (i), e.g., as assessed by the number of viable cells. In some embodiments, the population of cells from step (iii) is reduced from the number of viable cells in the population of cells at the start of step (i), e.g., as assessed by the number of viable cells. In some embodiments, the population of cells from step (iii) is expanded 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% relative to the population of cells at the start of step (i), e.g., as assessed by the number of viable cells. In some embodiments, the population of cells from step (iii) is not expanded or is expanded for less than 0.5, 1, 1.5, or 2 hours, e.g., less than 1 or 1.5 hours, relative 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 comprising 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) comprising IL-7, IL-21, or a combination thereof. In some embodiments, steps (i) and (ii) are performed in a cell culture medium comprising 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 comprising 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 comprising 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) comprising 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) comprising IL-7, IL-21, or a combination thereof. In some embodiments, step (i) is performed in a cell culture medium comprising 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 comprising 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 serum-free medium comprising a serum replacement.In some embodiments, the serum replacement is CTS™ Immune Cell Serum Replacement (ICSR).

[0097] In some embodiments, the aforementioned method further comprises, prior to step (i), (iv) receiving 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 a thymus removal)) from an entity, e.g., a laboratory, hospital, or healthcare provider.

[0098] In some embodiments, the aforementioned methods further comprise, prior to step (i), (v) isolating the population of cells (e.g., T cells, e.g., 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, a fresh bone marrow product, or a fresh tumor or organ biopsy or resection (e.g., a fresh product from a thymus removal)). In some embodiments, step (iii) is performed within 35 hours after the initiation of step (v), e.g., within 27, 28, 29, 30, 31, 32, 33, 34, or 35 hours after the initiation of step (v), e.g., within 30 hours after the initiation of step (v). In some embodiments, 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%, e.g., no more than 10%, relative to the population of cells at the end of step (v), as assessed by the number of viable cells.

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

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

[0101] In some embodiments, the aforementioned methods further comprise, prior to step (i), (v) isolating the population of cells (e.g., T cells, e.g., CD8+ and / or CD4+ T cells) to be contacted in step (i) from a cryopreserved leukapheresis product (or an alternative source of hematopoietic tissue, such as a cryopreserved whole blood product, a cryopreserved bone marrow product, or a cryopreserved tumor or organ biopsy or resection (e.g., a cryopreserved product from a thymus removal)). In some embodiments, step (iii) is performed within 35 hours after the initiation of step (v), e.g., within 27, 28, 29, 30, 31, 32, 33, 34, or 35 hours after the initiation of step (v), e.g., within 30 hours after the initiation of step (v). In some embodiments, 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%, e.g., no more than 10%, relative to the population of cells at the end of step (v), as assessed by the number of viable cells.

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

[0103] In some embodiments, the invention features a method of producing a population of cells (e.g., T cells) that express a chimeric antigen receptor (CAR), the method including the steps of: (1) contacting a population of cells (e.g., T cells, e.g., 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 (e.g., a DNA or RNA molecule) encoding a CAR, thereby providing a population of cells (e.g., T cells) comprising the nucleic acid molecule; and (3) preserving the cells (e.g., T cells) for storage (e.g., reformulating the population of cells in cryopreservation medium) or administration (e.g., (a) step (2) is performed together with step (1) or within 5 hours after the initiation of step (1), e.g., within 1, 2, 3, 4, or 5 hours after the initiation of step (1); and step (3) is performed within 26 hours after the initiation of step (1), e.g., within 22, 23, 24, or 25 hours after the initiation of step (1), e.g., within 24 hours after the initiation 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 assessed, e.g., by the number of viable cells, compared to the population of cells at the initiation 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, 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 (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 a vector. In some embodiments, step (2) 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, the nucleic acid molecule encoding the 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, e.g., the first and second nucleic acid sequences 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, wherein 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 a first antigen, and VH2 and VL2 bind a second antigen, and wherein VH1, VL1, VH2, and VL2 are arranged from N-terminus to C-terminus as follows: VH1-optional linker 1 ("L1")-VH2-optional linker 2 ("L2")-VL2-optional linker 3 ("L3")-VL1; VH1-optional L1-VL2-optional 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 performed together with step (1). In some embodiments, step (2) is performed within 5 hours after the initiation of step (1). In some embodiments, step (2) is performed within 1, 2, 3, 4, or 5 hours after the initiation of step (1). In some embodiments, step (3) is performed within 26 hours after the initiation of step (1). In some embodiments, step (3) is performed within 22, 23, 24, or 25 hours after the initiation of step (1). In some embodiments, step (3) is performed within 24 hours after the initiation of step (1).

[0105] In some embodiments, the population of cells from step (3) is not expanded, e.g., as assessed by the number of viable cells, compared to the population of cells at the start of step (1). In some embodiments, the population of cells from step (3) is expanded 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% compared to the population of cells at the start of step (1), e.g., as assessed by the number of viable cells. In some embodiments, the population of cells from step (3) is expanded by no more than 10% compared to the population of cells at the start of step (1), e.g., as assessed by the number of viable cells.

[0106] In some embodiments, step (1) comprises contacting the population of cells (e.g., T cells) with IL-2. In some embodiments, step (1) comprises contacting the population of cells (e.g., T cells) with IL-7. In some embodiments, step (1) comprises contacting the population of cells (e.g., T cells) with IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)). In some embodiments, step (1) comprises contacting the population of cells (e.g., T cells) with IL-21. In some embodiments, step (1) comprises contacting the population of cells (e.g., T cells) with IL-6 (e.g., IL-6 / sIL-6Ra). In some embodiments, step (1) comprises contacting the population of cells (e.g., T cells) with IL-2 and IL-7. In some embodiments, step (1) comprises contacting the 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 the population of cells (e.g., T cells) with IL-2 and IL-21. In some embodiments, step (1) comprises contacting the 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 the 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 the 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 among the CAR-expressing cells (e.g., at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40% higher) than cells produced by a similar method but further including contacting the population of cells with, for example, an anti-CD3 antibody.

[0108] In some embodiments, the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, in the population of cells from step (3) is the same as the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ cells, in the population of cells at the start of step (1). In some embodiments, the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, in the population of cells from step (3) differs by no more than 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12% from the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ cells, in the population of cells at the start of step (1). In some embodiments, the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, in the population of cells from step (3) differs from the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ cells, in the population of cells at the start of step (1) by no more than 5 or 10%. In some embodiments, the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, in the population of cells from step (3) is increased compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ cells, in the population of cells at the start of step (1). In some embodiments, the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, in the population of cells 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, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ cells, in the population of cells at the start of step (1).In some embodiments, the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ T cells, in the population of cells from step (3) is increased by at least 10 or 20% compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+CD45RO-CCR7+ cells, in the population of cells at the start of step (1).

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

[0110] In some embodiments, the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the population of cells from step (3) is the same as the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the population of cells at the start of step (i). In some embodiments, the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the population of cells at the start of step (i) differs by no more than 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12% from the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the population of cells at the start of step (i). In some embodiments, the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the population of cells from step (3) differs by no more than 5 or 10% from the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the population of cells at the start of step (i). In some embodiments, the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the population of cells from step (3) is reduced compared to the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the population of cells at the start of step (1). In some embodiments, the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the population of cells from step (3) is reduced by at least 10 or 20% compared to the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the population of cells at the start of step (1).In some embodiments, the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the population of cells from step (3) is reduced by at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20% compared to the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the population of cells at the start of step (1).

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

[0112] In some embodiments, the population of cells from step (3), after administration in vivo, persists longer or proliferates to a greater extent (e.g., at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90% greater) than cells produced by a similar method, except that step (3) is performed more than 26 hours after the initiation of step (1), e.g., more than 5, 6, 7, 8, 9, 10, 11, or 12 days after the initiation of step (1). In some embodiments, the population of cells from step (3), after administration in vivo, persists longer or proliferates to a greater extent (e.g., at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90% greater) than cells produced by a similar method but further including expanding the population of cells (e.g., T cells) in vitro after step (2) and before step (3) for more than 3 days, e.g., 5, 6, 7, 8, or 9 days.

[0113] In some embodiments, the population of cells from step (3) is not expanded relative to the population of cells at the start of step (1), e.g., as assessed by the number of viable cells. In some embodiments, the population of cells from step (3) is expanded 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% relative to the population of cells at the start of step (1), e.g., as assessed by the number of viable cells. In some embodiments, the population of cells from step (3) is expanded by no more than 10% relative to the population of cells at the start of step (1), e.g., as assessed by the number of viable cells. In some embodiments, the number of viable cells in the population of cells from step (3) is reduced from the number of viable cells in the population of cells at the start of step (1), e.g., as assessed by the number of viable cells.

[0114] In some embodiments, the population of cells from step (3) is not expanded, e.g., as assessed by the number of viable cells, relative to the population of cells at the start of step (1). In some embodiments, the population of cells from step (3) is expanded by less than 0.5, 1, 1.5, or 2 hours, e.g., less than 1 or 1.5 hours, relative to the population of cells at the start of step (1).

[0115] In some embodiments, the population of cells is not contacted in vitro with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of those cells, or if contacted, the contacting step is for less than 2 hours, e.g., 1 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 a costimulatory 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 costimulatory 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 an scFv), a small molecule, or a ligand (e.g., a naturally occurring, recombinant, or chimeric ligand).

[0116] In some embodiments, steps (1) and / or (2) are performed in a cell culture medium comprising 5, 4, 3, 2, 1, or 0% or less serum. In some embodiments, steps (1) and / or (2) are performed in a cell culture medium comprising 2% or less serum. In some embodiments, steps (1) and / or (2) are performed in a cell culture medium comprising about 2% serum. In some embodiments, steps (1) and / or (2) are performed in a cell culture medium comprising an LSD1 inhibitor or a MALT1 inhibitor. In some embodiments, step (1) is performed in a cell culture medium comprising 5, 4, 3, 2, 1, or 0% or less serum. In some embodiments, step (1) is performed in a cell culture medium comprising 2% or less serum. In some embodiments, step (1) is performed in a cell culture medium comprising about 2% serum. In some embodiments, step (2) is performed in a cell culture medium comprising 5, 4, 3, 2, 1, or 0% or less serum. In some embodiments, step (2) is performed in a cell culture medium comprising 2% or less serum. In some embodiments, step (2) is performed in a cell culture medium containing about 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), (iv) receiving 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 a thymus removal)) from an entity, e.g., a laboratory, hospital, or healthcare provider.

[0118] In some embodiments, the aforementioned methods further comprise, prior to step (i), the step of (v) isolating the population of cells (e.g., T cells, e.g., CD8+ and / or CD4+ T cells) to be contacted in step (i) from 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 a thymus removal)). In some embodiments, step (iii) is performed within 35 hours after the initiation of step (v), e.g., within 27, 28, 29, 30, 31, 32, 33, 34, or 35 hours after the initiation of step (v), e.g., within 30 hours after the initiation of step (v). In some embodiments, 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%, e.g., no more than 10%, relative to the population of cells at the end of step (v), as assessed by the number of viable cells.

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

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

[0121] In some embodiments, the aforementioned methods further comprise, prior to step (i), (v) isolating the population of cells (e.g., T cells, e.g., CD8+ and / or CD4+ T cells) to be contacted in step (i) from a cryopreserved leukapheresis product (or an alternative source of hematopoietic tissue, such as a cryopreserved whole blood product, a cryopreserved bone marrow product, or a cryopreserved tumor or organ biopsy or resection (e.g., a cryopreserved product from a thymus removal)). In some embodiments, step (iii) is performed within 35 hours after the initiation of step (v), e.g., within 27, 28, 29, 30, 31, 32, 33, 34, or 35 hours after the initiation of step (v), e.g., within 30 hours after the initiation of step (v). In some embodiments, 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%, e.g., no more than 10%, relative to the population of cells at the end of step (v), as assessed by the number of viable cells.

[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 containing IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)). In some embodiments, the IL-15 increases the ability of the population of cells to proliferate, e.g., after 10, 15, 20, or 25 days. In some embodiments, the IL-15 increases the percentage of IL6Rβ-expressing cells in the population of cells.

[0124] In some embodiments of the above-described methods, 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 above-described methods, the methods are performed in separate devices. In some embodiments, T cell separation, activation and transduction, incubation, and washing are performed in separate devices.

[0125] In some embodiments of the aforementioned methods, the method further comprises adding an adjuvant or transduction-enhancing reagent to the cell culture medium to increase 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 aforementioned methods, the step of transducing the population of cells (e.g., T cells) with a viral vector comprises subjecting the population of cells and the viral vector to centrifugation under conditions that enhance transduction efficiency. In one embodiment, the cells are transduced by spinoculation.

[0127] In some embodiments of the aforementioned methods, cells (e.g., T cells) are activated and transduced in cell culture flasks containing a gas-permeable membrane at the bottom, which supports a large medium volume without substantially compromising gas exchange. In some embodiments, cell growth is achieved by convective access to nutrients, e.g., uninterrupted access.

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

[0129] In some embodiments, the antigen binding domain is selected from the group consisting of 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-beta, 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-binding domain binds to an antigen 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 an MHC-presented peptide of any of these antigens. 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 a VH and a VL, wherein the VH and VL are connected 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 a 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, wherein 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, wherein 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, wherein 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 costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is selected from the group consisting of an MHC class I molecule, a TNF receptor protein, an immunoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocyte activation molecule (SLAM protein), an activating NK cell receptor, BTLA, a Toll ligand receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, and 4-1BB (CD137). , B7-H3, ICOS(CD278), GITR, BAFFR, LIGHT, HVEM(LIGHTR), KIRDS2, SLAMF7, NKp80(KLRF1), NKp44, NKp30, NKp46, C D19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD 11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, NKG 2C, 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 CD83. In some embodiments, the costimulatory signaling domain comprises a functional signaling domain derived from 4-1BB. 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 nucleic acid molecule comprises a nucleic acid sequence encoding a costimulatory signaling domain, wherein the nucleic acid sequence comprises 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) First cell population containing anti-BCMA CAR but no 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 containing both anti-BCMA and anti-CD19 CARs. Includes:

[0138] In some embodiments, (i) the total number of viable cells in the second and third populations combined 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 first and third populations combined 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 in the second and third populations combined; and / or (iii) the total number of viable cells in the first and third populations combined 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 populations.

[0139] In some embodiments, the population further comprises a fourth population of cells that does not comprise 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 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.

[0141] In some embodiments, in the final CAR cell product produced using the methods described herein, the total amount of beads (e.g., CD4 beads, CD8 beads and / or TransACT beads) is no more than 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, or 0.5% 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 comprises one or more of the following characteristics: (a) about the same percentage of naive cells, e.g., naive T cells, e.g., CD45RO-CCR7+ cells, as compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RO-CCR7+ cells, in the same population of cells before they were engineered to express a CAR; (b) about the same percentage of naive cells, e.g., naive T cells, in the same population of cells before they were engineered to express a CAR; (c) an increased, e.g., at least a 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, or 3-fold increase in the percentage of naive cells, e.g., naive T cells, e.g., CD45RO-CCR7+ cells, compared to the percentage of naive cells, e.g., naive T cells, e.g., CD45RO-CCR7+ cells, in the same population of cells before they were engineered to express a CAR. (d) about the same percentage of central memory cells, e.g., central memory T cells, e.g., CCR7CD45RO T cells, compared to the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7CD45RO T cells, in the same population of cells before they are engineered to express a CAR; (e) compared to the percentage of central memory cells, e.g., central memory T cells, e.g., CCR7CD45RO T cells, in the same population of cells before they are engineered to express a CAR. (f) a decreased percentage of central memory cells, e.g., central memory T cells, e.g., CCR7+CD45RO+ T cells, 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 they were engineered to express a CAR;(g) Approximately the same percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, compared to the percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in the same population of cells before they were engineered to express a CAR; (h) Approximately the same percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor β+CCR7+CD62L+ T cells, in the same population of cells before they were engineered to express a CAR. or (i) an increased percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor beta+CCR7+CD62L+ T cells, compared to the percentage of stem memory T cells, e.g., CD45RA+CD95+IL-2 receptor beta+CCR7+CD62L+ T cells, in the same population of cells prior to 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), wherein (a) the median GeneSetScore (Up TEM vs. Down TSCM) of the population of cells is about the same as or differs (e.g., is increased by no more than) about 25, 50, 75, 100, or 125% from the median GeneSetScore (Up TEM vs. Down TSCM) of the same population of cells before they were engineered to express a CAR; (b) the median GeneSetScore (Up Treg vs. Down Teff) of the population of cells is about the same as or differs (e.g., is increased by no more than) about 25, 50, 100, 150, or 200% from the median GeneSetScore (Up Treg vs. Down Teff) of the population of cells before they were engineered to express a CAR; (c) the median GeneSetScore (Down TEM vs. Down TSCM) of the population of cells is about the same as or differs (e.g., is increased by no more than) about 25, 50, 100, 150, or 200% from the median GeneSetScore (Up Treg vs. Down Teff) of the population of cells before they were engineered to express a CAR; (d) the median GeneSetScore (Up hypoxia) of the population of cells is about the same as or differs by (e.g., is increased by) no more than about 125, 150, 175, or 200% from (e.g., is increased by) the median GeneSetScore (Up hypoxia) of the population of cells before being engineered to express a CAR; or (e) the median GeneSetScore (Up autophagy) of the population of cells is about the same as or differs by (e.g., is increased by) no more than about 180, 190, 200, or 210% from (e.g., is increased by) the median GeneSetScore (Up autophagy) of the population of cells before being engineered to express a CAR.

[0144] In some embodiments, the invention features a method of increasing an immune response in a subject, comprising 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, methods of treating cancer in a subject are disclosed, comprising administering to the subject a population of CAR-expressing cells disclosed herein or a pharmaceutical composition disclosed herein, thereby treating the cancer in the subject. In some embodiments, the cancer is a solid cancer or metastasis thereof selected from, for example, one or more of 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, colon cancer, prostate cancer, cervical cancer, skin cancer, melanoma, renal cancer, liver cancer, brain tumor, thymoma, sarcoma, carcinoma, uterine cancer, kidney cancer, gastrointestinal cancer, urothelial cancer, pharyngeal cancer, head and neck cancer, rectal cancer, esophageal cancer, or bladder cancer. In some embodiments, the cancer is selected from the group consisting of, for example, chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), multiple myeloma, acute lymphocytic leukemia (ALL), Hodgkin's lymphoma, B-cell acute lymphocytic leukemia (BALL), T-cell acute lymphocytic leukemia (TALL), small lymphocytic leukemia (SLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, Diffuse large B-cell lymphoma (DLBCL), DLBCL with chronic inflammation, chronic myeloid leukemia, myeloproliferative neoplasms, follicular lymphoma, childhood follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma (mucosa-associated lymphoid tissue type extranodal marginal zone lymphoma), marginal zone lymphoma, myelodysplasia, myelodysplastic syndrome, non- Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, splenic marginal zone lymphoma, splenic lymphoma / leukemia, splenic diffuse red pulp small B-cell lymphoma, hairy cell leukemia-variant, lymphoplasmacytic lymphoma, heavy chain disease, plasma cell myeloma, isolated bone plasmacytoma, extraskeletal plasmacytoma, nodal marginal zone lymphoma, childhood nodal marginal zone lymphoma The liquid cancer is selected from lymphoma, primary cutaneous follicle center lymphoma, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK+ large B-cell lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, primary effusion lymphoma, B-cell lymphoma, acute myeloid leukemia (AML), or unclassifiable lymphoma.

[0146] In some embodiments, the method further includes 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 an 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, comprising: (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), wherein HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 comprise the amino acid sequences of SEQ ID NOs: 86, 87, 88, 95, 96, and 97, respectively; (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 the HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 comprise the amino acid sequences of SEQ ID NOs: 295, 304, and 297 to 300, respectively; Provided herein is an isolated cell or population of cells comprising:

[0148] In some embodiments, the isolated cell comprises: (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), wherein HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 comprise the amino acid sequences of SEQ ID NOs: 86, 87, 88, 95, 96, and 97, respectively; (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 the HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3 comprise the amino acid sequences of SEQ ID NOs: 295, 304, and 297 to 300, respectively; Provided herein is an isolated cell comprising:

[0149] In some embodiments, the VH and VL of the anti-BCMA binding domain comprise the amino acid sequences of SEQ ID NOs: 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 NOs: 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 NOs: 93 and 102, respectively, and the VH and VL of the anti-CD19 binding domain comprise the amino acid sequences of SEQ ID NOs: 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; and 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, provided herein are pharmaceutical compositions comprising the cells or populations of cells described herein.

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

[0152] In some embodiments, the disease associated with BCMA expression is a hematological cancer or solid cancer, e.g., a hematological cancer or solid cancer 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 myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndromes, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, The cancer is selected from Waldenstrom's macroglobulinemia, prostate cancer (e.g., castration-resistant or treatment-resistant prostate cancer or metastatic prostate cancer), pancreatic cancer, lung cancer, plasma cell proliferative disorder (e.g., asymptomatic myeloma (smoldering multiple myeloma or asymptomatic myeloma), monoclonal gammopathy of undetermined significance (MGUS)), Waldenstrom's macroglobulinemia, plasmacytoma (e.g., dysplasmocytoma, 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 a combination 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 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 references multiple sequence accession numbers, all sequence variants are encompassed.

[0156] Furthermore, the materials, methods, and examples are illustrative only and not intended to be limiting. Headings, subheadings, or numbered or lettered elements, e.g., (a), (b), (i), etc., are provided merely for ease of reading. The use of headings or numbered or lettered elements herein does not require the steps or elements to be performed in alphabetical order or that the steps or elements are necessarily distinct from one another. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. [Brief explanation of the drawings]

[0157] [Figure 1-1]The function of BCMA CAR was tested using a Jurkat NFAT luciferase (JNL) reporter assay using an automated system. CAR clones were evaluated for antigen-dependent activity in the JNL reporter assay. JNL cells containing the indicated CAR clones or untransduced JNL cells (UTD) were co-cultured with medium alone (Figures 1G and 1H) or with target cell lines (KMS11 (Figures 1A and 1C) as a BCMA-positive cell line and NALM6 (Figures 1E and 1F) as a BCMA-negative cell line) at various ratios, and luciferase activity was measured as luminescence intensity. Clones were considered active if their luminescence intensity was more than twice the level of UTD cells in the presence of antigen-expressing cells. The luminescence readout is a direct measure of CAR stimulation. Figures 1B and 1D are graphs showing the expression levels of BCMA CAR in JNL cells as detected by flow cytometry using human recombinant (r)BCMA_Fc-AF647. The 1x or 2x platform represented 40,000 H293 cells or 80,000 H293 cells inoculated for virus production. [Figure 1-2] Same as above. [Figure 1-3] Same as above. [Figure 1-4] Same as above. [Figure 2] Expression levels of BCMA CARs on primary human T cells. Cells were stained with human rBCMA_Fc-AF647 reagent and then assayed by flow cytometry. The percentage of CAR+ cells and MFI are shown graphically for days 5 and 9 of cell culture. The data are summarized in Table 27, including the viral titers achieved for each CAR. [Figure 3-1]The ability of T cells expressing the indicated CARs to mediate cytolysis and cytokine production was assessed against the KMS11 target cell line expressing firefly luciferase (KMS11-luc). Figure 3A: CART cells were cocultured with KMS11-luc target cells at the indicated E:T ratios. % cell killing was determined by the difference in luciferase between target cells without effector T cells (control) and target cells with effector T cells (experimental) and expressed as a percentage of the control. UTD indicates untransduced T cells. Figure 3B: Background killing was observed for the BCMA-negative line NALM6. Figure 3C: IFNγ was measured by MSD in supernatants collected from these cocultures at 24 h at an E:T ratio of 2.5. All data are presented as mean + / - standard deviation. [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Figure 4-1] CAR expression in T cells transduced at MOI=5 (viral titer determined by the first CAR expressed in SupT1 cells). Figure 4A is a table summarizing CAR19 (%), BCMA CAR (%), double positive (%), CAR19 only (%), and BCMA-CAR only (%) for various constructs. Figure 4B is a series of flow cytometry plots showing staining of cells for surface BCMA CAR expression (x-axis) and surface CD19 CAR expression (y-axis). Figure 4C is a pair of bar graphs showing BCMA CAR MFI (upper panel) and CD19 CAR MFI (lower panel). [Figure 4-2] Same as above. [Figure 4-3] Same as above. [Figure 5] In vitro killing assay with CART cells on day 8. Figures 5A-5C are a series of graphs showing the % killing of BCMA-positive KMS11 cells, CD19-positive Nalm6 cells, or BCMA / CD19-negative cells, respectively, at the indicated E:T ratios. [Figure 6]In vitro cytokine production using CART cells on day 8. Figures 6A-6D are a series of bar graphs showing IFNγ production by CART cells when co-cultured with BCMA-positive KMS11 cells or CD19-positive Nalm6 cells. [Figure 7-1] Individual CAR expression in cells manufactured using the ARM process. Figures 7A-7B are histograms showing the expression patterns of both anti-BCMA and anti-CD19 CARs 24 h or 72 h after transduction of human primary T cells manufactured using the ARM process. These studies used an MOI of 1 based on SupT1 titer determined by upstream CAR expression. In each of Figures 7A and 7B, the left section is a panel of histograms showing staining with rBCMA-Fc, and the right section is a panel of histograms showing staining with an anti-idiotypic antibody that binds to the CD19 CAR. Constructs #244 ("c244") and #245 ("c245") are mono-anti-CD19 CAR and anti-BCMA CAR, respectively. Figure 7C is a panel of flow cytometry plots showing anti-BCMA and anti-CD19 CARs 72 h after transduction of human primary T cells using an MOI of 1 based on upstream CAR titer. [Figure 7-2] Same as above. [Figure 7-3] Same as above. [Figure 8]In vivo antitumor activity of construct #236 ("c236") and construct #238 ("c238") was demonstrated using three mouse models: a disseminated KMS-11 (BCMA+CD19-) multiple myeloma model expressing a luciferase reporter gene (KMS11-Luc) (Figure 8A), a Nalm6-Luc (CD19+BCMA-) xenograft mouse model (Figure 8B), and a 95% KMS-luc mixed model containing 5% NALM6-Luc cells. Tumor burden is expressed as total body luminescence (p / s) and shown as the mean tumor burden + SEM. On day 7 or 8 after tumor inoculation, mice were treated with the indicated amounts of c236 and c238 BCMA CAR+ or CD19 CAR+ T cells (approximate number of surviving CAR+ T cells), as shown in Table 30. Vehicle (PBS) and untransduced T cells (UTD) were used as negative controls. The mono-anti-BCMA CAR PI61 and the mono-anti-CD19 CAR CTL119 were also used as controls. [Figure 9] Graft-versus-host reaction-induced weight loss. All mice were individually monitored for weight loss as a readout for X-GvHD by measuring body weight over time. Body weight (BWT) is plotted as % change from baseline. [Figure 10] In vivo expansion of peripheral blood CD3+ T cells was analyzed by flow cytometry up to 4 weeks after infusion. [Figure 11] In vivo expansion of CAR+ T cells (BCMA CAR+ percentage) was analyzed by flow cytometry up to 4 weeks after infusion. [Figure 12] In vivo expansion of CAR+ T cells (double CAR+ cell numbers) was analyzed by flow cytometry up to 4 weeks after infusion. [Figure 13] In vivo plasma IFNγ kinetics. Plasma IFNγ levels from all three mouse models treated with c236 and c238, as well as the monoCAR control at each CAR-T dose, were plotted on a graph. Mice were bled and plasma cytokines were measured by MSD assay. [Figure 14]Figure 14A: In vivo efficacy and cell expansion of cells generated using 236 and c238 in a multiple myeloma xenograft mouse model. Figure 14A: NSG mice were injected with the multiple myeloma line KMS11, which expressed a luciferase reporter gene. Tumor burden is expressed as total body luminescence (p / c) and shown as the mean tumor burden + SEM. On day 8 after tumor inoculation, mice were treated with c236 and c238 at a dose of 9e4 BCMA-CD19 dual CAR T cells (approximate number of surviving CAR T cells). Vehicle (PBS) and untransduced T cells (UTD) were used as negative controls. Figure 14B: Peripheral blood CAR T cell expansion was analyzed by flow cytometry up to 4 weeks after injection. Dual anti-BCMA and CD19 CAR T cell expansion was observed in the c236 and c238 CAR-T Rx groups. [Figure 15-1] CAR expression in cells manufactured using the ARM process. Flow cytometry plots showing expression of dual-positive anti-BCMA and anti-CD19 CARs 96 h (FIG. 15A) and 7 days (FIG. 15B) after virus addition to human primary T cells manufactured using the ARM process. In these studies, an MOI of 2 was used based on SupT1 titer determined by expression of dual CARs (positive for PI61 or R1G5 clones and CTL119) detected by anti-idiotypic antibodies binding to CD19 CAR and recombinant BCMA_Fc (AF647) binding to PI61 or R1G5. Mono anti-BCMA CARs PI61 and R1G5 and mono anti-CD19 CAR CTL119 were also used as controls. [Figure 15-2] Same as above. [Figure 16] CAR expression at day 7 with the TM process using an MOI of 5. Flow cytometry plot showing expression of dual positive anti-BCMA and anti-CD19 CARs at day 7 after virus addition to human primary T cells manufactured using the TM process. In these studies, an MOI of 5 was used based on SupT1 titer determined by expression of dual CARs (positive for PI61 or R1G5 clones and CTL119) detected by anti-idiotypic antibodies that bind to the CD19 CAR and recombinant BCMA_Fc (AF647) that binds to PI61 or R1G5. [Figure 17] Specific killing of BCMA- or CD19-expressing tumor cells in vitro by T cells engineered with anti-BCMACAR and CD19CAR diabody constructs. The ability of T cells expressing the PI61 / CTL119 clone to mediate cytolysis was assessed against KMS11-Luc or NALM6-Luc target cell lines. CART cells were cocultured with BCMA+KMS-11-luc or BCMA-NALM6-Luc target cells at the indicated E:T ratios for 20 hours, and the percent cell killing (determined by the difference in luciferase signal between target cells without effector T cells (control) and target cells with effector T cells (experimental)) was measured as a surrogate for target cell lysis and expressed as a percentage of the control. UTD indicates untransduced T cells. Mono-PI61 or CTL119 was also used as a control. [Figure 18] Cytokine production of T cells engineered with anti-BCMACAR and CD19CAR diabody constructs in response to BCMA- or CD19-expressing tumor cells. IFNγ (FIG. 18A) and IL-2 (FIG. 18B) were measured by MSD in supernatants from killing assay co-cultures at a ratio of 1.25:1. [Figure 19] Percentage of double CAR positive population, BCMA CAR positive population and CD19 CAR on day 4. [Figure 20] Flow cytometry plot showing staining of cells with rBCMA-Fc and anti-idiotypic antibodies binding to CD19 CAR. [Figure 21] Percentage of total CAR-positive population on days 4 and 7 under the conditions indicated. [Figure 22] Cell numbers (left panel) and percentage of viable cells (left panel) at days 0, 1, 3, and 7 under the indicated conditions. [Figure 23] Single-cell RNA-seq data for input cells (Figure 23A), day 1 cells (Figure 23B), and day 9 cells (Figure 23C). The "nGene" graph shows the number of expressing cells per cell. The "nUMI" graph shows the number of unique molecular identifiers (UMIs) per cell. [Figure 24-1] Figure 24D is a violin plot showing the distribution of gene set scores for gene sets containing genes characteristic of resting versus activated T cell states for day 1, day 9, and input cells. In Figure 24D, a higher gene set score (Up resting vs. Down activated) indicates an increased resting T cell phenotype, whereas a lower gene set score (Up resting vs. Down activated) indicates an increased activated T cell phenotype. Input cells were generally more quiescent than day 9 and day 1 cells, with day 1 cells showing the highest activated gene set score. [Figure 24-2] Same as above. [Figure 25-1]Gene set analysis for input cells, day 1 cells, and day 9 cells. In Figure 25A, a higher gene set score for the gene set, "Up TEM vs. Down TSCM," indicates an increase in the effector memory T cell (TEM) phenotype of the cells in that sample, while a lower gene set score indicates an increase in the stem cell memory T cell (TSCM) phenotype. In Figure 25B, a higher gene set score for the gene set, "Up Treg vs. Down Teff," indicates an increase in the regulatory T cell (Treg) phenotype, while a lower gene set score indicates an increase in the effector T cell (Teff) phenotype. In Figure 25C, a lower gene set score for the gene set, "Down stemness," indicates an increase in the stemness phenotype. In Figure 25D, a higher gene set score for the gene set, "Up hypoxia," indicates an increase in the hypoxic phenotype. In Figure 25E, a higher gene set score for the gene set, "Up autophagy," indicates an increase in the autophagy phenotype. Day 1 cells appeared similar to input cells with respect to memory, stem-like, and differentiation signatures, whereas day 9 cells showed high enrichment for metabolic stress. [Figure 25-2] Same as above. [Figure 25-3] Same as above. [Figure 25-4] Same as above. [Figure 25-5] Same as above. [Figure 26]Gene cluster analysis of input cells. Figures 26A-26C are violin plots showing gene set scores from gene set analysis of four clusters of input cells. Each dot overlaid on the violin plot in Figures 26A-26C represents the gene set score of a cell. In Figure 26A, a higher gene set score for the gene set "Up Treg vs. Down Teff" indicates an increase in the Treg cell phenotype, and a lower gene set score for the gene set "Up Treg vs. Down Teff" indicates an increase in the Teff cell phenotype. In Figure 26B, a higher gene set score for the gene set "Progressively up in memory differentiation" indicates an increase in the late memory T cell phenotype, and a lower gene set score for the gene set "Progressively up in memory differentiation" indicates an increase in the early memory T cell phenotype. In Figure 26C, a higher gene set score "Up TEM vs. Down TN" for a gene set indicates an increased effector memory T cell phenotype, while a lower gene set score "Up TEM vs. Down TN" for a gene set indicates an increased naive T cell phenotype. Compared to cells in Cluster 1 and Cluster 2, which are in an early memory, less differentiated T cell state, cells in Cluster 3 appear to be in a late memory, more differentiated T cell state. Cluster 0 is considered to be in an intermediate T cell state. Collectively, this data indicates a significant level of heterogeneity in the input cells. [Figure 27] TCR sequencing and measurement of clonotype diversity. Day 9 cells have a flatter distribution of clonotype frequencies (higher diversity). [Figure 28-1]Flow cytometry analysis of CAR expression 4 and 7 days after transduction. Flow cytometry analysis of CAR-T cells generated by co-transducing cells with BCMA and CD19 CARs at various MOI combinations using the ARM process in 24-well plates. Figure 28A: Flow cytometry plots show mono anti-BCMA CAR, mono anti-CD19 CAR, and dual + CAR expression 4 and 7 days after transduction under four different MOI conditions, in addition to controls (UTD and single vector). Figure 28B: Quantification of subsets of the CAR+ population, including total anti-BCMA CAR+ T cells, total anti-CD19 CAR+ T cells, and total CAR+ T cells (the sum of the two mono-CAR+ T cells and dual + CAR+ T cells) under each condition described in Figure 28A. Data shown are representative from three donor T cells with consistent results. CAR+ cell percentages are gated on the viable CD3+ T cell population. [Figure 28-2] Same as above. [Figure 28-3] Same as above. [Figure 29] Flow cytometry analysis of CAR expression on day 4 after transduction. Flow cytometry analysis of the final products of dual targeting cocktail CART, mono-BCMACART, and mono-CD19CART for CAR expression on day 4 after transduction. A small aliquot of each product at the 24 h harvest was re-cultured for 3 days before flow cytometry staining. [Figure 30-1]In vivo efficacy of dual CART compared with mono-BCMA CART and mono-CD19 CART in a xenograft model. NSG mice were injected with cell lines expressing luciferase reporter genes (KMS-11 or Nalm-6, or 5% Nalm-6-luc and a mixture of both). Tumor burden is expressed as total body luminescence (p / s), shown as the mean tumor burden + SEM. On day 7 or 8 after tumor inoculation, mice were treated with the respective doses (approximate number of surviving CAR+ T cells) of dual targeting cocktail CART, BCMACART, or CD19CART. Vehicle (PBS) and untransduced T cells (UTD) were used as negative controls. N=5 for all groups. BCMACART and CD19CART served as respective controls using the highest dose level. All experiments were terminated on day 23 after CAR-T administration. [Figure 30-2] Same as above. [Figure 30-3] Same as above. [Figure 30-4] Same as above. [Figure 30-5] Same as above. [Figure 31] Figure 31 is a bar graph showing mono CD19 CAR+ cells (%), mono BCMA CAR+ cells (%), and dual BCMA / CD19 CAR+ cells on day 4 post-transduction (day 3 post-harvest). [Figure 32] Characterization of T cell subsets. Figure 32 is a graph showing the % of CD4+ T cells, CD8+ T cells, naive T cells (Tn), central memory T cells (Tcm), effector memory T cells (Tem), and effector memory T cells re-expressing CD45RA (Temra) in the input material, the enriched material, and the material at day 1 after collection. [Figure 33-1] Plasma IFNγ kinetics in BCMA / CD19 dual CART cell product, BCMA CART, and CD19 CART-treated mice. Animals were treated with PBS, UTD, BCMA / CD19 dual CART cell product, BCMA CART, or CD19 CART at the respective CAR-T doses. Mice were bled and plasma cytokines were measured by MSD assay. [Figure 33-2] Same as above. DETAILED DESCRIPTION OF THE INVENTION

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

[0159] The terms "a" and "an" refer to one or to more than one (i.e., to at least one) of the grammatical referent 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, or the like, means that variations of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% from the specified value are encompassed, as such variations are appropriate for the practice of the methods of the present disclosure.

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

[0162] The term "substantially identical," in the context of nucleotide sequences, is used herein to refer to a nucleotide sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a first nucleic acid 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 aligned nucleotides in a second nucleic acid sequence, such that the first and second nucleotide sequences encode polypeptides having a common functional activity, or encode a common structural polypeptide domain or a common functional polypeptide activity.

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

[0164] The term "functional variant" refers to a polypeptide that has substantially the same amino acid sequence as a reference amino acid sequence, or is encoded by substantially the same 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, e.g., functional variants (including fragments and functional variants thereof having at least 10%, 30%, 50%, or 80% of the activity, e.g., immunomodulatory activity, of a naturally occurring cytokine). In some embodiments, a cytokine has an amino acid sequence that is substantially identical (e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical) to a naturally occurring cytokine or 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% identical) to a naturally occurring nucleotide sequence encoding the cytokine. In some embodiments, as understood from the context, the cytokine further comprises a receptor domain, eg, a cytokine receptor domain (eg, IL-15 / IL-15R).

[0166] As used herein, the term "BCMA" refers to a 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 primarily expressed on terminally differentiated B cells, such as memory B cells and plasma cells. Its ligands are called B-cell-activating factor of the TNF family (BAFF) and proliferation-inducing ligand (APRIL). BCMA is involved in mediating the survival of plasma cells to maintain long-term humoral immunity. The BCMA gene is encoded on chromosome 16 and produces a 994-nucleotide primary mRNA transcript (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 role in regulating BCMA expression. (Laabi Y. et al., Nucleic Acids Res., 1994, 22:1147-1154). Additional transcript variants have been described, but the significance is unknown (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 containing mutations of full-length wild-type BCMA, e.g., point mutations, fragments, insertions, deletions, and splice variants.

[0167] The phrase "disease associated with BCMA expression" includes, but is not limited to, a disease or condition associated with cells expressing BCMA (e.g., wild-type or mutant BCMA), such as a proliferative disease, such as a cancer or malignant disease, or a precancerous condition, such as myelodysplasia, myelodysplastic syndrome, or preleukemia; or a non-cancer-related indication associated with cells expressing BCMA (e.g., wild-type or mutant BCMA). For the avoidance of doubt, a disease associated with BCMA expression may include a condition associated with cells that previously expressed BCMA, but no longer express BCMA, e.g., due to downregulation of BCMA expression, e.g., by treatment with a molecule that targets BCMA (e.g., a BCMA inhibitor as described herein). In one aspect, the cancer associated with BCMA (e.g., wild-type or mutant BCMA) expression is a hematological cancer. In one aspect, the hematological cancer is leukemia or lymphoma. In one aspect, the cancer associated with expression of BCMA (e.g., wild-type or mutant BCMA) is a malignancy of differentiated plasma B cells. In one aspect, the cancer associated with expression of BCMA (e.g., wild-type or mutant BCMA) includes, but is not limited to, cancers and malignancies including acute leukemias such as, for example, B-cell acute lymphoblastic leukemia ("BALL"), T-cell acute lymphoblastic leukemia ("TALL"), acute lymphoblastic leukemia (ALL); chronic leukemias such as, for example, but not limited to, chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL).Other cancers or hematological cancers associated with expression of BCMA (e.g., wild-type or mutant BCMA) include, but are not limited to, B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplastic and myelodysplastic syndromes, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, and "preleukemias," a diverse collection of hematological conditions consistent with insufficient production (or dysplasia) of myeloid blood cells. In some embodiments, the cancer is multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or glioblastoma. In some embodiments, diseases associated with BCMA expression include plasma cell proliferative disorders, such as asymptomatic myeloma (smoldering multiple myeloma or asymptomatic myeloma), monoclonal gammopathy of undetermined significance (MGUS), Waldenstrom's macroglobulinemia, plasmacytomas (e.g., dysplasmocytosis, 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). Further diseases associated with expression of BCMA (e.g., wild-type or mutant BCMA) include, but are not limited to, for example, atypical and / or non-classical cancers, malignancies, precancerous conditions, or proliferative diseases associated with expression of BCMA (e.g., wild-type or mutant BCMA), such as a cancer described herein, for example, prostate cancer (e.g., castration-resistant or therapy-resistant prostate cancer or metastatic prostate cancer), pancreatic cancer, or lung cancer.

[0168] Non-cancer-related conditions associated with expression of BCMA (e.g., wild-type or mutant BCMA) include viral infections such as HIV, fungal infections such as C. neoformans, autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus (SLE or lupus), pemphigus vulgaris, and Sjogren's syndrome, inflammatory bowel disease, ulcerative colitis, transplant-associated allospecific immune disorders involving mucosal immunity, and unwanted immune responses to biologics (e.g., factor VIII) where humoral immunity is important. In some embodiments, non-cancer-related indications associated with expression of BCMA include, but are not limited to, autoimmune diseases (e.g., lupus), inflammatory diseases (allergies and asthma), and transplantation. In some embodiments, tumor antigen-expressing cells express, or have at any time expressed, mRNA encoding a tumor antigen. In one embodiment, 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 detectable levels of the tumor antigen protein at one time, but subsequently produced substantially no 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 an "intracellular signaling domain") comprising a functional signaling domain derived from a stimulatory molecule, as defined below. In some embodiments, the domains in a CAR polypeptide construct comprise within the same polypeptide chain, e.g., a chimeric fusion protein. In some embodiments, the domains in a CAR polypeptide construct are not contiguous to one another, e.g., provided in different polypeptide chains, e.g., within a RCAR as described herein.

[0170] In some embodiments, the cytoplasmic signaling domain comprises a primary signaling domain (e.g., a primary signaling domain of CD3ζ). In some embodiments, the cytoplasmic signaling domain further comprises one or more functional signaling domains from at least one costimulatory molecule, as defined below. In some embodiments, the costimulatory 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 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 from a costimulatory molecule and a functional signaling domain 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 from one or more costimulatory molecules and a functional signaling domain 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 costimulatory 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 is optionally cleaved from the antigen recognition domain (e.g., scFv) during cellular processing and localization of the CAR to the cell membrane.

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

[0172] The term "signaling domain" refers to a functional portion of a protein that acts by transmitting information within the cell to regulate cellular activity through a specified signaling pathway, either by generating second messengers or by functioning as an effector by responding to such messengers.

[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. Antibodies can be polyclonal or monoclonal, multi-chain or single-chain or intact immunoglobulins, and can be derived from natural or recombinant sources. An antibody can be a tetramer of immunoglobulin molecules.

[0174] The term "antibody fragment" refers to at least a portion of an intact antibody or a recombinant variant thereof, and refers to an antigen-binding domain, e.g., the antigenic determining variable region of an intact antibody, sufficient to confer recognition and specific binding of the antibody fragment to a target, e.g., an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, scFv antibody fragments, linear antibodies, single domain antibodies (either VL or VH) such as sdAbs, camelid VHH domains, and multispecific antibodies formed from antibody fragments such as two or more, e.g., two Fab fragments, linked by a disulfide bridge at the hinge region, or a bivalent fragment comprising two or more linked antibodies, e.g., two isolated CDRs or other epitope fragments. Antibody fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antibody fragments can also be grafted onto polypeptide-based scaffolds, such as fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide minibodies). The term "scFv" refers to a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light and heavy chain variable regions are closely linked by a short flexible polypeptide linker, capable of being expressed as a single polypeptide chain, and wherein 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, e.g., relative to the N- and C-terminal ends of the polypeptide, and can comprise a VL-linker-VH or a VH-linker-VL.

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

[0176] As used herein, the term "complementarity-determining region" or "CDR" refers to the sequence of amino acids within an antibody variable region that confers antigen specificity and binding affinity. For example, there are typically three CDRs in each heavy chain variable region (e.g., HCDR1, HCDR2, and HCDR3), and three CDRs in each light chain variable region (LCDR1, LCDR2, and LCDR3). 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 ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme), or a combination thereof. In the combined Kabat and Chothia numbering scheme, in some embodiments, the CDRs correspond to amino acid residues that are part of a Kabat CDR, part of a Chothia CDR, or both.

[0177] The portion of the CAR composition of the invention comprising an antibody or antibody fragment thereof can exist in various forms, for example, where the antigen-binding domain is expressed as part of a polypeptide chain, including, for example, a single-domain antibody fragment (sdAb), a single-chain antibody (scFv), or a humanized 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 a CAR of the invention comprises an antibody fragment. In some embodiments, a CAR comprises an antibody fragment, including an scFv.

[0178] As used herein, the term "binding domain" or "antibody molecule" (also referred to herein as "anti-target binding domain") refers to a protein, e.g., an immunoglobulin chain or fragment thereof, that comprises at least one immunoglobulin variable domain sequence. The term "binding domain" or "antibody molecule" encompasses antibodies and antibody fragments. In some embodiments, an antibody molecule is a multispecific antibody molecule, e.g., it comprises a plurality of immunoglobulin variable domain sequences, where a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In certain embodiments, a 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 that has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope.

[0179] The terms "bispecific antibody" and "bispecific antibodies" refer to a molecule that combines the antigen-binding sites of two antibodies in a single molecule. Thus, a bispecific antibody can bind to two different antibodies simultaneously or sequentially. Methods for producing bispecific antibodies are known in the art. Various formats for combining two antibodies are also known in the art. Forms of bispecific antibodies of the present invention include, but are not limited to, diabodies, single-chain antibodies, Fab dimerization (Fab-Fab), Fab-scFv, and tandem antibodies, as are well known to those skilled in the art.

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

[0181] The term "antibody light chain" refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. 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, for example, an antibody expressed in a bacteriophage or yeast expression system. The term should also be construed to mean an antibody made by synthesis of a DNA molecule encoding an antibody and expressing the antibody protein, or an amino acid sequence specifying that antibody, where the DNA or amino acid sequence was 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 immunologically competent cells. Those skilled in the art will understand that any macromolecule can be an antigen, including virtually any protein or peptide. Furthermore, antigens can be derived from recombinant or genomic DNA. Those skilled in the art will therefore understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response encodes an "antigen" as that term is used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of two or more genes, and that these nucleotide sequences are arranged in various combinations to encode a polypeptide that elicits a desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be synthetically produced, obtained from a biological sample, or be a macromolecule other than a polypeptide. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells or bodily 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 be manifested by various means, including, but not limited to, a reduction in tumor or cancer volume, a reduction in the number of tumor or cancer cells, a reduction in the number of metastases, an increase in life expectancy, a reduction in tumor or cancer cell proliferation, a reduction in tumor or cancer cell survival, or an improvement in various physiological symptoms associated with a cancerous condition. An "anti-tumor effect" or "anti-cancer effect" can also be primarily manifested by the ability of the peptides, polynucleotides, cells, and antibodies of the invention to prevent the development of tumors or cancers.

[0185] The term "autologous" refers to any material originating from the same individual that is later 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 one another when the genes at one or more loci are not identical. In some embodiments, allogeneic material from individuals of the same species may be sufficiently genetically different to interact antigenically.

[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 art-recognized extracorporeal procedure in which a donor's or patient's blood is withdrawn from the donor or patient, passed through a device that separates certain selected components, and then the remainder is returned to the donor or patient, e.g., by reinfusion. Thus, reference to an "apheresis sample" 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 to other parts of the body through the bloodstream and lymphatic system. 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, etc. In some embodiments, the cancer treated by the methods described herein comprises multiple myeloma, Hodgkin's lymphoma, or non-Hodgkin's lymphoma.

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

[0191] "Derived from," as the term is used herein, refers to the relationship between a first molecule and a second molecule. This generally refers to the structural similarity between the first and second molecules and does not imply or include a limitation on the manner or source by which the first molecule is derived from the second molecule. For example, in the case of an intracellular signaling domain derived from the CD3ζ molecule, the intracellular signaling domain retains sufficient CD3ζ structure such that it has the desired function, i.e., the ability to generate a signal under appropriate conditions. This does not imply or include a limitation to a particular method of making the intracellular signaling domain, e.g., it does not mean that one must start with the CD3ζ sequence and delete unnecessary sequence or make mutations to arrive at the intracellular signaling domain in order to provide the intracellular signaling domain.

[0192] The term "conservative sequence modifications" refers to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody or antibody fragment containing that amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into an antibody or antibody fragment 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 have been defined in the art. These families include amino acids with 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), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within a CAR of the 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, e.g., a primary or secondary response, induced by the binding of a stimulatory molecule (e.g., TCR / CD3 complex) and / or costimulatory molecule (e.g., C28 or 4-1BB) to its cognate ligand, thereby mediating a signaling event, such as, but not limited to, signaling through the TCR / CD3 complex. Stimulation can mediate, for example, changes in the expression of certain molecules and / or reorganization of cytoskeletal structures.

[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 primary activation of the TCR complex for at least some aspects of the T cell signaling pathway. In some embodiments, an ITAM-containing domain within a CAR recapitulates primary TCR signaling independent of the endogenous TCR complex. In some embodiments, the primary signal is, for example, a primary signal initiated by binding of the TCR / CD3 complex to a peptide-bearing MHC molecule, which results in mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, etc. The primary cytoplasmic signaling sequence (also referred to as a "primary signaling domain") that acts in a stimulatory manner may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif, or 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 CARS of the present invention comprises an intracellular signaling sequence, e.g., a primary signaling sequence of CD3-ζ. The term "antigen-presenting cell" or "APC" refers to immune system cells such as accessory cells (e.g., B cells, dendritic cells, etc.) that present foreign antigens complexed with major histocompatibility complexes (MHC) on their surface. T cells can recognize such complexes using their T cell receptors (TCRs). APCs process antigens and present them to T cells.

[0195] "Intracellular signaling domain," as the term is used herein, refers to the intracellular portion of a molecule. In embodiments, the intracellular signaling domain transduces an effector function signal, instructing the cell to perform a specific function. While the entire intracellular signaling domain can be used, it is often not necessary to use the entire chain. To the extent a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the complete chain, so long as it transmits the effector function signal. The term intracellular signaling domain, therefore, is intended to include any truncated portion of the intracellular signaling domain sufficient to transmit the effector function signal.

[0196] The intracellular signaling domain generates a signal that promotes immune effector function of the CAR-containing cell, e.g., a CART cell, such as helper activity, including cytolytic activity and cytokine secretion.

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

[0198] A primary intracellular signaling domain can contain a signaling motif known as an immunoreceptor tyrosine-based activation motif, or ITAM. Examples of primary cytoplasmic signaling sequences containing ITAMs 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 "zeta" or alternatively "zeta chain," "CD3-zeta," or "TCR-zeta" refers to CD247. Swiss-Prot Accession No. P20963 provides an exemplary human CD3zeta amino acid sequence. "zeta stimulatory domain" or alternatively "CD3-zeta stimulatory domain" or "TCR-zeta stimulatory domain" refers to the stimulatory domain of CD3zeta 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 zeta comprises residues 52-164 of GenBank Accession No. 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 "zeta stimulatory domain" or "CD3-zeta stimulatory 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 a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response of the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are necessary for an efficient immune response. Costimulatory molecules include MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD5, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), B7-H3, CD S, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD1 9, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, IT GAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, T NFR2, 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] A costimulatory intracellular signaling domain refers to the intracellular portion of a costimulatory molecule.

[0202] The intracellular signaling domain can comprise the entire intracellular portion of the molecule from which it is derived or the entire naturally occurring 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 No. P20963 provides an exemplary human 4-1BB amino acid sequence. A "4-1BB costimulatory domain" refers to the costimulatory 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 costimulatory 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] "Immune effector cells," as that term is used herein, refer to cells that are involved in an immune response, e.g., promoting 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] "Immune effector function or immune effector response," as the term is used herein, refers to a function or response, e.g., of an immune effector cell, that enhances or promotes an immune attack of a target cell. For example, an immune effector function or response refers to a property of a T cell or NK cell that promotes the killing or growth or proliferation inhibition of a target cell. In the case of T cells, primary stimulation and costimulation 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 secretion of cytokines.

[0207] The term "encoding" refers to the inherent property of a specific nucleotide sequence within a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes that have either a defined nucleotide sequence (e.g., rRNA, tRNA, and mRNA) or a defined amino acid sequence. 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 usually provided in a sequence listing, and the non-coding strand used as a transcription template for a gene or cDNA can be said to encode the protein or other product of that gene's cDNA.

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

[0209] The terms "effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an 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 from or produced within an organism, cell, tissue, or system.

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

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

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

[0214] The term "expression vector" refers to a vector containing a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in 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 Retroviridae family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells, and they can deliver large amounts of genetic information into the DNA of host cells, making them one of the most efficient 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 a lentiviral genome, including, inter alia, self-inactivating lentiviral vectors as provided in Milone et al., Mol. Ther. 17(8):1453-1464 (2009). Other examples of lentiviral vectors that may be used clinically include, but are not limited to, the LENTIVECTOR® gene delivery technology from Oxford BioMedica and the LENTIMAX™ vector system from Lentigen. Non-clinical types of lentiviral vectors are also available and would be known to those skilled in the art.

[0217] The term "homologous" or "identity" refers to the 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 a subunit position in both of the two molecules is occupied by the same monomer subunit, e.g., if a position in each of the 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 are homologous (e.g., 5 positions in a polymer 10 subunits long), the two sequences are 50% homologous. If 90% of the positions (e.g., 9 out of 10) are matched or homologous, the two sequences are 90% homologous.

[0218] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies and antibody fragments thereof are those in which residues from a recipient complementarity-determining region (CDR) of a human immunoglobulin (recipient antibody or antibody fragment) are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies / antibody fragments can comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications can further refine and optimize antibody or antibody fragment performance. Generally, a humanized antibody or antibody fragment thereof will comprise 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 those of a human immunoglobulin sequence. The humanized antibody or antibody fragment can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that 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, whose entire molecule is of human origin or consists of an amino acid sequence identical to a human form of an antibody or immunoglobulin.

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

[0221] In the context of the present invention, the following abbreviations are used for 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 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 functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be contiguous with each other and, for example, in the same reading frame, as necessary to join two protein-coding regions.

[0223] The term "parenteral" administration of an immunogenic composition includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), 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) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the terms encompass nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. In some embodiments, a "nucleic acid," "nucleic acid molecule," "polypeptide," or "polynucleotide molecule" includes nucleotide / nucleoside derivatives or analogs. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions, e.g., conservative substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions, e.g., conservative substitutions, can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (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 interchangeably 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, with no limit on the maximum number of amino acids that can comprise a protein or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, commonly referred to in the art as peptides, oligopeptides, and oligomers, and to the numerous types of longer chains 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. A polypeptide includes 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 introduced synthetic machinery necessary to initiate the specific transcription of a polynucleotide sequence.

[0227] The term "promoter / regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product operably linked to that promoter / regulatory sequence. In some instances, this sequence may be a core promoter sequence, and in other instances, this sequence may also include an enhancer sequence and other regulatory elements required for expression of the gene product. The promoter / regulatory sequence may, for example, be one that confers tissue-specific expression of the gene product.

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

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

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

[0231] The terms "cancer-associated antigen," "tumor antigen," "hyperproliferative disorder antigen," and "antigen associated with a hyperproliferative disorder" interchangeably refer to antigens common to a particular hyperproliferative disorder. In some embodiments, these terms refer to molecules (typically proteins, carbohydrates, or lipids) expressed on the surface of cancer cells, either intact or as fragments (e.g., MHC / peptides), that are useful for 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, e.g., CD19 on B cells. In some embodiments, a tumor antigen is a cell surface molecule that is overexpressed on cancer cells relative to normal cells (e.g., 1-fold overexpression, 2-fold overexpression, 3-fold or more overexpression relative to normal cells). In some embodiments, a tumor antigen is a cell surface molecule that is inappropriately synthesized on cancer cells, such as a molecule that contains a deletion, addition, or mutation relative to the molecule expressed on normal cells. In some embodiments, tumor antigens will be expressed entirely or as fragments (e.g., MHC / peptides) only on the cell surface of cancer cells and not synthesized or expressed on the surface of normal cells. In some embodiments, the hyperproliferative disorder antigens of the invention are derived from primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin's lymphoma, Hodgkin's 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, and the like, or plasma cell proliferative disorders such as asymptomatic myeloma (smoldering multiple myeloma or asymptomatic myeloma), monoclonal gammopathy of undetermined significance (MGUS), Waldenstrom's macroglobulinemia, plasmacytoma (e.g., plasmacytosis, 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, a CAR of the invention comprises a CAR that comprises 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 pocket of major histocompatibility complex (MHC) class I molecules and are recognized by T cell receptors (TCRs) on CD8+ T lymphocytes. MHC class I complexes are constitutively expressed by all nucleated cells. In cancer, virus-specific and / or tumor-specific peptide / MHC complexes represent a unique class of cell surface targets for immunotherapy. TCR-like antibodies that target peptides derived from viral or tumor antigens in the context of human leukocyte antigen (HLA)-A1 or HLA-A2 have been described (see, e.g., 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 a library, such as a human scFv phage display library.

[0232] The terms "tumor-supporting antigen" or "cancer-supporting antigen" refer 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 supports cancer cells, for example, by promoting their growth or survival, e.g., resistance to immune cells. Exemplary cells of this type include stromal cells and myeloid-derived immunosuppressive cells (MDSCs). The tumor-supporting antigen itself need not play a role in supporting tumor cells, as long as the antigen is present on cells that support the cancer cells.

[0233] The term "flexible polypeptide linker" or "linker," when used in the context of 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 the variable heavy and variable light chain regions together. In some embodiments, the flexible polypeptide linker is a Gly / Ser linker and comprises the amino acid sequence (Gly-Gly-Gly-Ser)n, where n is a positive integer equal to or greater than 1, e.g., 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, flexible polypeptide linkers include, but are not limited to, (Gly4Ser)4 (SEQ ID NO:27) or (Gly4Ser)3 (SEQ ID NO:28). In some embodiments, the linker comprises 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, which is incorporated herein by reference.

[0234] As used herein, a 5' cap (RNA cap, RNA 7-methylguanosine cap or RNA m 7 A 5' cap (also called a G-cap) is a modified guanine nucleotide added to the "pre" or 5' end of eukaryotic messenger RNA immediately after transcription initiation. The 5' cap consists of a terminal group attached to the first transcribed nucleotide. Its presence is important for ribosome recognition and protection from RNases. Capping is coupled to transcription and occurs cotranscriptionally, with each cap affecting the other. Shortly after transcription initiation, a cap-synthesizing complex associated with RNA polymerase binds to the 5' end of the mRNA being synthesized. This enzyme complex catalyzes the chemical reactions required for mRNA capping. Synthesis proceeds as a multistep biochemical reaction. The capping moiety can be modified to adjust mRNA functions such as its stability or translation efficiency.

[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 to generate the in vitro transcribed RNA.

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

[0237] As used herein, "polyadenylation" refers to the covalent attachment of a polyadenylyl moiety or its modified variants 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 (often several hundred) of adenine nucleotides added to pre-mRNA by the action of the 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 its associated proteins help protect the mRNA from exonuclease degradation. Polyadenylation is also important for transcription termination, mRNA nuclear export, and translation. Polyadenylation occurs in the nucleus immediately after DNA-to-RNA transcription, but can also occur later in the cytoplasm. After transcription is 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 is cleaved, an adenosine residue is added to the free 3' end of the cleavage site.

[0238] As used herein, "transient" refers to expression of a non-integrated transgene for a period of hours, days, or weeks, which is shorter than the period of expression of the gene when integrated into the genome or contained in a stable plasmid replicon in the host cell.

[0239] As used herein, the terms "treat," "treatment," and "treating" refer to a reduction or amelioration of the progression, severity, and / or duration of a proliferative disorder or an amelioration of one or more symptoms (preferably one or more discernible symptoms) of a proliferative disorder resulting from the administration of one or more therapies (e.g., one or more therapeutic agents, such as a CAR of the invention). In specific embodiments, the terms "treat," "treatment," and "treating" refer to an improvement in at least one measurable physical parameter of a proliferative disorder, such as tumor growth, that is not necessarily discernible to the patient. In other embodiments, the terms "treat," "treatment," and "treating" refer to either or both physical, e.g., by stabilization of discernible symptoms, inhibition of the progression of a proliferative disorder, physiological, e.g., by stabilization of physical parameters. In other embodiments, the terms "treat," "treatment," and "treating" refer to a reduction or stabilization of tumor size or cancerous cell number.

[0240] The term "signal transduction pathway" refers to the biochemical relationships between various signaling molecules that play a role in transmitting a signal 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 membrane of a cell.

[0241] The term "subject" is intended to include living organisms in which an immune response can be generated (eg, mammals, such as humans).

[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 have been separated from other cell types with which they are normally associated in their naturally occurring state. In some instances, a substantially purified cell population refers to a homogenous cell population. In other instances, the term simply refers to cells that have been separated from the cells with which they are naturally associated in their natural state. In some embodiments, the cells are cultured in vitro. In some embodiments, the cells are not cultured in vitro.

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

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

[0245] The terms "transfected" or "transformed" or "transduced" refer to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0246] The term "specifically binds" refers to an antibody or ligand that recognizes and binds to a cognate binding partner protein present in a sample (e.g., a stimulatory and / or costimulatory molecule present on a T cell) 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 set of polypeptides, typically two polypeptides in the simplest embodiment, that, when present in an immune effector cell, confers specificity for a target cell, typically a cancer cell, and intracellular signal generation to the cell. In some embodiments, an 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 comprises a functional signaling domain derived from a stimulatory molecule and / or a costimulatory molecule as defined herein in the context of the CAR molecule. In some embodiments, the set of polypeptides in an RCAR are not adjacent to each other, e.g., in different polypeptide chains. In some embodiments, an RCAR comprises a dimerization switch, which, in the presence of a dimerization molecule, allows the polypeptides to bind to each other, e.g., link an antigen-binding domain to an intracellular signaling domain. In some embodiments, an RCAR is expressed in a cell (e.g., an immune effector cell) described herein, e.g., an RCAR-expressing cell (also referred to as an "RCARX cell"). In some embodiments, an RCARX cell is a T cell and is referred to as an RCART cell. In some embodiments, the RCARX is a NK cell, referred to as an RCARN cell. The RCAR can confer specificity for target cells, typically cancer cells, and regulatable intracellular signal generation or proliferation to the RCAR-expressing cell, which can optimize the immune effector properties of the RCAR-expressing cell. In several embodiments, the RCAR cell relies, at least in part, on the antigen-binding domain to acquire specificity for target cells that contain an antigen bound to the antigen-binding domain.

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

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

[0250] The term "dimerization molecule," as that term is used herein, e.g., in reference to an RCAR, refers to a molecule that promotes association of a first switch domain with a second switch domain. In embodiments, the dimerization molecule is not naturally present in a subject or is not present at concentrations that result in significant dimerization. In embodiments, the dimerization molecule is a small molecule, e.g., rapamycin or a rapalog, e.g., RAD001.

[0251] The dose "low immune enhancing dose," when used in conjunction with an mTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g., RAD001 or rapamycin, or a catalytic mTOR inhibitor, refers to a dose of the mTOR inhibitor that partially, but not completely, inhibits mTOR activity, e.g., as measured by inhibition of P70 S6 kinase activity. Methods for assessing mTOR activity, e.g., by inhibition of P70 S6 kinase activity, are detailed herein. This dose is insufficient to result in complete immune suppression, but is sufficient to enhance the immune response. In some embodiments, the low immune enhancing dose of the 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, the low immune enhancing dose of the mTOR inhibitor results in an increase in the number of naive T cells. In some embodiments, the low immune enhancing dose of the mTOR inhibitor For example, the following markers on memory T cells, e.g., memory T cell precursors: CD62L high , CD127 high , CD27 + and increased expression of one or more of BCL2; For example, a decrease in the expression of KLRG1 in memory T cells, e.g., memory T cell precursors; and For example, the following characteristics: CD62L high Increased CD127 high Increased CD27 + an increase in the number of memory T cell precursors with any one or combination of increased KLRG1, increased BCL2, decreased KLRG1, and increased BCL2 wherein any of the aforementioned changes occur at least transiently, for example, compared to an untreated subject.

[0252] "Refractory," as used herein, refers to a disease, e.g., cancer, that does not respond to treatment. In embodiments, a refractory cancer may be resistant to treatment before or at the time of treatment initiation. 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, "relapsed" or "recurrence" refers to the recurrence or reappearance of signs and symptoms of a disease (e.g., cancer) or of a disease such as cancer, after a period of improvement or responsiveness, e.g., after previous treatment with a therapy (e.g., cancer therapy). The initial period of responsiveness can include a decrease in the level of cancer cells below a certain threshold, e.g., below 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1%. Reappearance can include an increase in the level of cancer cells above a certain threshold, e.g., above 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1%. For example, reappearance can include the reappearance of blasts in, e.g., the blood, bone marrow (>5%), or any extramedullary site after a complete response, e.g., in the context of B-ALL. In this context, a complete response can include <5% BM blasts. More generally, in some embodiments, a response (e.g., complete response or partial response) can include the absence of detectable MRD (minimal residual disease). In one embodiment, the initial period of responsiveness lasts for 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 a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be construed to include all the possible subranges specifically disclosed as well as individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be construed to include specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as 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 includes those with 95%, 96%, 97%, 98%, or 99% identity, and includes 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] "Gene editing system," as that term is used herein, refers to a system, e.g., one or more molecules, that direct and carry out the modification, e.g., deletion, of one or more nucleic acids at or near a site in genomic DNA targeted by the system. Gene editing systems are known in the art and are described in more detail below.

[0256] As used herein, "administered in combination" means that two (or more) different treatments are delivered to a subject over the course of the subject's illness, e.g., after the subject is diagnosed with an illness and before the illness is cured or eliminated, or before treatment is discontinued for other reasons. In some embodiments, the delivery of one treatment is still ongoing when the delivery of the second treatment begins, thereby resulting in an overlap in 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 when administered in combination. For example, the second treatment is more effective, e.g., a comparable effect is seen with less of the second treatment, or the second treatment alleviates symptoms to a greater extent than, or a similar condition to, that seen when the second treatment is administered without the first treatment. In some embodiments, the delivery is such that the reduction in symptoms or other parameters related to the disorder is greater than that observed when one treatment is delivered without the other. The effects of the two treatments can be partially additive, fully additive, or greater than additive. The delivery can be such that the effect of the first treatment delivered is still detectable when the second treatment is delivered.

[0257] The terms "depletion" or "depleting," as used interchangeably herein, refer to a reduction or decrease in the level or amount of cells, proteins, or macromolecules in a sample after a process, e.g., a selection step, e.g., negative selection, has been performed. Depletion can be a complete or partial depletion of cells, proteins, or macromolecules. In some embodiments, depletion is a reduction or decrease of 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% of 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, "naive T cells" refer to antigen-naive T cells. In some embodiments, antigen-naive T cells have encountered their cognate antigen in the thymus but not in the periphery. In some embodiments, naive T cells are precursors of memory T cells. In some embodiments, naive T cells express both CD45RA and CCR7, but do not express CD45RO. In some embodiments, naive T cells may be characterized by expression of CD62L, CD27, CCR7, CD45RA, CD28, and CD127, and the absence of CD95 or CD45RO isoforms. In some embodiments, naive T cells express CD62L, IL-7 receptor alpha, IL-6 receptor, and CD132, but do not express CD25, CD44, CD69, or CD45RO. In some embodiments, naive T cells express CD45RA, CCR7, and CD62L, but do not express CD95 or IL-2 receptor β. In some embodiments, the surface expression levels of the markers are assessed using flow cytometry.

[0259] The term "central memory T cells" refers to a subset of T cells in humans that are CD45RO positive and express CCR7. 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 beta, CCR7, and CD62L. In some embodiments, the surface expression levels of markers are assessed using flow cytometry.

[0260] The terms "stem memory T cells," "stem cell memory T cells," "stem cell-like memory T cells," "memory stem T cells," "T memory stem cells," "T stem cell memory cells," or "TSCM cells" refer to a subset of memory T cells with stem cell-like capabilities, e.g., the ability to self-renew and / or multipotency to reconstitute memory and / or effector T cell subsets. In some embodiments, stem memory T cells express CD45RA, CD95, IL-2 receptor beta, CCR7, and CD62L. In some embodiments, the surface expression levels of markers are assessed 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 contents of which are incorporated herein by reference.

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

[0262] As used herein, the term "GeneSetScore (Up TEM vs. Down TSCM)" 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 stem cell memory T cell (TSCM) phenotype. A higher GeneSetScore (Up TEM vs. Down TSCM) indicates an increase in the TEM phenotype, whereas a lower GeneSetScore (Up TEM vs. Down TSCM) indicates an increase in the TSCM phenotype. In some embodiments, the GeneSetScore (Up TEM vs. Down TSCM) TSCM) is a gene that is upregulated in TEM cells and / or downregulated in TSCM, e.g., MXRA7, CLIC1, NAT13, TBC1D2B, GLCCI1, DUSP10, APOBEC3D, CACNB3, ANXA2P2, TPRG1, EOMES, MATK, ARHGAP10, ADAM8, MAN1A1, SLFN12L, SH2D2A, EIF2C4, CD58, MYO1F, RA The GeneSetScore (Up TEM vs. Down TSCM) is determined by measuring the expression of one or more genes selected from the group consisting of B27B, 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, the GeneSetScore (Up TEM vs. Down TSCM) is determined for each cell using RNA-seq, e.g., single-cell RNA-seq (scRNA-seq), e.g., as exemplified in Example 7 with reference to Figure 25A. In some embodiments, the GeneSetScore (Up TEM vs. Down TSCM) is calculated by taking 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 extent to which the cell exhibits a regulatory T cell (Treg) versus an effector T cell (Teff) phenotype. A higher GeneSetScore (Up Treg vs. Down Teff) indicates an increase in the Treg phenotype, whereas a lower GeneSetScore (Up Treg vs. Down Teff) indicates an increase in the Teff phenotype. In some embodiments, the GeneSetScore (Up Treg vs. Down Teff) can be expressed as 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, CDKN2 A, SKAP2, GPR55, CDCA7, S100A4, GDPD5, PMAIP1, ACOT9, CEP55, SGMS1, ADPRH, AKAP2, HDAC9, IKZF4, CARD17, VAV 3, OBFC2A, ITGB1, CIITA, SETD7, HLA-DMA, CCR10, KIAA0101, SLC14A1, PTTG3P, DUSP10, FAM164A, PYHIN1, MYO1 F, 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,

[0013] In some embodiments, the GeneSetScore (Up Treg vs. Down Teff) is determined using RNA-seq, e.g., single-cell RNA-seq (scRNA-seq), as exemplified in Example 7, e.g., with reference to Figure 25B. In some embodiments, the GeneSetScore (Up Treg vs. Down Teff) is calculated by taking the average log-normalized gene expression value of all genes in the gene set.

[0264] As used herein, the term "GeneSetScore(Down stemness)" of a cell refers to a score that represents the extent to which the cell exhibits a stem cell phenotype. A lower GeneSetScore(Down stemness) indicates an increased 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, e.g., 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, e.g., single-cell RNA-seq (scRNA-seq), e.g., as exemplified in Example 7 with reference to Figure 25C. In some embodiments, GeneSetScore(Down stemness) is calculated by taking the average log-normalized gene expression value of all the genes in a gene set.

[0265] As used herein, the term "GeneSetScore(UP hypoxia)" of a cell refers to a score that represents the degree to which the cell exhibits a hypoxic phenotype. A higher GeneSetScore(UP hypoxia) indicates an increased hypoxic phenotype. In some embodiments, the GeneSetScore(UP hypoxia) is determined by the expression of one or more genes that are upregulated in cells experiencing 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, B NIP3L, 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, HA P1, 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, LG ALS1, LONP1, LOX, LRP1, MAP4, MET, MIF, MMP13, MMP2, MMP7, MPI, MT1L, MTL3P, ​​MUC1, MXI1, NDRG1, NFIL3, NFKB1, NFKB2, NOS1, NOS2,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, P LOD2, 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, e.g., single-cell RNA-seq (scRNA-seq), e.g., as illustrated in Example 7 with reference to Figure 25D. In some embodiments, GeneSetScore(UP hypoxia) is calculated by taking the average log-normalized gene expression value of all genes in a gene set.

[0266] As used herein, the term "GeneSetScore(UP autophagy)" of a cell refers to a score that represents the degree to which the cell exhibits an autophagic phenotype. A higher GeneSetScore(UP autophagy) indicates an increased autophagic phenotype. In some embodiments, the GeneSetScore(UP autophagy) indicates 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, C11o rf2, C11orf41, C12orf44, C12orf5, C14orf133, C1orf210, C5, C6orf106, C7orf59, C7orf68, C8orf59, C9orf72, CA7, CALCB, CAL COCO2, CAPS, CCDC36, CD163L1, CD93, CDC37, CDKN2A, CHAF1B, CHMP2A, CHMP2B, CHMP3, CHMP4A, CHMP4B, CHMP4C, CHMP6, CHST3, C ISD2, CLDN7, CLEC16A, CLN3, CLVS1, COX8A, CPA3, CRNKL1, CSPG5, CTSA, CTSB, CTSD, CXCR7, DAP, DKKL1, DNAAF2, DPF3, DRAM1, DRA M2, DYNLL1, DYNLL2, DZANK1, EI24, EIF2S1, EPG5, EPM2A, FABP1, FAM125A, FAM131B, FAM134B, FAM13B, FAM176A, FAM176B, FAM48 A, 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、KIAA022 6、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、MT MR14、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、P RKAA1、PRKAA2、PRKAB1、PRKAB2、PRKAG1、PRKAG2、PRKAG3、PRKD2、PRKG1、PSEN1、PTPN22、RAB12、RAB1A、RAB1B、RAB23、RAB24、RAB33B、RAB39、RAB7A、RB1 CC1、RBM18、REEP2、REP15、RFWD3、RGS19、RHEB、RIMS3、RNF185、RNF41、RPS27A、RPTOR、RRAGA、RRAGB、RRAGC、RRAGD、S100A8、S100A9、SCN1A、SERPIN10、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, ST K11, STK32A, STOM, STX12, STX17, SUPT3H, TBC1D17, TBC1D25, TBC1D5, TCIRG1, TEAD4, TECPR1, TECPR2, TFEB, TM9SF1, TMBIM 6, TMEM203, TMEM208, TMEM39A, TMEM39B, TMEM59, TMEM74, TMEM93, TNIK, TOLLIP, TOMM20, TOMM22, TOMM40, TOMM5, TOMM6, TOM M7, TOMM70A, TP53INP1, TP53INP2, TRAPPC8, TREM1, TRIM17, TRIM5, TSG101, TXLNA, UBA52, UBB, UBC, UBQLN1, UBQLN2, UBQLN 4, ULK1, ULK2, ULK3, USP10, USP13, USP30, UVRAG, VAMP7, VAMP8, VDAC1, VMP1, VPS11, VPS16, VPS18, VPS25, VPS28, VPS33A, VP In some embodiments, GeneSetScore(UP autophagy) is determined using RNA-seq, e.g., single-cell RNA-seq (scRNA-seq), e.g., as exemplified in Example 7 with reference to Figure 25E. In some embodiments, GeneSetScore(UP autophagy) is determined using RNA-seq, e.g., single-cell RNA-seq (scRNA-seq), e.g., as exemplified in Example 7 with reference to Figure 25E. In some embodiments, GeneSetScore(UP autophagy) is determined using RNA-seq, e.g., single-cell RNA-seq (scRNA-seq), e.g., as exemplified in Example 7 with reference to Figure 25E.It is calculated by taking the average log-normalized gene expression value of all the 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 degree 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, whereas a lower GeneSetScore (Up resting vs. Down activated) indicates an increase in the activated T cell phenotype. In some embodiments, the GeneSetScore (Up resting vs. Down activated) is determined by the expression of 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, DLG 1, DUSP1, EAPP, ECE1, ECHDC2, ERBB2IP, FAM117A, FAM134B, FAM134C, FAM169A, FAM190B, FAU, FLJ10038, FOXJ 2, FOXJ3, FOXL1, FOXO1, FXYD5, FYB, HLA-E, HSPA1L, HYAL2, ICAM2, IFIT5, IFITM1, IKBKB, IQSEC1, IRS4, KIAA 0664L3, KIAA0748, KLF3, KLF9, KRT18, LEF1, LINC00342, LIPA, LIPT1, LLGL2, LMBR1L, LPAR2, LTBP3, LYPD3, L ZTFL1, MAMBA, MAP2K6, MAP3K1, MARCH8, MAU2, MGEA5, MMP8, MPO, MSL1, MSL3, MYH3, MYLIP, NAGPA, NDST2, NISC H, NKTR, NLRP1, NOSIP, NPIP, NUMA1, PAIP2B, PAPD7, PBXIP1, PCIF1, PI4KA, PLCL2, PLEKHA1, PLEKHF2, PNISR,PPFIBP2, PRKCA, PRKCZ, PRKD3, PRMT2, PTP4A3, PXN, RASA2, RASA3, RASGRP2, RBM38, REPIN1, RNF3 8, 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, SNP H, SP140L, SPATA6, SPG7, SREK1IP1, SRSF5, STAT5B, SVIL, SYF2, SYNJ2BP, TAF1C, TBC1D4, TCF20, T The determination is made by measuring the expression of one or more genes selected from the group consisting of ECTA, 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, e.g., single-cell RNA-seq (scRNA-seq), e.g., as illustrated in Example 7 with reference to Figure 24D. In some embodiments, GeneSetScore (Up resting vs. Down activated) is calculated by taking the average log-normalized gene expression value of all genes in a 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 late memory T cell phenotype, whereas a lower GeneSetScore (Progressively up in memory differentiation) indicates an increase in early memory T cell phenotype. In some embodiments, the GeneSetScore (UP autophagy) indicates one or more genes 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, MAP 1LC3B2, PIP4K2A, HCN3, GTPBP1, TLN1, C4orf34, KIF3B, TCIRG1, PPP3CA, ATG4D, TYMP, TRAF6, C17orf76, WI PF1, FAM108A1, MYL6, NRM, SPCS2, GGT3P, GALK1, CLIP4, ARL4C, YWHAQ, LPCAT4, ATG2A, IDS, TBC1D5, DMPK, S T6GALNAC6, REEP5, ABHD6, KIAA0247, EMB, TSEN54, SPIRE2, PIWIL4, ZSCAN22, ICAM1, CHD9, LPIN2, SETD8, Z C3H12A, 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, CN NM3, 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、P ARP8、RNF166、UEVLD、LIMK1、CACNB1、TMX4、SLC6A6、LBA1、SV2A、LLGL2、IRF 1、PPP2R5C、CD99、RAPGEF1、PPP4R1、OSBPL7、FOXP4、SLA2、TBC1D2B、ST7、JAZ F1、GGA2、PI4K2A、CD68、LPGAT1、STX11、ZAK、FAM160B1、RORA、C8orf80、APO BEC3F、TGFBI、DNAJC1、GPR114、LRP8、CD69、CMIP、NAT13、TGFB1、FLJ00049、A NTXR2、NR4A3、IL12RB1、NTNG2、RDX、MLLT4、GPRIN3、ADCY9、CD300A、SCD5、A BI3、PTPN22、LGALS1、SYTL3、BMPR1A、TBK1、PMAIP1、RASGEF1A、GCNT1、GABAR APL1, STOM, CALHM2, ABCA2, PPP1R16B, SYNE2, PAM, C12orf75, CLCF1, MXRA7, APOBEC3C, CLSTN3, ACOT9, HIP1, LAG3, TNFAIP3, DCBLD1, KLF6, CACNB3, RNF 19A、RAB27A、FADS3、DLG5、APOBEC3D、TNFRSF1B、ACTN4、TBKBP1、ATXN1、ARA P2、ARHGEF12、FAM53B、MAN1A1、FAM38A、PLXNC1、GRLF1、SRGN、HLA-DRB5、B4G ALT5、WIPI1、PTPRJ、SLFN11、DUSP2、ANXA5、AHNAK、NEO1、CLIC1、EIF2C4、MA P3K5、IL2RB、PLEKHG1、MYO6、GTDC1、EDARADD、GALM、TARP、ADAM8、MSC、HNRPL L、SYT11、ATP2B4、NHSL2、MATK、ARHGAP18、SLFN12L、SPATS2L、RAB27B、PIK3 R3、TP53INP1、MBOAT1、GYG1、KATNAL1、FAM46C、ZC3HAV1L、ANXA2P2、CTNNA1、NPC1, C3AR1, CRIM1, SH2D2A, ERN1, YPEL1, TBX21, SLC1A4, FASLG, PHACTR2, GALNT3, ADRB2, PIK3AP1, TLR3, PLEKHA5, DUSP10, GNAO1, PTGDR, FRMD4B, A NXA2, 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, e.g., single-cell RNA-seq (scRNA-seq), e.g., as exemplified in Example 7 with reference to Figure 26B. In some embodiments, the GeneSetScore (Progressively up in memory differentiation) is calculated by taking the average log-normalized gene expression value of all of the 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, whereas a lower GeneSetScore (Up TEM vs. Down TN) indicates an increase in the TN phenotype.In some embodiments, the GeneSetScore (Up TEM vs. Down TN) is calculated based on 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, OS BPL3, 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, MAN 1A1, 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, NBE It is determined by measuring the expression of one or more genes selected from the group consisting of AL2, 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.In some embodiments, GeneSetScore (Up TEM vs. Down TN) is determined using RNA-seq, e.g., single-cell RNA-seq (scRNA-seq), e.g., as illustrated in Example 7 with reference to Figure 26C. In some embodiments, GeneSetScore (Up TEM vs. Down TN) is calculated by taking the average log-normalized gene expression value of all genes in a gene set.

[0270] Relative to a GeneSetScore value (e.g., median GeneSetScore), a 100% decrease in a positive GeneSetScore results in a value of 0. A 100% increase in a negative GeneSetScore results in a value of 0. For example, in Figure 25A, the median GeneSetScore for day 1 samples is -0.084; the median GeneSetScore for day 9 samples is 0.035; and the median GeneSetScore for input samples is -0.1. In Figure 25A, a 100% increase in the median GeneSetScore for input samples results in a GeneSetScore value of 0; a 200% increase in the median GeneSetScore for input samples results in a GeneSetScore value of 0.1. In Figure 25A, a 100% decrease in the median GeneSetScore for the day 9 samples results in a GeneSetScore value of 0; a 200% decrease in the median GeneSetScore for the day 9 samples results in a GeneSetScore value of -0.035.

[0271] As used herein, the term "bead" refers to a discrete particle with a solid surface ranging in size from about 0.1 μm to several millimeters in diameter. Beads can be spherical (e.g., microspheres) or irregularly shaped. Beads can comprise a variety of materials, including, but not limited to, paramagnetic materials, ceramic, plastic, glass, polystyrene, methylstyrene, acrylic polymers, titanium, latex, Sepharose™, cellulose, nylon, and the like. In some embodiments, the beads are relatively uniform, approximately 4.5 μm in diameter, spherical, superparamagnetic polystyrene beads, coated with, e.g., coupled to, a mixture of antibodies against CD3 (e.g., CD3ε) and CD28. In some embodiments, the beads are Dynabeads®. In some embodiments, both anti-CD3 and anti-CD28 antibodies are coupled to the same beads to mimic 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, see, e.g., 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 comprising a matrix of flexible polymer chains. The nanomatrix is ​​sized between 1 and 500 nm, e.g., between 10 and 200 nm. In some embodiments, the matrix of flexible polymer chains is coupled to one or more agonists, e.g., agonistic anti-CD3 and / or anti-CD28 antibodies, that provide activation signals to T cells. In some embodiments, the nanomatrix comprises a colloidal polymer nanomatrix coupled, e.g., covalently attached, to one or more agonists of stimulatory molecules and / or one or more agonists of costimulatory molecules. In some embodiments, the one or more agonists of stimulatory molecules are CD3 agonists (e.g., anti-CD3 agonist antibodies). In some embodiments, the one or more agonists of stimulatory molecules are CD28 agonists (e.g., anti-CD28 agonist antibodies). In some embodiments, the nanomatrix is ​​characterized by the absence of a solid surface as a binding point for agonists, e.g., anti-CD3 and / or anti-CD28 antibodies. In some embodiments, the nanomatrix is ​​the nanomatrix disclosed in WO 2014 / 048920 A1 or as provided in the MACS® GMP T Cell TransAct™ kit from Miltenyi Biotcc GmbH, the entire contents of which are incorporated herein by reference. MACS® GMP T Cell TransAct™ is composed of a colloidal polymer nanomatrix covalently linked to humanized recombinant agonistic antibodies against human CD3 and CD28.

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

[0274] Provided herein are compositions of matter and methods of use for the treatment of diseases such as cancer using cells expressing one or more chimeric antigen receptors (CARs). In some embodiments, the invention provides cells (e.g., immune effector cells, e.g., T cells or NK cells) engineered to express one or more CARs, wherein 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 a first antigen and a second CAR that binds 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, e.g., an anti-BCMA CAR disclosed herein, comprising a CDR, VH, VL, scFv, or CAR sequence disclosed herein, e.g., a sequence 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. In some embodiments, the second antigen is CD19. In some embodiments, the second antigen is an anti-CD19 CAR, e.g., an anti-CD19 CAR disclosed herein, comprising a CDR, VH, VL, scFv, or CAR sequence disclosed herein, e.g., a sequence disclosed in Tables 2, 19, and 22, or an amino acid sequence having at least about 85%, 90%, 95%, or 99% sequence identity thereto. In some embodiments, the first CAR and the second CAR are expressed by nucleic acid sequences located on 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-cleavage site, e.g., a P2A site, a T2A site, an E2A site, or an F2A site. In some embodiments, the cell is a cell expressing a dual CAR disclosed herein. In some embodiments, the first CAR and the second CAR are expressed by nucleic acid sequences located on separate nucleic acid molecules. In some embodiments, the cell is engineered using a co-transduction system disclosed herein.

[0276] In some embodiments, the cells express a CAR that binds 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-optional linker 1 ("L1")-VH2-optional linker 2 ("L2")-VL2-optional linker 3 ("L3")-VL1, VH1-optional L1-VL2-optional L2-VH2-optional L3-VL1, VL1-optional L1-VH2-optional L2-VL2-optional L3-VH1, VL1-optional or VL2-optionally L1-VL1-optionally L2-VH1-optionally L3-VH2; 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 CARs of the invention link the antigen binding domain to an intracellular signaling molecule, for example, in some embodiments, the intracellular signaling molecule includes, but is not limited to, the CD3 zeta chain, 4-1BB, and CD28 signaling modules, and combinations thereof.

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

[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 CARs, which direct the immune effector cells to cancer. This is accomplished through an antigen-binding domain on the CAR that is specific for a cancer-associated antigen. There are two classes of cancer-associated antigens (tumor antigens) that can be targeted by the CARs described herein: (1) cancer-associated antigens that are expressed on the surface of cancer cells; and (2) cancer-associated antigens that are themselves intracellular, but where fragments (peptides) of such antigens are presented on the surface of cancer cells by the MHC (major histocompatibility complex).

[0280] Thus, 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, ephrinB2, IGF-I receptor, CAIX, LMP2, gp100, bcr-a bl, tyrosinase, 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 mutant, prostein, survivin and telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoints, 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 mutated hsp70-2.

[0281] Non-limiting example sequences of various components that can be part of the CAR molecules described herein are listed in Table 1, where "aa" represents an amino acid and "na" represents a nucleic acid that encodes the corresponding peptide.

[0282] [Table 1]

[0283] [Table 2]

[0284] [Table 3]

[0285] [Table 4]

[0286] [Table 5]

[0287] In some embodiments, the antigen-binding domain comprises the extracellular domain of a molecule that binds to a counter-ligand on the surface of a target cell, or a counter-ligand-binding fragment thereof.

[0288] The immune effector cells can comprise a recombinant DNA construct comprising sequences encoding a CAR, where the CAR comprises an antigen binding domain (e.g., an antibody or antibody fragment, TCR or TCR fragment) that specifically binds to a tumor antigen, e.g., a tumor antigen described herein, and an intracellular signaling domain. The intracellular signaling domain can comprise a costimulatory signaling domain and / or a primary signaling domain, e.g., a zeta chain. As described elsewhere, the methods described herein can include transducing cells (e.g., from a population of T regulatory-depleted cells) with a nucleic acid encoding a CAR, e.g., a CAR described herein.

[0289] In some embodiments, the CAR comprises an scFv domain, where the scFv can be preceded by an optional leader sequence such as provided in SEQ ID NO: 1 and can be followed by an optional hinge sequence such as provided in SEQ ID NO: 2 or SEQ ID NO: 36 or SEQ ID NO: 38, a transmembrane region such as provided in SEQ ID NO: 6, an intracellular signaling domain comprising SEQ ID NO: 7 or SEQ ID NO: 16, and a CD3ζ sequence comprising SEQ ID NO: 9 or SEQ ID NO: 10, e.g., where these domains are contiguous and in the same reading frame to form a single fusion protein.

[0290] In some embodiments, exemplary CAR constructs comprise an optional leader sequence (e.g., a leader sequence described herein), an extracellular antigen binding domain (e.g., an antigen binding domain described herein), a hinge (e.g., a hinge region described herein), a transmembrane domain (e.g., a transmembrane domain described herein), and an intracellular stimulatory domain (e.g., an intracellular stimulatory domain described herein). In some embodiments, exemplary CAR constructs comprise an optional leader sequence (e.g., a leader sequence described herein), an extracellular antigen binding domain (e.g., an antigen binding domain described herein), a hinge (e.g., a hinge region described herein), a transmembrane domain (e.g., a transmembrane domain described herein), an intracellular costimulatory signaling domain (e.g., a costimulatory signaling domain described herein), and / or an intracellular primary signaling domain (e.g., a primary signaling domain described herein).

[0291] An exemplary leader sequence is provided as SEQ ID NO: 1. Another exemplary leader includes that provided in SEQ ID NO: 351 or encoded by SEQ ID NO: 352 or 353. An exemplary hinge / spacer sequence is provided as SEQ ID NO: 2, or SEQ ID NO: 36, or SEQ ID NO: 38. An exemplary transmembrane domain sequence is provided as SEQ ID NO: 6. An exemplary 4-1BB protein intracellular signaling domain sequence is provided as SEQ ID NO: 7. An exemplary CD27 intracellular signaling domain sequence is provided as ...

Claims

1. A pharmaceutical composition for use in treating an autoimmune disease in a subject, comprising an isolated immune effector cell or population of immune effector cells comprising a chimeric antigen receptor (CAR), wherein the CAR is: (a) a first CAR (BCMA CAR) comprising: a first antigen-binding domain comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) that binds to BCMA; a first transmembrane domain; and a first intracellular signaling domain comprising a first costimulatory signaling domain and a first primary signaling domain; and (b) a second CAR (CD19 CAR) comprising a second antigen-binding domain comprising a VH and a VL that binds to CD19; a second transmembrane domain; and a second intracellular signaling domain comprising a second costimulatory signaling domain and a second primary signaling domain; Including, the first CAR and the second CAR each comprise an HCDR1, an HCDR2, an HCDR3, an LCDR1, an LCDR2, and an LCDR3 set forth in any one of SEQ ID NOs: 214, 216, 218, 220, or 222; and 1. A pharmaceutical composition, wherein the CAR comprises the amino acid sequence of SEQ ID NO: 214, 216, 218, 220, or 222, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity thereto.

2. Use of an isolated immune effector cell or population of immune effector cells comprising a CAR for the manufacture of a medicament for treating an autoimmune disease in a subject, wherein the CAR is: (a) a first CAR (BCMA CAR) comprising: a first antigen-binding domain comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) that binds to BCMA; a first transmembrane domain; and a first intracellular signaling domain comprising a first costimulatory signaling domain and a first primary signaling domain; and (b) a second CAR (CD19 CAR) comprising a second antigen-binding domain comprising a VH and a VL that binds to CD19; a second transmembrane domain; and a second intracellular signaling domain comprising a second costimulatory signaling domain and a second primary signaling domain; Including, the first CAR and the second CAR each comprise an HCDR1, an HCDR2, an HCDR3, an LCDR1, an LCDR2, and an LCDR3 set forth in any one of SEQ ID NOs: 214, 216, 218, 220, or 222; and The use, wherein the CAR comprises the amino acid sequence of SEQ ID NO: 214, 216, 218, 220 or 222, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto.

3. 3. The pharmaceutical composition of claim 1 or the use of claim 2, 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 nucleic acid sequence and the second nucleic acid sequence are located on a single nucleic acid molecule. (a) the single nucleic acid molecule has, in a 5' to 3' direction, the following composition: (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, and a nucleic acid sequence encoding the second intracellular signaling domain; or (ii) 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; and (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% 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% identity thereto; A pharmaceutical composition according to claim 3 or a use according to claim 3.

5. A linker comprising a P2A site, (I) encoded by the nucleic acid sequence of SEQ ID NO: 209, or encoded by a nucleic acid sequence having at least 90%, 95%, or 99% identity thereto; or (II) comprising the amino acid sequence of SEQ ID NO: 208, or an amino acid sequence having at least 90%, 95%, or 99% identity thereto; A pharmaceutical composition according to claim 4 or a use according to claim 4.

6. 3. The pharmaceutical composition of claim 1, or the use of claim 2, wherein the first antigen-binding domain or the second antigen-binding domain comprises a VH and a VL, wherein the VH and the VL are connected 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.

7. (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% 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% 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 signaling domain comprises a functional signaling domain derived from CD3ζ, wherein: (a) the primary signaling domain comprises the amino acid sequence of SEQ ID NO: 9 or 10, or an amino acid sequence having at least 90%, 95%, or 99% identity thereto; or (b) 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 90%, 95%, or 99% identity thereto; (v) the costimulatory signaling domain comprises a functional signaling domain derived from 4-1BB (CD137), wherein: (a) the costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having at least 90%, 95%, or 99% identity thereto; or (b) the costimulatory signaling domain is encoded by the nucleic acid sequence of SEQ ID NO: 18 or 204, or by a nucleic acid sequence having at least 90%, 95%, or 99% identity thereto; (vi) 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, wherein: (a) 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 90%, 95%, or 99% identity thereto, and the amino acid sequence of SEQ ID NO: 9 or 10, or an amino acid sequence having at least 90%, 95%, or 99% identity thereto; or (b) 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; or (vii) the first CAR further comprises a first leader sequence, or the second CAR further comprises a second leader sequence, wherein: (a) the first leader sequence or the second leader sequence comprises the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 90%, 95%, or 99% identity thereto; or 4. The pharmaceutical composition of claim 3, or the use of claim 3, wherein (b) the first leader sequence or the second leader sequence is encoded by a nucleic acid sequence of SEQ ID NO: 199 or 210, or by a nucleic acid sequence having at least 90%, 95% or 99% identity thereto.

8. (a) the hinge region comprises the amino acid sequence of SEQ ID NO:2, or an amino acid sequence having at least 90%, 95%, or 99% identity thereto; (b) the hinge region is encoded by the nucleic acid sequence of SEQ ID NO: 13, or a nucleic acid sequence having at least 90%, 95%, or 99% identity thereto; (c) 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 90%, 95%, or 99% identity thereto; or 8. The pharmaceutical composition of claim 7, or the use of claim 7, wherein (d) the hinge region and the transmembrane domain are encoded by the nucleic acid sequence of SEQ ID NO: 203 or 213, or by a nucleic acid sequence having at least 90%, 95% or 99% identity thereto.

9. (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 comprise different amino acid sequences; (ii) the first hinge region and the second hinge region comprise the same amino acid sequence or comprise different amino acid sequences; (iii) the first transmembrane domain and the second transmembrane domain comprise the same amino acid sequence or comprise 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 comprise different amino acid sequences; and / or (c): (i) the first leader sequence and the second leader sequence are encoded by a nucleic acid sequence comprising SEQ ID NOs: 199 and 210, respectively, or a nucleic acid sequence having at least 90%, 95%, or 99% identity thereto, or are encoded by a nucleic acid sequence comprising SEQ ID NOs: 210 and 199, respectively, or a nucleic acid sequence having at least 90%, 95%, or 99% identity thereto; (ii) the first hinge region and the second hinge region are encoded by a nucleic acid sequence comprising SEQ ID NOs: 337 and 13, respectively, or a nucleic acid sequence having at least 90%, 95%, or 99% identity thereto; or are encoded by a nucleic acid sequence comprising SEQ ID NOs: 13 and 337, respectively, or a nucleic acid sequence having at least 90%, 95%, or 99% identity thereto; (iii) the first transmembrane domain and the second transmembrane domain are encoded by a nucleic acid sequence comprising SEQ ID NOs: 338 and 17, respectively, or a nucleic acid sequence having at least 90%, 95%, or 99% identity thereto; or are encoded by a nucleic acid sequence comprising SEQ ID NOs: 17 and 338, respectively, or a nucleic acid sequence having at least 90%, 95%, or 99% identity thereto; (iv) the first costimulatory signaling domain and the second costimulatory signaling domain are encoded by a nucleic acid sequence comprising SEQ ID NOs: 204 and 18, respectively, or a nucleic acid sequence having at least 90%, 95%, or 99% identity thereto; or are encoded by a nucleic acid sequence comprising SEQ ID NOs: 18 and 204, respectively, or a nucleic acid sequence having at least 90%, 95%, or 99% identity thereto; and / or (v) the first primary signaling domain and the second primary signaling domain are encoded by a nucleic acid sequence comprising SEQ ID NOs: 205 and 21, respectively, or a nucleic acid sequence having at least 90%, 95%, or 99% identity thereto; or are encoded by a nucleic acid sequence comprising SEQ ID NOs: 21 and 205, respectively, or a nucleic acid sequence having at least 90%, 95%, or 99% identity thereto; or (d) the first CAR or the second CAR is encoded by a nucleic acid molecule comprising a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

10. (a) the first primary signaling domain and the second primary signaling domain are encoded by different nucleic acid sequences; (b) the first costimulatory signaling domain and the second costimulatory signaling domain are encoded by different nucleic acid sequences; (c) 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 90%, 95%, or 99% identity thereto; (d) 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 90%, 95%, or 99% identity thereto; (e) 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 90%, 95%, or 99% identity thereto; (f) 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 90%, 95%, or 99% identity thereto; or (g) the first costimulatory signaling domain and the second costimulatory signaling domain comprise the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having at least 90%, 95%, or 99% identity thereto.

11. A pharmaceutical composition for use in treating an autoimmune disease in a subject, comprising an isolated nucleic acid molecule encoding a CAR, wherein the nucleic acid molecule is: (a) a first nucleic acid sequence encoding a first CAR (BCMA CAR) comprising: a first antigen-binding domain comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) that binds BCMA; a first transmembrane domain; and a first intracellular signaling domain comprising a costimulatory signaling domain and a first primary signaling domain; and (b) a second nucleic acid sequence encoding a second CAR (CD19 CAR) comprising: a second antigen-binding domain comprising a VH and a VL that binds to CD19; a second transmembrane domain; and a second intracellular signaling domain comprising a second costimulatory signaling domain and a second primary signaling domain; Including, the first CAR and the second CAR each comprise an HCDR1, an HCDR2, an HCDR3, an LCDR1, an LCDR2, and an LCDR3 set forth in any one of SEQ ID NOs: 214, 216, 218, 220, or 222; and 1. A pharmaceutical composition wherein the encoded CAR comprises the amino acid sequence of SEQ ID NO: 214, 216, 218, 220, or 222, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity thereto, and wherein the first nucleic acid sequence and the second nucleic acid sequence are located on a single nucleic acid molecule.

12. Use of an isolated nucleic acid molecule encoding a CAR for the manufacture of a medicament for treating an autoimmune disease in a subject, wherein the nucleic acid molecule is: (a) a first nucleic acid sequence encoding a first CAR (BCMA CAR) comprising: a first antigen-binding domain comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) that binds BCMA; a first transmembrane domain; and a first intracellular signaling domain comprising a costimulatory signaling domain and a first primary signaling domain; and (b) a second nucleic acid sequence encoding a second CAR (CD19 CAR) comprising: a second antigen-binding domain comprising a VH and a VL that binds to CD19; a second transmembrane domain; and a second intracellular signaling domain comprising a second costimulatory signaling domain and a second primary signaling domain; Including, the first CAR and the second CAR each comprise an HCDR1, an HCDR2, an HCDR3, an LCDR1, an LCDR2, and an LCDR3 set forth in any one of SEQ ID NOs: 214, 216, 218, 220, or 222; and 21. The use of claim 1, wherein the encoded CAR comprises the amino acid sequence of SEQ ID NO: 214, 216, 218, 220, or 222, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity thereto, and wherein the first nucleic acid sequence and the second nucleic acid sequence are located on a single nucleic acid molecule.

13. A pharmaceutical composition for use in treating an autoimmune disease in a subject, comprising an isolated CAR, wherein the CAR is: (a) a first CAR (BCMA CAR) comprising: a first antigen-binding domain comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) that binds to BCMA; a first transmembrane domain; and a first intracellular signaling domain comprising a costimulatory signaling domain and a first primary signaling domain; and (b) a second CAR (CD19 CAR) comprising a second antigen-binding domain comprising a VH and a VL that binds to CD19; a second transmembrane domain; and a second intracellular signaling domain comprising a second costimulatory signaling domain and a second primary signaling domain; Including, the first CAR and the second CAR each comprise an HCDR1, an HCDR2, an HCDR3, an LCDR1, an LCDR2, and an LCDR3 set forth in any one of SEQ ID NOs: 214, 216, 218, 220, or 222; and The pharmaceutical composition, wherein the CAR comprises the amino acid sequence of SEQ ID NO: 214, 216, 218, 220, or 222, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity thereto.

14. Use of an isolated CAR for the manufacture of a medicament for treating an autoimmune disease in a subject, wherein the CAR is: (a) a first CAR (BCMA CAR) comprising: a first antigen-binding domain comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) that binds BCMA; and a first transmembrane domain; a first intracellular signaling domain comprising a costimulatory signaling domain and a first primary signaling domain; and (b) a second CAR (CD19 CAR) comprising a second antigen-binding domain comprising a VH and VL that binds to CD19 and a second transmembrane domain; a second intracellular signaling domain comprising a second costimulatory signaling domain; and a second primary signaling domain; Including, the first CAR and the second CAR each comprise an HCDR1, an HCDR2, an HCDR3, an LCDR1, an LCDR2, and an LCDR3 set forth in any one of SEQ ID NOs: 214, 216, 218, 220, or 222; and The use, wherein the CAR comprises the amino acid sequence of SEQ ID NO: 214, 216, 218, 220 or 222, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto.

15. The pharmaceutical composition of claim 11, or the use of claim 12, wherein the nucleic acid molecule is contained in a vector.

16. The pharmaceutical composition described in claim 15 or the use described in claim 15, wherein the vector is selected from the group consisting of a DNA vector, an RNA vector, a plasmid, a lentiviral vector, an adenoviral vector, or a retroviral vector.

17. The pharmaceutical composition of claim 11 or the use of claim 12, wherein the nucleic acid molecule is contained in an isolated immune effector cell or a population of immune effector cells.

18. The immune effector cell population of about 1 x 10 ６ 20. The pharmaceutical composition of any one of claims 1, 3-10 or 17, or the use of any one of claims 2-10 or 17, administered to the subject at a dose of up to about 1 x 10 viable CAR-positive cells.

19. The pharmaceutical composition of claim 18, or the use of claim 18, wherein the immune effector cell population is administered to the subject at a dose of about 5 x 10 6 to about 2 x 10 7 viable CAR-positive cells.

20. A pharmaceutical composition according to any one of claims 1, 3-10 or 17-19, or a use according to any one of claims 2-10 or 17-19, wherein the immune effector cell population is formulated for administration in combination with a second therapeutic agent.

21. A pharmaceutical composition according to any one of claims 1, 3 to 11, 13 or 15 to 20, or a use according to any one of claims 2 to 10, 12 or 14 to 20, wherein the autoimmune disease is associated with BCMA.

22. The pharmaceutical composition of any one of claims 1, 3 to 11, 13 or 15 to 21, or the use of any one of claims 2 to 10, 12 or 14 to 21, wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis, lupus, pemphigus vulgaris, Sjogren's syndrome, inflammatory bowel disease, and ulcerative colitis.

23. The pharmaceutical composition described in claim 22, or the use described in claim 22, wherein the lupus is systemic lupus erythematosus (SLE).