Cells and compositions for treating cancer

Abstract

The presently disclosed subject matter provides cells, compositions, and methods for enhancing immune responses to tumor antigens. These cells comprise a first antigen-recognizing receptor (e.g., a chimeric antigen receptor (CAR)) and a second antigen-recognizing receptor (e.g., a TCR-like fusion molecule). These cells have improved activity and / or efficiency. The presently disclosed subject matter further provides uses of these compositions in reducing tumor burden, treating and / or preventing neoplasms or tumors, preventing and / or treating pathogen infections, preventing and / or treating autoimmune diseases, and / or preventing and / or treating infectious diseases.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 418,302, filed October 21, 2022, the contents of which are incorporated by reference in their entirety and to which priority is claimed.

[0002] Sequence Listing A Sequence Listing conforming to the rules of WIPO Standard ST.26 is incorporated herein by reference. The Sequence Listing has been submitted via the PatentCenter as an electronic document encoded as XML in UTF-8 text. The electronic document, created on October 23, 2023, is entitled "072734_1497_SL.xml" and is 235,322 bytes in size.

[0003] Introduction The presently disclosed subject matter provides compositions and methods for targeting an immune response against tumor antigen-bearing cells, which relate to compositions, e.g., modified immunoresponsive cells, comprising a first antigen-recognizing receptor (e.g., a chimeric antigen receptor (CAR)) and a second antigen-recognizing receptor (e.g., a TCR-like fusion molecule), wherein the composition mediates an immune response against cells bearing the first and / or second antigen. [Background technology]

[0004] Target heterogeneity can limit the therapeutic success of certain CAR therapies in cancer (e.g., B-cell and other malignancies). Several approaches to address target heterogeneity in CAR-based therapies have been described, including the use of dual CARs (T cells co-expressing two CARs), tandem CARs (T cells expressing a single bispecific CAR), and pooled single CAR-T cells (T cells expressing a single CAR). The clinical efficacy of such CAR therapies is still under investigation, and emerging preclinical data suggest that these approaches may require fine-tuning of combinatorial CAR signaling to balance effector and memory functions and avoid premature differentiation and eventual exhaustion. Therefore, novel concepts for addressing target heterogeneity are urgently needed. Summary of the Invention [Means for solving the problem]

[0005] The presently disclosed subject matter provides compositions, e.g., modified immunoresponsive cells, comprising (1) a CAR that targets a first antigen and (2) a TCR-like fusion molecule that targets a second antigen. The presently disclosed subject matter further provides uses of these compositions in reducing tumor burden, treating and / or preventing neoplasms or tumors, preventing and / or treating pathogen infections, preventing and / or treating autoimmune diseases, and / or preventing and / or treating infectious diseases.

[0006] In certain embodiments, the CAR comprises an extracellular antigen-binding domain that binds to a first antigen and an intracellular signaling domain that can deliver an activation signal to a cell. In certain embodiments, the intracellular signaling domain of the CAR comprises a CD3ζ polypeptide. In certain embodiments, the CD3ζ polypeptide is a native CD3ζ polypeptide or a modified CD3ζ polypeptide. In certain embodiments, the modified CD3ζ polypeptide comprises a native ITAM1, an ITAM2 variant consisting of two loss-of-function mutations, and an ITAM3 variant consisting of two loss-of-function mutations. In certain embodiments, the modified CD3ζ polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 22. In certain embodiments, the intracellular signaling domain of the CAR further comprises at least one costimulatory signaling region. In certain embodiments, the at least one costimulatory signaling region comprises at least the intracellular domain of a costimulatory molecule or a portion thereof. In certain embodiments, the costimulatory molecule is selected from the group consisting of CD28, 4-1BB, OX40, CD27, CD40, CD154, CD97, CD11a / CD18, ICOS, DAP-10, CD2, CD150, CD226, and NKG2D. In certain embodiments, the costimulatory molecule comprises amino acids 180-220 of SEQ ID NO: 7. In certain embodiments, the CAR comprises a transmembrane domain.

[0007] In certain embodiments, the TCR-like fusion molecule comprises i) a first antigen-binding chain comprising an antigen-binding fragment of an antibody heavy chain variable region (VH), and ii) a second antigen-binding chain comprising an antigen-binding fragment of an antibody light chain variable region (VL), wherein the first and second antigen-binding chains a) each comprise a TRAC polypeptide or a TRBC polypeptide, and b) bind to a second antigen, and the TCR-like fusion molecule binds to the second antigen in an HLA-independent manner. In certain embodiments, at least one of the TRAC polypeptide and the TRBC polypeptide is endogenous. In certain embodiments, the first and second antigen-binding chains are about 1×10 -8In certain embodiments, the first and second antigen-binding chains bind to the second antigen with a dissociation constant (KD) of about 5×10 M or less. -9 It binds to a second antigen with a dissociation constant (KD) of M or less.

[0008] In certain embodiments, the first antigen binding chain comprises an antigen-binding fragment of an antibody VH and a TRBC polypeptide, and the second antigen binding chain comprises an antigen-binding fragment of an antibody VL and a TRAC polypeptide. In certain embodiments, the first antigen binding chain comprises an antigen-binding fragment of an antibody VH and a TRAC polypeptide, and the second antigen binding chain comprises an antigen-binding fragment of an antibody VL and a TRBC polypeptide. In certain embodiments, the first and second antigen binding chains are capable of associating with a CD3ζ polypeptide. In certain embodiments, the first and second antigen binding chains can activate the CD3ζ polypeptide upon binding to the second antigen. In certain embodiments, activation of the CD3ζ polypeptide can activate cells.

[0009] In certain embodiments, the cell further comprises a gene disruption at the TRAC locus and / or the TRBC locus. In certain embodiments, the cell further comprises a gene disruption at the CD70 locus. In certain embodiments, the cell further comprises a gene disruption at the TRAC locus, the TRBC locus, and / or the CD70 locus.

[0010] In certain embodiments, the cells further comprise a genetic modification of the TRAC gene and / or the TRBC gene. In certain embodiments, the cells further comprise a genetic modification of the CD70 gene. In certain embodiments, the cells further comprise a genetic modification of the TRAC gene, the TRBC gene, and / or the CD70 gene.

[0011] In certain embodiments, the immunoresponsive cell is a cell of the lymphoid lineage or a cell of the myeloid lineage. In certain embodiments, the cell of the lymphoid lineage is selected from the group consisting of a T cell, a B cell, a natural killer (NK) cell, and a dendritic cell. In certain embodiments, the cell is a T cell. In certain embodiments, the T cell is derived from an induced pluripotent stem cell. In certain embodiments, the T cell is a CD8 + In certain embodiments, CD8 + T cells are CD4-independent.

[0012] In certain embodiments, the T cells are selected from the group consisting of cytotoxic T lymphocytes (CTLs), γδ T cells, tumor-infiltrating lymphocytes (TILs), regulatory T cells, and natural killer T (NKT) cells. + In certain embodiments, the T cells are CD45RA + In certain embodiments, the T cells are CD45RA + and CD62L + is.

[0013] In certain embodiments, the CAR and / or TCR-like fusion molecule is integrated into a locus in the genome of an immunoresponsive cell. In certain embodiments, the locus is selected from the group consisting of the TRAC locus, the TRBC locus, the TRDC locus, and the TRGC locus. In certain embodiments, the locus is the TRAC locus or the TRBC locus. In certain embodiments, the locus is the TRAC locus.

[0014] In certain embodiments, the first antigen and / or the second antigen is a tumor antigen or a pathogen antigen. In certain embodiments, the tumor antigen is selected from the group consisting of CD19, CD70, IL1RAP, ABCG2, AChR, ACKR6, ADAMTS13, ADGRE2, ADGRE2 (EMR2), ADORA3, ADRA1D, AGER, ALS2, an antigen of a cytomegalovirus (CMV)-infected cell (e.g., a cell surface antigen), ANO9, AQP2, ASIC3, ASPRV1, ATP6V0A4, B3GNT4, B7-H3, BCMA, BEST4, C3orf35, CADM3, CAIX, CAPN3, CCDC155, CCR1, CD10, CD117, CD123, CD133, CD135(FLT3), CD138, CD20, CD22, CD244(2B4), CD25, CD26, CD30, CD300LF, CD312, CD32, CD321, CD33, CD34, CD36, CD38, CD 41, CD44, CD44V6, CD47, CD49f, CD56, CD7, CD71, CD74, CD8, CD82, CD96, CD98, CD99, CDH13, CDHR1, CEA, CEACAM6, CHST3, CLEC12A, CLEC1A, CLL1, C NIH2, COL15A1, COLEC12, CPM, CR1, CX3CR1, CXCR4, CYP4F11, DAGLB, DARC, DFNB31, DGKI, EGF1R, EGFR-VIII, EGP-2, EGP-40, ELOVL6, EMB, EMC10, EMR2, ENG, EpCAM, EphA2, EPHA4, ERBB, ERBB2, Erb-B3, Erb-B4, E-selectin, EXOC3L4, EXTL3, FAM186B, FBP, FCGR1A, FKBP1B, FLRT1, folate receptor-α, FOLR2, FRMD5, GABRB2, GAS2, GD2, GD3, GDPD3, GNA14, GNAZ, GPR153, GPR56, GYPA, HEPHL1, HER-2, hERT, HILPDA, HLA-DR, HOOK1, hTERT, HTR2A, ICAM1, IGFBP3, IL10RB, IL20RB, IL23R, ILDR1, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), ITFG3, ITGA4, ITGA5, ITGA8, ITGAX, ITGB5, ITGB8, JAM3, KCND1,KCNJ5, KCNK13, KCNN4, KCNV2, KDR, KIF19, KIF26B, κ-light chain, L1CAM, LAX1, LEPR, Lewis Y (CD174), Lewis Y (LeY), LILRA2, LILRA6, LILRB2, LILRB3, LILRB4, LOXL4, LPAR2, LRRC37A3, LRRC8E, LRRN2, LRRTM2, LTB4R, MAGE-A1, MAGEA3, MANSC1, MART1, GP100, MBOAT1, MBOAT7, melanoma antigen family A, mesothelin (MSLN), MFAP3L, MMP25, MRP1, MT-ND1, mucin 1 (MUC1), mucin 16 (MUC16), MYADM, MYADML2, NGFR, NKCS1, NKG2D ligand, NLGN3, NPAS2, NY-ESO-1, oncofetal antigen (h5T4), OTOA, P2RY13, p53, PDE3A, PEAR1, PIEZO1, PLXNA4, PLXNC1, PNPLA3, PPFIA4, PPP2R5B, PRAME, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), proteinase 3 (PR1), PSD2, PTPRJ, RDH16, receptor tyrosine-protein kinase Erb-B2, R HBDL3, RNF173, RNF183, ROR1, RYR2, SCIN, SCN11A, SCN2A, SCNN1D, SEC31B, SEMA4A, SH3PXD2A, SIGLEC11, SIRPB1, SLC16A6, SLC19A1, SL C22A5, SLC25A36, SLC25A41, SLC30A1, SLC34A3, SLC43A3, SLC44A1, SLC44A3, SLC45A3, SLC6A16, SLC6A6, SLC8A3, SLC9A1, SLCO2B1, SPAG 17, STC1, STON2, SUN3, survivin, SUSD2, SYNC, TACSTD2, TAS1R3, TEX29, TFR2, TIM-3 (HAVCR2), TLR2, TMEFF2, TMEM145, TMEM27, TMEM40, TMEM59L, TMEM89, TMPRSS5, TNFRSF14, TNFRSF1B, TRIM55, TSPEAR, TTYH3, tumor-associated glycoprotein 72 (TAG-72), tyrosinase, vascular endothelial growth factor R2 (VEGF-R2), VLA-4,Selected from the group consisting of Wilms tumor protein (WT-1), WNT4, WT1, and ZDHHC11.

[0015] In certain embodiments, the first antigen is selected from the group consisting of CD312, CD19, CD20, CD22, CD276, and CAIX, hi certain embodiments, the second antigen is selected from the group consisting of CD70, CD19, CD20, and CD22.

[0016] In certain embodiments, the first antigen and the second antigen are CD312 and CD70. In certain embodiments, the extracellular antigen-binding domain of a CAR targeting CD312 comprises a VH comprising a CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 73, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 74, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 75, and a VL comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 76, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 77, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 78. In certain embodiments, a TCR-like fusion molecule targeting CD70 comprises a first antigen-binding chain comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 133, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 134, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 135, and a second antigen-binding chain comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 136, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 137, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 138.

[0017] In certain embodiments, the first and second antigens are CD276 and CD70. In certain embodiments, the extracellular antigen-binding domain of a CAR targeting CD276 comprises a VH comprising a CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 85, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 86, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 87, and a VL comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 88, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 89, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 90. In certain embodiments, a TCR-like fusion molecule targeting CD70 comprises a first antigen-binding chain comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 133, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 134, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 135, and a second antigen-binding chain comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 136, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 137, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 138.

[0018] In certain embodiments, the first antigen and the second antigen are CAIX and CD70. In certain embodiments, the first antigen and the second antigen are CD19 and CD22. In certain embodiments, the first antigen and the second antigen are CD19 and CD20. In certain embodiments, the first antigen and the second antigen are CD20 and CD22. In certain embodiments, the first antigen and the second antigen are CD20 and CD19. In certain embodiments, the first antigen and the second antigen are CD22 and CD20.

[0019] In certain embodiments, the first antigen and the second antigen are CD22 and CD 19. In certain embodiments, the extracellular antigen-binding domain of a CAR targeting CD22 comprises: a) a VH comprising a CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 97, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 98, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 99, and a VL comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 100, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 101, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 102; b) a VH comprising a CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 97, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 153, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 99, and a VL comprising a CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 100. a VL comprising a CDR1 having an amino acid sequence set forth in SEQ ID NO: 101, a CDR2 having an amino acid sequence set forth in SEQ ID NO: 102, a VH comprising a CDR1 having an amino acid sequence set forth in SEQ ID NO: 119, a CDR2 having an amino acid sequence set forth in SEQ ID NO: 120, and a CDR3 having an amino acid sequence set forth in SEQ ID NO: 121, and a VL comprising a CDR1 having an amino acid sequence set forth in SEQ ID NO: 122, a CDR2 having an amino acid sequence set forth in SEQ ID NO: 123, and a CDR3 having an amino acid sequence set forth in SEQ ID NO: 124, or d) a VH comprising a CDR1 having an amino acid sequence set forth in SEQ ID NO: 97, a CDR2 having an amino acid sequence set forth in SEQ ID NO: 154, and a CDR3 having an amino acid sequence set forth in SEQ ID NO: 99. H and a V comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 100, CDR2 having the amino acid sequence set forth in SEQ ID NO: 101, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 102. LIn certain embodiments, a TCR-like fusion molecule that targets CD19 comprises a first antigen binding chain comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 143, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 144, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 145, and a second antigen binding chain comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 146, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 147, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 148.

[0020] In certain embodiments, the first antigen and the second antigen are selected from Table 8.

[0021] In certain embodiments, the cell further comprises a chimeric costimulatory receptor (CCR). In certain embodiments, the CCR comprises an extracellular antigen-binding domain that binds to a third antigen and an intracellular domain that can deliver a costimulatory signal to the cell but does not alone deliver an activating signal to the cell. In certain embodiments, the intracellular domain of the CCR comprises at least the intracellular domain of a costimulatory molecule, or a portion thereof. In certain embodiments, the costimulatory molecule is selected from the group consisting of CD28, 4-1BB, OX40, CD27, CD40, CD154, CD97, CD11a / CD18, ICOS, DAP-10, CD2, CD150, CD226, and NKG2D.

[0022] In certain embodiments, the cells further comprise at least one exogenous costimulatory ligand. In certain embodiments, the at least one exogenous costimulatory ligand is selected from the group consisting of a tumor necrosis factor (TNF) family member, an immunoglobulin (Ig) superfamily member, and a combination thereof. In certain embodiments, the TNF family member is selected from the group consisting of 4-1BBL, OX40L, CD70, FasL, GITRL, TNF-related apoptosis-inducing ligand (TRAIL), CD30L, LIGHT (TNFSF14), and CD40L. In certain embodiments, the Ig superfamily member is selected from the group consisting of CD80, CD86, ICOSLG, and a combination thereof. In certain embodiments, the at least one exogenous costimulatory ligand comprises CD80. In certain embodiments, the at least one exogenous costimulatory ligand comprises 4-1BBL. In certain embodiments, the cells comprise two exogenous costimulatory ligands. In certain embodiments, the at least two exogenous costimulatory ligands comprise CD80 and 4-1BBL, hi certain embodiments, the at least two exogenous costimulatory ligands comprise the amino acid sequence set forth in SEQ ID NO:67 and / or the amino acid sequence set forth in SEQ ID NO:69.

[0023] In certain embodiments, the cells further comprise a fusion polypeptide comprising a) the extracellular domain and transmembrane domain of a costimulatory ligand and b) the intracellular domain of a first costimulatory molecule. In certain embodiments, the costimulatory ligand is selected from the group consisting of a tumor necrosis factor (TNF) family member, an immunoglobulin (Ig) superfamily member, and a combination thereof. In certain embodiments, the TNF family member is selected from the group consisting of 4-1BBL, OX40L, CD70, GITRL, CD40L, and a combination thereof. In certain embodiments, the Ig superfamily member is selected from the group consisting of CD80, CD86, ICOSLG, and a combination thereof. In certain embodiments, the costimulatory ligand is CD80. In certain embodiments, the first costimulatory molecule is selected from the group consisting of CD28, 4-1BB, OX40, ICOS, DAP-10, CD27, CD40, NKG2D, CD2, and a combination thereof. In certain embodiments, the first costimulatory molecule is 4-1BB. In certain embodiments, the costimulatory ligand is CD80 and the first costimulatory molecule is 4-1BB. In certain embodiments, the fusion polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 71. In certain embodiments, the fusion polypeptide further comprises the intracellular domain of a second costimulatory molecule. In certain embodiments, the second costimulatory molecule is selected from the group consisting of CD28, 4-1BB, OX40, ICOS, DAP-10, CD27, CD40, NKG2D, CD2, and combinations thereof. In certain embodiments, the second costimulatory molecule is CD28. In certain embodiments, the costimulatory ligand is CD80, the first costimulatory molecule is 4-1BB, and the second costimulatory molecule is CD28. In certain embodiments, the fusion polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 72.

[0024] In certain embodiments, the cells are autologous. In certain embodiments, the cells are allogeneic.

[0025] In certain embodiments, the presently disclosed subject matter provides a composition comprising the cells disclosed herein, hi certain embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable excipient.

[0026] In certain embodiments, the presently disclosed subject matter provides a nucleic acid composition, vector, or lipid nanoparticle comprising a first polynucleotide encoding a chimeric antigen receptor (CAR) that targets a first antigen and a second polynucleotide encoding a TCR-like fusion molecule that targets a second antigen. In certain embodiments, the presently disclosed subject matter provides a vector comprising a first polynucleotide encoding a chimeric antigen receptor (CAR) that targets a first antigen and a second polynucleotide encoding a TCR-like fusion molecule that targets a second antigen. In certain embodiments, the vector is a lentiviral vector. In certain embodiments, the vector is a gamma-retroviral vector.

[0027] In certain embodiments, the presently disclosed subject matter provides polynucleotides encoding a chimeric antigen receptor (CAR) that targets a first antigen and a TCR-like fusion molecule that targets a second antigen. The presently disclosed subject matter also provides vectors and lipid nanoparticles comprising the polynucleotides disclosed herein.

[0028] In certain embodiments, the presently disclosed subject matter provides a composition comprising a polynucleotide, a vector, and a lipid nanoparticle disclosed herein, hi certain embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable excipient.

[0029] In certain embodiments, the presently disclosed subject matter provides methods for producing an immunoresponsive cell as disclosed herein, in certain embodiments, the method comprising introducing into the immunoresponsive cell a nucleic acid composition, polynucleotide, vector, or lipid nanoparticle as disclosed herein.

[0030] In certain embodiments, the method further comprises generating a gene disruption at the CD70 locus. In certain embodiments, the method further comprises generating a gene disruption at the TRAC locus. In certain embodiments, the gene disruption comprises a substitution, deletion, insertion, mutation, or a combination thereof. In certain embodiments, the mutation comprises a missense mutation, a nonsense mutation, or a combination thereof. In certain embodiments, the deletion comprises a non-frameshift deletion, a frameshift deletion, or a combination thereof. In certain embodiments, the insertion comprises a non-frameshift insertion, a frameshift insertion, or a combination thereof. In certain embodiments, the gene disruption at the CD70 locus results in a non-functional CD70 protein or results in knockout of CD70 gene expression. In certain embodiments, the gene disruption at the CD70 locus results in a non-functional TRAC protein or results in knockout of TRAC gene expression.

[0031] In certain embodiments, generating the gene disruption comprises a gene editing method comprising homologous recombination, zinc finger nucleases, meganucleases, transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR) systems, or a combination thereof.

[0032] In certain embodiments, the gene disruption of the CD70 locus or the TRAC locus is generated before activation of the cells. In certain embodiments, the gene disruption of the CD70 locus or the TRAC locus is generated after activation of the cells. In certain embodiments, a) the gene disruption of the CD70 locus is generated before activation of the cells, and b) the gene disruption of the TRAC locus is generated after activation of the cells. In certain embodiments, a) the gene disruption of the TRAC locus is generated before activation of the cells, and b) the gene disruption of the CD70 locus is generated after activation of the cells.

[0033] In certain embodiments, the method further comprises generating a genetic modification of the CD70 gene. In certain embodiments, the method further comprises generating a genetic modification of the TRAC gene and / or the TRBC gene. In certain embodiments, the method further comprises genetic modification of the CD70 gene, resulting in a non-functional CD70 protein or resulting in knockdown of CD70 gene expression. In certain embodiments, the method further comprises genetic modification of the TRAC locus, resulting in a non-functional TRAC protein or resulting in knockdown of TRAC gene expression. In certain embodiments, the method further comprises genetic modification of the TRBC locus, resulting in a non-functional TRBC protein or resulting in knockdown of TRBC gene expression.

[0034] In certain embodiments, the method further comprises introducing a chimeric costimulatory receptor (CCR). In certain embodiments, the method further comprises introducing at least one exogenous costimulatory ligand. In certain embodiments, the method further comprises introducing a fusion polypeptide comprising a) the extracellular domain and transmembrane domain of the costimulatory ligand and b) the intracellular domain of a first costimulatory molecule.

[0035] In certain embodiments, the presently disclosed subject matter provides immunoresponsive cells produced by the methods disclosed herein.

[0036] In certain embodiments, the presently disclosed subject matter provides methods for reducing tumor burden in a subject, treating and / or preventing a neoplasm or tumor, preventing and / or treating a pathogen infection, preventing and / or treating an autoimmune disease, and / or preventing and / or treating an infectious disease using the immunoresponsive cells or compositions disclosed herein.

[0037] In certain embodiments, the neoplasm or tumor is cancer. In certain embodiments, the neoplasm or tumor comprises antigenic heterogeneity of the first antigen and the second antigen. In certain embodiments, the second antigen has low antigen density. In certain embodiments, the second antigen is expressed on tumor cells with low tumor cell frequency. In certain embodiments, the first antigen and the second antigen are independently selected from CD19, CD70, IL1RAP, ABCG2, AChR, ACKR6, ADAMTS13, ADGRE2, ADGRE2 (EMR2), ADORA3, ADRA1D, AGER, ALS2, an antigen of cytomegalovirus (CMV)-infected cells, ANO9, AQP2, ASIC3, ASPRV1, ATP6V0A4, B3GNT4, B7-H3, BCMA, BEST4, C3orf35, CADM3, CAI X, CAPN3, CCDC155, CCR1, CD10, CD117, CD123, CD133, CD135(FLT3), CD138, CD20, CD22, CD244(2B4), CD25, CD26, CD30, CD300LF, C D312, CD32, CD321, CD33, CD34, CD36, CD38, CD41, CD44, CD44V6, CD47, CD49f, CD56, CD7, CD71, CD74, CD8, CD82, CD96, CD98, CD99, CDH13, CDHR1, CEA, CEACAM6, CHST3, CLEC12A, CLEC1A, CLL1, CNIH2, COL15A1, COLEC12, CPM, CR1, CX3CR1, CXCR4, CYP4F11, DAGLB, DARC, DFNB31, DGKI, EGF1R, EGFR-VIII, EGP-2, EGP-40, ELOVL6, EMB, EMC10, EMR2, ENG, EpCAM, EphA2, EPHA4, ERBB, ERBB2, Erb-B3 , Erb-B4, E-selectin, EXOC3L4, EXTL3, FAM186B, FBP, FCGR1A, FKBP1B, FLRT1, folate receptor-α, FOLR2, FRMD5, GABRB2, GAS2, GD2, GD3, GDPD3 , GNA14, GNAZ, GPR153, GPR56, GYPA, HEPHL1, HER-2, hERT, HILPDA, HLA-DR, HOOK1, hTERT, HTR2A, ICAM1, IGFBP3, IL10RB, IL20RB,IL23R, ILDR1, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), ITFG3, ITGA4, ITGA5, ITGA8, ITGAX, ITGB5, ITGB8, JAM3, KCND1, KCNJ5, KCNK13, KCNN4, KCNV2, KDR, KIF19, KIF26B, κ-light chain, L1CAM, LAX1, LEPR, Lewis Y (CD174), Lewis Y (LeY), LILRA2, LILRA6, LILRB2, LILRB3, LILRB4, LOXL4, LPAR2, LRRC37A3, LRRC8E, LRRN2, LRRTM2, LTB4R, MAGE-A1, MAGEA3, MANSC1, MART1, GP100, MBOAT1, MBOAT7, melanoma antigen family A, mesothelin (MSLN), MFAP3L, MMP25, MRP1, MT-ND1, mucin 1 (MUC1), mucin 1 6 (MUC16), MYADM, MYADML2, NGFR, NKCS1, NKG2D ligand, NLGN3, NPAS2, NY-ESO-1, oncofetal antigen (h5T4), OTOA, P2RY13, p53, PDE3A, PEAR1, PIEZO1, PLXNA4, PLXNC1, PNPLA3, PPFIA4, PPP2R5B, PRAME, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), proteinase 3 (P R1), PSD2, PTPRJ, RDH16, receptor tyrosine-protein kinase Erb-B2, RHBDL3, RNF173, RNF183, ROR1, RYR2, SCIN, SCN11A, SCN2A, SCNN1D, SEC31B, SEMA4A, SH3PXD2A, SIGLEC11, SIRPB1, SLC16A6, SLC19A1, SLC22A5, SLC25A36, SLC25A41, SLC30A1, SLC34A3, SLC4 3A3, SLC44A1, SLC44A3, SLC45A3, SLC6A16, SLC6A6, SLC8A3, SLC9A1, SLCO2B1, SPAG17, STC1, STON2, SUN3, Survivin, SUSD2, S YNC, TACSTD2, TAS1R3, TEX29, TFR2, TIM-3(HAVCR2), TLR2, TMEFF2, TMEM145, TMEM27, TMEM40, TMEM59L, TMEM89, TMPRSS5,The first antigen is selected from the group consisting of TNFRSF14, TNFRSFlB, TRIM55, TSPEAR, TTYH3, tumor-associated glycoprotein 72 (TAG-72), tyrosinase, vascular endothelial growth factor R2 (VEGF-R2), VLA-4, Wilms tumor protein (WT-1), WNT4, WT1, and ZDHHC11. In certain embodiments, the first antigen is selected from the group consisting of CD312, CD19, CD20, CD22, CD276, and CAIX. In certain embodiments, the second antigen is selected from the group consisting of CD70, CD19, CD20, and CD22. In certain embodiments, the first antigen and the second antigen are selected from a) CD312 and CD70, b) CD276 and CD70, c) CAIX and CD70, d) CD19 and CD22, e) CD19 and CD20, f) CD20 and CD22, g) CD20 and CD19, h) CD22 and CD20, or i) CD22 and CD19. In certain embodiments, the first antigen and the second antigen are selected from Table 8.

[0038] In certain embodiments, the neoplasm or tumor is a solid tumor. In certain embodiments, the solid tumor is selected from the group consisting of melanoma, renal cell carcinoma, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, lung neuroendocrine carcinoma, small cell lung cancer, pancreatic cancer, breast cancer, astrocytoma, glioblastoma, laryngeal / pharyngeal cancer, EBV-associated nasopharyngeal carcinoma, and ovarian cancer. In certain embodiments, the solid tumor is melanoma.

[0039] In certain embodiments, the neoplasm or tumor is a hematological cancer. In certain embodiments, the neoplasm or tumor is a bone marrow disorder. In certain embodiments, the bone marrow disorder is selected from the group consisting of myelodysplastic syndrome, myeloproliferative neoplasm, chronic myelomonocytic leukemia, or acute myeloid leukemia (AML), blastic plasmacytoid dendritic cell neoplasm, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, chronic myelocytic leukemia, and polycythemia vera. In certain embodiments, the bone marrow disorder is acute myeloid leukemia (AML).

[0040] In certain embodiments, the neoplasm or tumor is a B-cell malignancy. In certain embodiments, the B-cell malignancy is selected from the group consisting of B-cell non-Hodgkin's lymphoma (NHL), B-cell Hodgkin's lymphoma, B-cell acute lymphocytic leukemia (ALL), B-cell chronic lymphocytic leukemia (CLL), multiple myeloma (MM), CLL with Richter's transformation, and CNS lymphoma. In certain embodiments, the B-cell malignancy is B-cell acute lymphocytic leukemia.

[0041] In certain embodiments, the neoplasm or tumor is a leukemia, hi certain embodiments, the leukemia is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute promyelocytic leukemia (APL), mixed phenotype acute leukemia (MLL), hairy cell leukemia, B-cell prolymphocytic leukemia, B-cell precursor acute lymphoblastic leukemia, and T-cell precursor acute lymphoblastic leukemia.

[0042] In certain embodiments, the neoplasm or tumor is a lymphoma, hi certain embodiments, the lymphoma is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell non-Hodgkin's lymphoma, T-cell non-Hodgkin's lymphoma, and T-cell precursor acute lymphoblastic lymphoma.

[0043] In certain embodiments, the subject has a recurrence of a neoplasm or tumor, hi certain embodiments, the subject has undergone treatment that results in residual tumor cells.

[0044] In certain embodiments, the presently disclosed subject matter provides kits comprising the cells or compositions disclosed herein, hi certain embodiments, the kits further comprise written instructions for reducing tumor burden, treating and / or preventing neoplasms or tumors, preventing and / or treating pathogen infection, preventing and / or treating autoimmune diseases, and / or preventing and / or treating infectious diseases.

[0045] The following detailed description is given by way of example and is not intended to limit the subject matter disclosed herein to the particular embodiments described, and may be understood in conjunction with the accompanying drawings, in which: [Brief explanation of the drawings]

[0046] [Figure 1] 1 shows a FACS analysis of a TCR-like fusion molecule ("70HIT") targeting CD70 expressed from the TRAC locus ("TRAC-70HIT") under the control of the endogenous TRAC promoter or expressed from an SFG vector ("SFG-70HIT"). [Figure 2A] 1 shows the effect of TRAC-70HIT and SFG-70HIT on a MOLM13 AML xenograft model. 2 shows bioluminescence-based tumor quantification. [Figure 2B] 1 shows the effect of TRAC-70HIT and SFG-70HIT on a MOLM13 AML xenograft model. Survival curves are shown. [Figure 3A] 1 shows the effect of SFG-70HIT in a patient-derived AML xenograft model. Analysis of peripheral blood cells by FACS analysis and AML load. [Figure 3B] Figure 1 shows the effect of SFG-70HIT in a patient-derived AML xenograft model. Total T cell counts are shown. [Figure 3C-1]

[0033] Figure 1 shows the effect of SFG-70HIT in a patient-derived AML xenograft model. FACS analysis is shown 20 days after injection. [Figure 3C-2] Same as above. [Figure 4] 1 shows FACS analysis of MOL13 wild-type and edited cells to model target heterogeneity in AML. [Figure 5A] Figure 1 shows the effect of 70H_312C-28z1XX in an AML heterogeneity model. Figure 2 shows FACS analysis of an AML heterogeneity model. [Figure 5B] Figure 1 shows the effect of 70H_312C-28z1XX in an AML heterogeneity model. Bioluminescence-based tumor quantification is shown. [Figure 5C] Figure 1 shows the effect of 70H_312C-28z1XX in an AML heterogeneity model. Survival curves are shown. [Figure 6A] Figure 1 shows the effect of 70H_312C-28z1XX in an AML heterogeneity model compared to alternative HIT+CAR designs. Figure 2 shows a FACS analysis of an AML heterogeneity model. [Figure 6B] 1 shows the efficacy of 70H_312C-28z1XX in an AML heterogeneity model compared to alternative HIT+CAR designs. 2 shows bioluminescence-based tumor quantification. [Figure 6C] 1 shows the effect of 70H_312C-28z1XX in an AML heterogeneity model compared to alternative HIT+CAR designs. Survival curves are shown. [Figure 7A] Figure 1 shows the effect of 70H_312C-28z1XX in an AML heterogeneity model compared to an alternative dual CAR approach. Figure 2 shows a FACS analysis of an AML heterogeneity model. [Figure 7B] Figure 1 shows the effect of 70H_312C-28z1XX in an AML heterogeneity model compared to an alternative dual CAR approach. Bioluminescence-based tumor quantification is shown. [Figure 7C] 1 shows the effect of 70H_312C-28z1XX in an AML heterogeneity model compared to an alternative dual CAR approach. Survival curves are shown. [Figure 8A-1] Figure 1 shows that SFG-70HIT+312CAR-1XX is associated with a less differentiated T cell phenotype compared to CAR+CAR. CD8 differentiation is shown. [Figure 8A-2] Same as above. [Figure 8A-3] Same as above. [Figure 8B-1] Figure 1 shows that SFG-70HIT+312CAR-1XX is associated with a less differentiated T cell phenotype compared to CAR+CAR. CD4 differentiation is shown. [Figure 8B-2] Same as above. [Figure 8B-3] Same as above. [Figure 9A-1]Figure 1 shows that SFG-70HIT+312CAR-1XX is effective across a range of xenophenotypes. FACS analysis of CD70 and CD312 in the different tested xenophenotypes is shown. [Figure 9A-2] Same as above. [Figure 9A-3] Same as above. [Figure 9B-1] SFG-70HIT+312CAR-1XX is effective across a range of heterogeneous phenotypes. Bioluminescence-based tumor quantification is shown. [Figure 9B-2] Same as above. [Figure 9B-3] Same as above. [Figure 9C-1] SFG-70HIT+312CAR-1XX is effective across a range of heterogeneous phenotypes. Survival curves are shown. [Figure 9C-2] Same as above. [Figure 9C-3] Same as above. [Figure 10A] This paper presents the editing strategy for the production of the 70H+312C platform. It also shows the current standard. [Figure 10B] The editing strategy for the fabrication of the 70H+312C platform is shown. The first alternative strategy involves electroporation on day 0. [Figure 10C] Figure 1 shows the editing strategy for the production of the 70H+312C platform. Figure 2 shows the sequential editing strategy to avoid translocations. [Figure 11A-1] Figure 1 shows the effect of different editing strategies for the production of the 70H+312C platform on AML tumor models. Figure 2 shows the effect on the MOLM13-WT model. [Figure 11A-2] Same as above. [Figure 11B-1] Figure 1 shows the effect of different editing strategies for the production of the 70H+312C platform on an AML tumor model. Figure 2 shows the effect on an AML heterogeneity model. [Figure 11B-2] Same as above. [Figure 12A]Figure 1 shows the effect of the HIT+CAR strategy in a solid tumor model. Figure 2 shows FACS analysis of CD70 and CD276 in the human melanoma cell line SK-MEL37. [Figure 12B] 1 shows the efficacy of the HIT+CAR strategy in a solid tumor model. 2 shows the efficacy in a lung metastatic melanoma model. [Figure 12C] Figure 1 shows the efficacy of the HIT+CAR strategy in a solid tumor model. Figure 2 shows the efficacy in an orthotopic cutaneous melanoma model. [Figure 12D] Figure 1 shows the effect of HIT+CAR strategy in a solid tumor model. Figure 2 shows FACS analysis of an AML heterogeneity model. [Figure 13A] Efficacy of the HIT+CAR strategy in a renal cell carcinoma model. Efficacy in a K5 orthotopic kidney tumor model. [Figure 13B] Shows the efficacy of the HIT+CAR strategy in a renal cell carcinoma model. Shows efficacy in a K7 orthotopic kidney tumor model. [Figure 13C] 1 shows the efficacy of a HIT+CAR strategy in a renal cell carcinoma model. 2 shows bioluminescence-based tumor quantification of K5 and K7 tumor models. [Figure 14A-1] 1 shows the efficacy of the cells (HIT+CAR) disclosed herein in the setting of B-ALL. Analyses are shown of Nalm6 xenograft cells with wild-type (WT) levels of CD22 and CD19, a first Nalm6 xenograft heterogeneous cell line comprising Nalm6 cells knocked out for CD19 (Nalm6-19KO) and CD22 (Nalm6-22KO) at a 1:1:1 ratio, and a second Nalm6 xenograft heterogeneous cell line comprising WT, Nalm6-19KO, and Nalm6-22KO at a 1:1:1 ratio. [Figure 14A-2] Same as above. [Figure 14B-1]1 shows the efficacy of the cells disclosed herein (HIT+CAR) in the setting of B-ALL. Survival curves are shown for animals challenged with the above-described Nalm6 xenograft cells and receiving 5E5 T cells containing a HIT receptor targeting CD19 and a 1XX CAR targeting CD22 (19-HIT+22-CAR-1XX), or a first CAR targeting CD19 and containing the 4-1BB / CD3zeta intracellular domain and a 1XX CAR targeting CD22 (19-CAR-BBz+22-CAR-1XX). [Figure 14B-2] Same as above. [Figure 14C-1]

[0023] Figure 1 shows the efficacy of the cells disclosed herein (HIT+CAR) in the setting of B-ALL. Analysis of Nalm6 xenograft cells is shown, with the indicated percentage values ​​for WT cells (WT), Nalm6-19KO cells (19-KO), and Nalm6-22KO cells (22-KO). [Figure 14C-2] Same as above. [Figure 14D-1] 14C shows the efficacy of the cells disclosed herein (HIT+CAR) in the setting of B-ALL. Figure 14D shows the survival curves for animals receiving 5E5 T cells containing Nalm6 xenograft cells and either 19-HIT+22-CAR-1XX or 19-CAR-BBz+22-CAR-1XX. [Figure 14D-2] Same as above. [Figure 15-1] 1 shows survival curves for animals receiving 1E6 T cells containing Nalm6 xenograft cells as described above and including 19-HIT+22-CAR-1XX, 19-CAR-BBz+22-CAR-1XX, or T cells expressing a single receptor. [Figure 15-2] Same as above. [Figure 16A]Figure 1 shows the phenotypic and biochemical characterization of T cells in mouse models receiving 19-HIT+22-CAR-1XX or 19-CAR-BBz+22-CAR-1XX. Figure 2 shows an analysis of tumor burden measured by bioluminescence imaging in animals challenged with 1:1:1 Nalm6 tumors and receiving different doses (5E5 or 1E6) of T cells containing 19-HIT+22-CAR-1XX or 19-CAR-BBz+22-CAR-1XX. [Figure 16B] Figure 1 shows the phenotypic and biochemical characterization of T cells in mouse models receiving 19-HIT+22-CAR-1XX or 19-CAR-BBz+22-CAR-1XX. Quantification of Nalm6-GFP+ expressing CD19 and / or CD22 in bone marrow 10 days after administration of 5E5 T cells with 19-HIT+22-CAR-1XX (H+C) or 19-CAR-BBz+22-CAR-1XX (C+C) is shown. [Figure 16C] Figure 1 shows the phenotypic and biochemical characterization of T cells in mouse models receiving 19-HIT+22-CAR-1XX or 19-CAR-BBz+22-CAR-1XX. Quantification of Nalm6-GFP+ expressing CD19 and / or CD22 in bone marrow 16 days after administration of 5E5 T cells with 19-HIT+22-CAR-1XX (H+C) or 19-CAR-BBz+22-CAR-1XX (C+C) is shown. [Figure 16D] Figure 1 shows the phenotypic and biochemical characterization of T cells in mouse models receiving 19-HIT+22-CAR-1XX or 19-CAR-BBz+22-CAR-1XX. CD8+ and CD4+ T cell counts in the bone marrow and spleen of mice challenged with 1:1:1 Nalm6 tumors 10 and 16 days after administration of 5E5 T cells containing 19-HIT+22-CAR-1XX (H+C) or 19-CAR-BBz+22-CAR-1XX (C+C). [Figure 16E]Figure 1 shows the phenotypic and biochemical characterization of T cells in mouse models receiving 19-HIT+22-CAR-1XX or 19-CAR-BBz+22-CAR-1XX. Figure 1 shows the cytophenotyping of CD8+ T cells in the bone marrow and spleen of mice challenged with 1:1:1 Nalm6 tumors 10 and 16 days after administration of 5E5 T cells containing 19-HIT+22-CAR-1XX (H+C) or 19-CAR-BBz+22-CAR-1XX (C+C). [Figure 16F] Figure 1 shows the phenotypic and biochemical characterization of T cells in mouse models receiving 19-HIT+22-CAR-1XX or 19-CAR-BBz+22-CAR-1XX. Figure 2 shows the cytophenotyping of CD4+ T cells in the bone marrow and spleen of mice challenged with 1:1:1 Nalm6 tumors 10 and 16 days after administration of 5E5 T cells containing 19-HIT+22-CAR-1XX (H+C) or 19-CAR-BBz+22-CAR-1XX (C+C). [Figure 16G] Figure 1 shows the phenotypic and biochemical characterization of T cells in mouse models receiving 19-HIT+22-CAR-1XX or 19-CAR-BBz+22-CAR-1XX. Figure 2 shows the cell depletion phenotyping of CD8+ T cells in the bone marrow and spleen of mice challenged with 1:1:1 Nalm6 tumors 10 and 16 days after administration of 5E5 T cells containing 19-HIT+22-CAR-1XX (H+C) or 19-CAR-BBz+22-CAR-1XX (C+C). [Figure 16H] Figure 1 shows the phenotypic and biochemical characterization of T cells in mouse models receiving 19-HIT+22-CAR-1XX or 19-CAR-BBz+22-CAR-1XX. Figure 2 shows the cell depletion phenotyping of CD4+ T cells in the bone marrow and spleen of mice challenged with 1:1:1 Nalm6 tumors 10 and 16 days after administration of 5E5 T cells containing 19-HIT+22-CAR-1XX (H+C) or 19-CAR-BBz+22-CAR-1XX (C+C). [Figure 16I]Figure 1 shows the phenotypic and biochemical characterization of T cells in mouse models receiving 19-HIT+22-CAR-1XX or 19-CAR-BBz+22-CAR-1XX. Expression of cell exhaustion markers in CD8+ T cells in the bone marrow and spleen of mice challenged with 1:1:1 Nalm6 tumors 10 and 16 days after administration of 5E5 T cells containing 19-HIT+22-CAR-1XX (H+C) or 19-CAR-BBz+22-CAR-1XX (C+C) is shown. [Figure 16J] Figure 1 shows the phenotypic and biochemical characterization of T cells in mouse models receiving 19-HIT+22-CAR-1XX or 19-CAR-BBz+22-CAR-1XX. Expression of cell depletion markers in CD4+ T cells in the bone marrow and spleen of mice challenged with 1:1:1 Nalm6 tumors 10 and 16 days after administration of 5E5 T cells containing 19-HIT+22-CAR-1XX (H+C) or 19-CAR-BBz+22-CAR-1XX (C+C) is shown. DETAILED DESCRIPTION OF THE INVENTION

[0047] The presently disclosed subject matter provides compositions, e.g., modified immune cells, useful for immunotherapy (e.g., T cell immunotherapy). The compositions, e.g., modified immune cells, disclosed herein include (1) a chimeric antigen receptor (CAR) that targets a first antigen and (2) a TCR-like fusion molecule that targets a second antigen. The presently disclosed subject matter also provides methods for producing such compositions and methods of using such compositions to treat and / or prevent tumors (e.g., cancer, e.g., solid tumors or hematological cancers, e.g., bone marrow disorders, e.g., acute myeloid leukemia (AML)). The presently disclosed subject matter is based, at least in part, on the discovery that an OR-gated targeting approach involving HITs and CARs can address antigen heterogeneity (i.e., the presence of a tumor cell population with a mixed phenotype, including tumor cells with either low antigen density or low tumor cell frequency). Co-expression of a HIT and a CAR directed against two independent target antigens can enhance at least one activity of a cell, such as cytotoxicity, cell proliferation, and / or cell persistence. Furthermore, co-expression of a HIT and a CAR reduces cumulative costimulation that drives excessive T cell differentiation and limits functional persistence.

[0048] Non-limiting embodiments of the presently disclosed subject matter are illustrated by the specification and examples.

[0049] For clarity of disclosure, and not by way of limitation, this detailed description is divided into the following subsections. 1. Definition, 2.Cells, 3. Nucleic acid compositions and vectors, 4. Formulation and Administration 5. Treatment method, 6. Kits, and 7. Exemplary embodiments.

[0050] 1.Definition Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art. The following references provide those skilled in the art with general definitions of many of the terms used in the subject matter disclosed herein: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994), The Cambridge Dictionary of Science and Technology (Walker ed., 1988), The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991), and Hale & Marham, The Harper Collins Dictionary of Biology (1991).

[0051] As used herein, the term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 3 or more than 3 standard deviations, in accordance with convention in the art. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude of a value, e.g., within 5-fold or within 2-fold.

[0052] As used herein, a "costimulatory molecule" refers to a cell surface molecule other than an antigen receptor or its ligand that can provide an efficient response of a lymphocyte to an antigen. In certain embodiments, a costimulatory molecule can provide optimal lymphocyte activation.

[0053] As used herein, a "costimulatory ligand" refers to a molecule that, upon binding to its receptor (e.g., a costimulatory molecule), generates a costimulatory response, e.g., an intracellular response that produces the stimulation provided when an antigen-recognizing receptor (e.g., a chimeric antigen receptor (CAR)) binds to its target antigen.

[0054] "Immunoresponsive cell" refers to a cell that functions in an immune response or a precursor, or progeny thereof. In certain embodiments, an immunoresponsive cell is a cell of the lymphoid lineage. Non-limiting examples of cells of the lymphoid lineage include T cells, natural killer (NK) cells, B cells, and stem cells from which lymphoid cells can differentiate. In certain embodiments, an immunoresponsive cell is a cell of the myeloid lineage.

[0055] "Activating an immunoresponsive cell" refers to the induction of intracellular signal transduction or changes in protein expression that result in the initiation of an immune response. For example, clustering of CD3 chains in response to ligand binding and immunoreceptor tyrosine-based inhibitory motifs (ITAMs) generates a signaling cascade. In certain embodiments, binding of an endogenous TCR or an exogenous CAR to an antigen results in the formation of an immunological synapse, which involves the clustering of many molecules near the bound receptor (e.g., CD4 or CD8, CD3γ / δ / ε / ζ, etc.). This clustering of membrane-bound signaling molecules allows the ITAM motifs contained within the CD3 chains to become phosphorylated. This phosphorylation, in turn, initiates the T cell activation pathway, ultimately activating transcription factors such as NF-κB and AP-1. These transcription factors induce global gene expression in T cells, increasing IL-2 production for proliferation and expression of master regulator T cell proteins, thereby initiating a T cell-mediated immune response.

[0056] "Stimulating immunoresponsive cells" refers to signals that result in a robust and sustained immune response. In various embodiments, this occurs after activation of immune cells (e.g., T cells) or is mediated simultaneously through receptors including, but not limited to, CD28, CD137 (4-1BB), OX40, CD40, ICOS, DAP-10, CD27, NKG2D, CD2, CD150, and CD226. Receiving multiple stimulatory signals can be important for mounting a robust and long-lasting T cell-mediated immune response. T cells can quickly become inhibited and unresponsive to antigen. While the effects of these costimulatory signals can vary, they generally result in increased gene expression to generate long-lived, proliferative, anti-apoptotic T cells that strongly respond to antigen for complete and sustained eradication.

[0057] As used herein, the term "antigenic heterogeneity" refers to the differential expression of numerous antigens (e.g., tumor antigens, e.g., CD70, CD312) that result in variation in tumor cell phenotype and distribution of tumor antigen-positive cells.

[0058] As used herein, the term "low antigen density" refers to target molecules (e.g., antigens) having a cell surface density of less than about 5,000 molecules per cell. In certain embodiments, low antigen density refers to a cell surface density of less than about 4,000 molecules per cell, less than about 3,000 molecules per cell, less than about 2,000 molecules per cell, less than about 1,500 molecules per cell, less than about 1,000 molecules per cell, less than about 500 molecules per cell, less than about 200 molecules per cell, or less than about 100 molecules per cell. In certain embodiments, low antigen density refers to a cell surface density of less than about 2,000 molecules per cell. In certain embodiments, low antigen density refers to a cell surface density of less than about 1,500 molecules per cell. In certain embodiments, low antigen density refers to a cell surface density of less than about 1,000 molecules per cell. In certain embodiments, low antigen density is a cell surface density of about 4,000 molecules per cell to about 2,000 molecules per cell, about 2,000 molecules per cell to about 1,000 molecules per cell, about 1,500 molecules per cell to about 1,000 molecules per cell, about 2,000 molecules per cell to about 500 molecules per cell, about 1,000 molecules per cell to about 200 molecules per cell, or about 1,000 molecules per cell to about 100 molecules per cell.

[0059] As used herein, the term "low tumor cell frequency" refers to target cells having a target cell frequency of less than about 50% per tumor. In certain embodiments, a low tumor cell frequency is less than about 40% per tumor, less than about 30% per tumor, less than about 20% per tumor, less than about 15% per tumor, less than about 10% per tumor, less than about 5% per tumor, less than about 2% per tumor, or less than about 1% per tumor. In certain embodiments, a low tumor cell frequency is less than about 2% per tumor. In certain embodiments, a low tumor cell frequency is less than about 1.5% per tumor. In certain embodiments, a low tumor cell frequency is less than about 1% per tumor. In certain embodiments, low tumor cell frequency is between about 40% per tumor and about 20% per tumor, between about 20% per tumor and about 10% per tumor, between about 15% per tumor and about 10% per tumor, between about 20% per tumor and about 5% per tumor, between about 10% per tumor and about 2% per tumor, or between about 10% per tumor and about 1% per tumor.

[0060] As used herein, the term "antigen-recognizing receptor" refers to a receptor that can activate an immune cell or immunoresponsive cell (e.g., a T cell) in response to its binding to an antigen.

[0061] As used herein, the term "antibody" refers not only to intact antibody molecules but also to fragments of antibody molecules that retain immunogen-binding ability. Such fragments are also well known in the art and are commonly used both in vitro and in vivo. Thus, as used herein, the term "antibody" refers not only to intact immunoglobulin molecules but also to the well-known active fragments F(ab')2 and Fab. F(ab')2 and Fab fragments, which lack the Fe fragment of intact antibodies, are cleared from the circulation more rapidly and may exhibit less nonspecific tissue binding than intact antibodies (Wahl et al., J. Nucl. Med. 24:316-325 (1983). As used herein, antibodies include natural whole antibodies, bispecific antibodies, chimeric antibodies, Fab, Fab', single-chain variable fragments (scFv), fusion polypeptides, and non-traditional antibodies. In certain embodiments, antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (referred to herein as V H ) and heavy chain constant (C H The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (referred to herein as V L ) and light chain constant C L The light chain constant region consists of one domain, C L It consists of V H Area and V L The regions can be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). H and V Lis composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0062] As used herein, "CDR" is defined as the complementarity-determining region amino acid sequence of an antibody, which is the hypervariable region of an immunoglobulin heavy chain and light chain. See, e.g., Kabat et al., "Sequences of Proteins of Immunological Interest," 4th USDapartment of Health and Human Services, National Institutes of Health (1987). Generally, an antibody contains three heavy chain and three light chain CDRs or CDR regions in the variable region. CDRs provide the majority of contact residues for antibody binding to an antigen or epitope. In certain embodiments, CDR regions are delineated using the Kabat system (Kabat, EA, et al. (1991) "Sequences of Proteins of Immunological Interest," Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242). In certain embodiments, the CDR regions are delineated using the PyIgClassify system (Adolf-Bryfogle et al., Nucleic acids research 43.D1(2015):D432-D438).

[0063] As used herein, the term "linker" is intended to mean a functional group (e.g., chemical or polypeptide) that covalently bonds two or more polypeptides or nucleic acids such that they are connected to one another. As used herein, a "peptide linker" is a peptide linker that couples two proteins together (e.g., V H and V L In certain embodiments, the linker is a G4S linker. In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1, as provided below: GGGGSGGGGSGGGGS [SEQ ID NO: 1]

[0064] In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO:2, provided below: GGGGSGGGGSGGGSGGGGS [SEQ ID NO: 2]

[0065] In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO:3, provided below: GGGGSGGGGSGGGGSGGGSGGGGS [SEQ ID NO: 3]

[0066] In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO:4, provided below: GGGGSGGGGSGGGGSGGGGSGGGSGGGGS [SEQ ID NO: 4]

[0067] In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO: 5, provided below: GGGGS [SEQ ID NO: 5]

[0068] In certain embodiments, the linker comprises or consists of the amino acid sequence set forth in SEQ ID NO:6, provided below: GGGGSGGGGS [SEQ ID NO: 6]

[0069] As used herein, the term "single-chain variable fragment" or "scFv" refers to a V H ::V L Covalently linked immunoglobulin heavy chains (V) to form heterodimers H ) and light chain (V L ) is a fusion protein of the variable region of V H and V L are either directly joined or joined by a peptide-encoding linker (e.g., 10, 15, 20, 25 amino acids), and V H N-terminus of V L or V H The C-terminus of V L The linker is usually rich in glycine for flexibility and rich in serine or threonine for solubility. Despite the removal of the constant region and the introduction of the linker, the scFv protein retains the specificity of the original immunoglobulin. Single-chain Fv polypeptide antibodies are composed of V, V, VH ... H and V LThe polypeptide can be expressed from a nucleic acid containing a sequence encoding the polypeptide. See also U.S. Patent Nos. 5,091,513, 5,132,405, and 4,956,778, and U.S. Patent Publication Nos. 2005 / 0196754 and 2005 / 0196754. Antagonist scFvs with inhibitory activity have been described (e.g., Zhao et al., Hyrbidoma (Larchmt) 2008 27(6):455-51; Peter et al., J Cachexia Sarcopenia Muscle 2012 August 12; Shieh et al., J Imunol 2009 183(4):2277-85; Giomarelli et al., Thromb Haemost 2007 97(6):955-63; Fife et al., J Clin Invst 2006 116(8):2252-61; Brocks et al., Immunotechnology 1997 3(3):173-84; Moosmayer et al., Ther Immunol 1995 2 (10:31-40). Agonist scFvs with stimulatory activity have been described (see, e.g., Peter et al., J Bioi Chern 2003 25278(38):36740-7; Xie et al., Nat Biotech 1997 15(8):768-71; Ledbetter et al., Crit Rev Immunol 1997 17(5-6):427-55; Ho et al., BioChim Biophys Acta 2003 1638(3):257-66).

[0070] As used herein, the term "affinity" refers to a measure of binding strength. Affinity may depend on the closeness of the stereochemical compatibility between the antibody binding site and the antigenic determinant, the size of the contact area between them, and / or the distribution of charged and hydrophobic groups. As used herein, the term "affinity" also includes "avidity," which refers to the strength of antigen-antibody binding after the formation of a reversible complex. Methods for calculating the affinity of an antibody for an antigen are known in the art, and include, but are not limited to, various antigen binding experiments, such as functional assays (e.g., flow cytometry assays).

[0071] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a molecule comprising an extracellular antigen-binding domain fused to an intracellular signaling domain capable of activating or stimulating an immune cell or immunoresponsive cell, and a transmembrane domain. In certain embodiments, the extracellular antigen-binding domain of a CAR comprises an scFv. An scFv can be derived by fusing the variable heavy and variable light regions of an antibody. Alternatively or additionally, an scFv can be derived from a Fab (e.g., obtained from a Fab library instead of from an antibody). In certain embodiments, an scFv is fused to a transmembrane domain and then to an intracellular signaling domain. In certain embodiments, a CAR is selected to have high binding affinity or avidity for an antigen.

[0072] As used herein, the terms "substantially identical" or "substantially homologous" refer to a polypeptide or nucleic acid molecule that exhibits at least about 50% identity or homology to a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or a reference nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). In certain embodiments, such a sequence is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% identical or homologous to the amino acid or nucleic acid sequence used for comparison.

[0073] Sequence identity is measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, the BLAST program may be used, with a probability score of e-3 to e-100 indicating closely related sequences.

[0074] The percent homology between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent homology between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) incorporated into the GAP program in the GCG software package (available at www.gcg.com) using either a Blossum 62 matrix or a PAM250 matrix, gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6. Additionally or alternatively, the amino acid sequences of the subject matter disclosed herein can be further used as a "query sequence" to perform searches against public databases, for example, to identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. To obtain amino acid sequences homologous to particular sequences disclosed herein (e.g., heavy and light chain variable region sequences), BLAST protein searches can be performed with the XBLAST program, score=50, word length=3. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0075] As used herein, the term "conservative sequence modification" refers to an amino acid modification that does not significantly affect or change the binding properties of the antigen-recognizing receptor (e.g., the extracellular antigen-binding domain of a CAR) disclosed herein, including the amino acid sequence. Conservative modifications can include amino acid substitutions, additions, and deletions. Modifications can be introduced into the extracellular antigen-binding domain of a CAR of the present disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids can be classified into groups according to physicochemical properties, such as charge and polarity. A conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid from the same group. For example, amino acids can be classified by charge, with positively charged amino acids including lysine, arginine, and histidine, negatively charged amino acids including aspartic acid and glutamic acid, and neutrally charged amino acids including alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Additionally, amino acids can be classified by polarity, with polar amino acids including arginine (basic polar), asparagine, aspartic acid (acidic polar), glutamic acid (acidic polar), glutamine, histidine (basic polar), lysine (basic polar), serine, threonine, and tyrosine, and nonpolar amino acids including alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Thus, one or more amino acid residues in a CDR region can be replaced with other amino acid residues from the same group, and the altered antibody can be tested for retained function (i.e., the functions set forth in (c)-(l) above) using the functional assays described herein. In certain embodiments, no more than one, no more than two, no more than three, no more than four, or no more than five residues in a designated sequence or CDR region are altered.

[0076] By "disease" is meant any condition, disease, or disorder that damages or interferes with the normal function of a cell, tissue, or organ, for example, neoplasms and pathogenic infection of cells.

[0077] "Effective amount" means an amount sufficient to have a therapeutic effect. In certain embodiments, an "effective amount" is an amount sufficient to prevent, ameliorate, or inhibit the continued proliferation, growth, or metastasis (e.g., invasion or migration) of a neoplasm.

[0078] "Endogenous" means a nucleic acid molecule or polypeptide that is normally expressed in a cell or tissue.

[0079] "Exogenous" refers to a nucleic acid molecule or polypeptide that is not endogenously present in a cell. Thus, the term "exogenous" will encompass foreign, heterologous, and any recombinant nucleic acid molecule or polypeptide expressed in a cell, such as overexpressed nucleic acid molecules and polypeptides. An "exogenous" nucleic acid refers to a nucleic acid that is not present in a native, wild-type cell; for example, an exogenous nucleic acid may differ from its endogenous counterpart by sequence, position / location, or both. For clarity, an exogenous nucleic acid may have the same or a different sequence compared to its native, endogenous counterpart, may be introduced into the cell itself or its precursor by genetic engineering, and may optionally be linked to alternative regulatory sequences, such as a non-native promoter or secretory sequence.

[0080] By "increase" is meant a positive change of at least about 5%. The change may be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, about 100%, or more.

[0081] By "decreasing" is meant a negative change of at least about 5%. The change may be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or even about 100%.

[0082] The terms "isolated," "purified," or "biologically pure" refer to material that is free to varying degrees from components that normally accompany it as found in its native state. "Isolated" refers to a degree of separation from the original source or surroundings. "Purified" refers to a degree of separation greater than isolation. A "purified" or "biologically pure" protein is sufficiently free from other substances so that any impurities do not substantially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide is purified when it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically measured using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term "purified" can indicate that a nucleic acid or protein yields essentially one band in an electrophoretic gel. For proteins that can be subjected to modifications, such as phosphorylation or glycosylation, different modifications can yield different isolated proteins that can be purified separately.

[0083] The term "isolated cell" means a cell that is separated from molecules and / or cellular components that naturally accompany the cell.

[0084] As used herein, the term "antigen-binding domain" refers to a domain that is capable of specifically binding to a particular antigenic determinant or set of antigenic determinants present on a cell.

[0085] "Neoplasm" or "malignancy" refers to a disease characterized by the pathological proliferation of cells or tissues and their subsequent migration or invasion into other tissues or organs. Neoplastic growth is typically uncontrolled and progressive, occurring under conditions that do not induce or cause the cessation of normal cell proliferation. Neoplasms can affect a variety of cell types, tissues, or organs, including, but not limited to, the bladder, bone, brain, breast, cartilage, glia, esophagus, fallopian tubes, gallbladder, heart, intestine, kidney, liver, lung, lymph nodes, nervous tissue, ovaries, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, urinary tract, ureter, urethra, uterus, and vagina, or organs selected from these tissues or cell types. Neoplasms include cancers, such as sarcomas, carcinomas, or plasmacytomas (malignant tumors of plasma cells). In certain embodiments, the neoplasm is cancer.

[0086] By "specifically binds" is meant a polypeptide or fragment thereof that recognizes and binds to a biological molecule (e.g., a polypeptide) of interest, but does not substantially recognize and bind to other molecules in a sample, e.g., a biological sample, which naturally includes the polypeptides disclosed herein.

[0087] As used herein, the term "tumor antigen" refers to an antigen (e.g., a polypeptide) that is uniquely or differentially expressed on tumor cells compared to normal or non-neoplastic cells. In certain embodiments, a tumor antigen includes any polypeptide expressed by a tumor that can activate or induce an immune response via an antigen-recognizing receptor, or that can suppress an immune response via receptor-ligand binding.

[0088] The terms "comprises" and "comprising" are intended to have the broad meaning ascribed to them in U.S. patent law and may mean "includes," "including," etc.

[0089] As used herein, the term "treatment" refers to clinical intervention in an attempt to alter the disease course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. The therapeutic effect of treatment includes, but is not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and remission or improved prognosis. By preventing the progression of a disease or disorder, treatment can not only prevent deterioration due to the disorder in a subject affected or diagnosed with, or suspected of having, the disorder, but also prevent the onset of the disorder or symptoms of the disorder in a subject at risk of, or suspected of having, the disorder.

[0090] As used herein, an "individual" or "subject" refers to a vertebrate, e.g., a human or a non-human animal, e.g., a mammal. Mammals include, but are not limited to, humans, primates, farm animals, sport animals, rodents, and pets. Non-limiting examples of non-human animal subjects include rodents, such as mice, rats, and hamsters, and non-human primates, such as guinea pigs, rabbits, dogs, cats, sheep, pigs, goats, cows, horses, and apes and monkeys. As used herein, the term "immunocompromised" refers to a subject with an immunodeficiency. The subject is highly vulnerable to opportunistic infections, infections caused by organisms that do not normally cause disease in humans with healthy immune systems but may affect people with dysfunctional or suppressed immune systems.

[0091] As used herein, a "functional fragment" of a molecule or polypeptide includes a fragment of a molecule or polypeptide that retains at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% of the primary function of the molecule or polypeptide.

[0092] Other aspects of the presently disclosed subject matter are described in the disclosure that follows and are within the scope of the presently disclosed subject matter.

[0093] 2.Cells The presently disclosed subject matter provides a cell comprising: a) a first antigen-recognizing receptor that targets a first antigen; and b) a second antigen-recognizing receptor that targets a second antigen.

[0094] In certain embodiments, the cell is selected from the group consisting of a cell of the lymphoid lineage and a cell of the myeloid lineage. In certain embodiments, the cell is an immunoresponsive cell. In certain embodiments, the immunoresponsive cell is a cell of the lymphoid lineage.

[0095] In certain embodiments, the cells are lymphoid lineage cells. Lymphoid lineage cells can provide antibody production, regulation of the cellular immune system, detection of foreign substances in the blood, detection of cells foreign to the host, etc. Non-limiting examples of lymphoid lineage cells include T cells, natural killer (NK) cells, B cells, dendritic cells, and stem cells from which lymphoid cells can be differentiated. In certain embodiments, the stem cells are pluripotent stem cells (e.g., embryonic stem cells).

[0096] In certain embodiments, the cell is a T cell. T cells can be lymphocytes that mature in the thymus and are primarily involved in cell-mediated immunity. T cells participate in the adaptive immune system. T cells of the presently disclosed subject matter include helper T cells, cytotoxic T cells, memory T cells (central memory T cells, stem cell-like memory T cells (or stem-like memory T cells)), as well as two types of effector memory T cells: e.g., T EM Cells and T EMRAThe T cells can be any type of T cell, including, but not limited to, CD4 T cells, regulatory T cells (also known as suppressor T cells), tumor-infiltrating lymphocytes (TIL), natural killer T cells, mucosal-associated invariant T cells, and γδ T cells. Cytotoxic T cells (CTL or killer T cells) are a subset of T lymphocytes that can induce the death of infected somatic cells or tumor cells. The patient's own T cells may be genetically modified to target specific antigens through the introduction of antigen-recognition receptors, such as CAR or TCR. T cells can be CD4 + T cells or CD8 + In certain embodiments, the T cells are CD4 + In certain embodiments, the T cells are CD8 + In certain embodiments, CD8 + The T cells are CD4-independent. In certain embodiments, the T cells are derived from induced pluripotent stem cells (iPSCs). In certain embodiments, the T cells are CD8 + T cells, CD8 + T cells are derived from iPSCs.

[0097] In certain embodiments, the T cells are CD62L + In certain embodiments, the T cells are CD45RA + In certain embodiments, the T cells are CD62L + / CD45RA + T cells.

[0098] In certain embodiments, the cells are NK cells. Natural killer (NK) cells are lymphocytes that are part of cell-mediated immunity and act during innate immune responses. NK cells do not require prior activation to exert cytotoxic effects on target cells.

[0099] Types of human lymphocytes of the presently disclosed subject matter include, but are not limited to, peripheral donor lymphocytes. For example, Sadelain, M., et al. 2003 Nat Rev Cancer 3:35-45 (disclosing peripheral donor lymphocytes genetically modified to express CAR), Morgan, RA, et al. 2006 Science 314:126-129 (disclosing peripheral donor lymphocytes genetically modified to express a full-length tumor antigen-recognizing T cell receptor complex containing α and β heterodimers), Panelli, MC, et al. 2000 J Immunol 164:495-504, Panelli, MC, et al. 2000 J Immunol 164:4382-4392 (disclosing lymphocyte cultures derived from tumor-infiltrating lymphocytes (TILs) in tumor biopsies), and Dupont, J., et al. 2005 Cancer Res 65:5417-5427, Papanicolaou, GA, et al. 2003 Blood 102:2498-2505 (disclosing selectively in vitro expanded antigen-specific peripheral blood leukocytes using artificial antigen presenting cells (AAPC) or pulsed dendritic cells).

[0100] In certain embodiments, the cells (e.g., T cells) are autologous. In certain embodiments, the cells (e.g., T cells) are non-autologous. In certain embodiments, the cells (e.g., T cells) are allogeneic. In certain embodiments, the cells (e.g., T cells) are derived from in vitro engineered progenitor or stem cells.

[0101] In certain embodiments, the cells are cells of the myeloid lineage. Non-limiting examples of cells of the myeloid lineage include monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes, and stem cells from which myeloid cells can differentiate.

[0102] In certain embodiments, the stem cells are pluripotent stem cells (eg, embryonic stem cells or induced pluripotent stem cells).

[0103] 2.1. First antigen-recognition receptor The first antigen-recognizing receptor targets a first antigen. The first antigen can be a tumor antigen or a pathogen antigen. In certain embodiments, the first antigen-recognizing receptor is a chimeric receptor. In certain embodiments, the chimeric receptor is a chimeric antigen receptor (CAR).

[0104] 2.1.1. First Antigen In certain embodiments, the first antigen is a tumor antigen. In certain embodiments, the tumor antigen is an antigen with low antigen density. In certain embodiments, the tumor antigen is expressed on cells with low tumor cell frequency.

[0105] Any tumor antigen (antigenic peptide) can be used in the tumor-related embodiments described herein. Sources of antigens include, but are not limited to, cancer proteins. The first antigen can be expressed as a peptide or as an intact protein or a portion thereof. The intact protein or portion thereof can be naturally occurring or mutagenized. Non-limiting examples of tumor antigens include CD19, CD70, IL1RAP, ABCG2, AChR, ACKR6, ADAMTS13, ADGRE2, ADGRE2 (EMR2), ADORA3, ADRA1D, AGER, ALS2, antigens of cytomegalovirus (CMV)-infected cells (e.g., cell surface antigens), ANO9, AQP2, ASIC3, ASPRV1, ATP6V0A4, B3GNT4, B7-H3, BCMA, BEST4, C3orf35, CADM3, CAIX, CAPN3, CCDC155, CCR1, CD10, CD117, CD123, CD133, CD135(FLT3), CD138, CD20, CD22, CD244(2B4), CD25, CD26, CD276, CD 30, CD300LF, CD312, CD32, CD321, CD33, CD34, CD36, CD38, CD41, CD44, CD44V6, CD47, CD49f, CD56, CD7, CD71, CD74, CD8, CD82, CD96, CD98, CD99, CDH13, CDHR1, CEA, CEACAM6, CHST3, CLEC12A, CLEC1A, CLL1, CNIH2, COL15A1, COLEC12, CPM, CR1, CX3CR1, CX CR4, CYP4F11, DAGLB, DARC, DFNB31, DGKI, EGF1R, EGFR-VIII, EGP-2, EGP-40, ELOVL6, EMB, EMC10, EMR2, ENG, EpCAM, EphA2, EP HA4, ERBB, ERBB2, Erb-B3, Erb-B4, E-selectin, EXOC3L4, EXTL3, FAM186B, FBP, FCGR1A, FKBP1B, FLRT1, folate receptor-a, FOLR2, FRMD5, G ABRB2, GAS2, GD2, GD3, GDPD3, GNA14, GNAZ, GPR153, GPR56, GYPA, HEPHL1, HER-2, hERT, HILPDA, HLA-DR, HOOK1, hTERT, HTR2A,ICAM1, IGFBP3, IL10RB, IL20RB, IL23R, ILDR1, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), ITFG3, ITGA4, ITGA5, ITGA8, ITGAX, ITGB5, ITGB8, JAM3, KCND1, KCNJ5, KCNK13, KCNN4, KCNV2, KDR, KIF19, KIF26B, κ-light chain, L1CAM, LAX1, LEPR, Lewis Y (CD174), Lewis Y (LeY), LILRA2, LILRA6, LILRB2, LILRB3, LILRB4, LOXL4, LPAR2, LRRC37A3, LRRC8E, LRRN2, LRRTM2, LTB4R, MAGE-A1, MAGEA3, MANSC1, MART1, GP100, MBOAT1, MBOAT7, melanoma antigen family A, mesothelin (MSLN), MFAP3L, MMP25, MRP1, MT-ND1, mucin 1 (MUC1) , mucin 16 (MUC16), MYADM, MYADML2, NGFR, NKCS1, NKG2D ligand, NLGN3, NPAS2, NY-ESO-1, oncofetal antigen (h5T4), OTOA, P2RY13, p53, PDE3A, PEAR1, PIEZO1, PLXNA4, PLXNC1, PNPLA3, PPFIA4, PPP2R5B, PRAME, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA). , proteinase 3 (PR1), PSD2, PTPRJ, RDH16, receptor tyrosine-protein kinase Erb-B2, RHBDL3, RNF173, RNF183, ROR1, RYR2, SCIN, SCN11A, SCN2A, SCNN1D, SEC31B, SEMA4A, SH3PXD2A, SIGLEC11, SIRPB1, SLC16A6, SLC19A1, SLC22A5, SLC25A36, SLC25A41, SLC 30A1, SLC34A3, SLC43A3, SLC44A1, SLC44A3, SLC45A3, SLC6A16, SLC6A6, SLC8A3, SLC9A1, SLCO2B1, SPAG17, STC1, STON 2, SUN3, Survivin, SUSD2, SYNC, TACSTD2, TAS1R3, TEX29, TFR2, TIM-3(HAVCR2), TLR2, TMEFF2, TMEM145, TMEM27, TMEM40,These include TMEM59L, TMEM89, TMPRSS5, TNFRSF14, TNFRSFlB, TRIM55, TSPEAR, TTYH3, tumor-associated glycoprotein 72 (TAG-72), tyrosinase, vascular endothelial growth factor R2 (VEGF-R2), VLA-4, Wilms tumor protein (WT-1), WNT4, WT1, and ZDHHC11.

[0106] In certain embodiments, the first antigen is selected from the group consisting of CD312, CLEC12A, CD33, CD123, IL1RAP, SIGLEC-6, GRP78, TIM3, CD70, CD20, CD22, CD19, GPRC5D, SLAMF7, BCMA, CD276, and CAIX. In certain embodiments, the first antigen is CD312. In certain embodiments, the first antigen is CD276.

[0107] In certain embodiments, the first antigen is a pathogen antigen. Non-limiting examples of viruses include Retroviridae (e.g., human immunodeficiency viruses, such as HIV-1 (also referred to as HDLV-III, LAVE, or HTLV-III / LAV, or other isolates such as HIV-III and HIV-LP), Picornaviridae (e.g., poliovirus, hepatitis A virus, enterovirus, human coxsackievirus, rhinovirus, echovirus), Calciviridae (e.g., strains that cause gastroenteritis), Togaviridae (e.g., equine encephalitis virus, rubella virus), Flaviviridae (e.g., dengue virus, encephalitis virus, yellow fever virus), Coronoviridae (e.g., coronavirus), Rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus), Filoviridae (e.g., Ebola virus), Paramyxoviridae (e.g., parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus), Orthomyxoviridae (e.g., influenza virus), Bungaviridae (e.g., Hantavirus, viruses, Bungaviruses, Phleboviruses, and Nairaviruses), Arenaviridae (hemorrhagic fever viruses), Reoviridae (e.g., reoviruses, orbivulus, and rotaviruses), Birnaviridae, Hepadnaviridae (hepatitis B virus), Parvoviridae (parvoviruses), Papovaviridae (papillomaviruses, polyomaviruses), Adenoviridae (most adenoviruses), Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella-zoster virus, saproviridae, varicella-zoster virus ... These include cytomegalovirus (CMV), herpesviruses, poxviridae (variola virus, vaccinia virus, poxvirus), and iridoviridae (e.g., African swine fever virus), as well as unclassified viruses such as the causative agent of hepatitis delta (thought to be a defective satellite of hepatitis B virus), non-A, non-B hepatitis agents (Class 1 = internally transmitted, Class 2 = parenterally transmitted (i.e., hepatitis C)), Norwalk and related viruses, and astroviruses.

[0108] Non-limiting examples of bacteria include Pasteurella, Staphylococci, Streptococcus, Escherichia coli, Pseudomonas species, and Salmonella species.The main ingredients of the fermentation product are Helicobacter pylori, Borelia burgdorferi, Legionella, Legionella pneumophilia, and Mycobacteria sps(from M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae, M. leprae) Staphylococcus aureus, Staphylococcus epidermidis, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (A Streptococcus), Streptococcus agalactiae (B, Streptococcus) faecalis anthracis, corynebacterium diphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium spp multocida、Bacteroides sp.、Fusobacterium nucleatum、Streptobacillus moniliformis、Treponema pallidium、Treponema pertenua、Leptospira、Rickettsia、Actinomyces israelli is a very good friend.Mycoplasma, Pseudomonas aeruginosa, Pseudomonas fluorescens, Corynobacteria diphtheriae, Bartonella henselae, Bartonella quintana, Coxiella burnetii, chlamydia, shigella, Yersinia enterocolitica, Yersinia pseudotuberculosis, Listeria monocytogenes, Mycoplasma spp., Vibrio cholerae, Borrelia, Francisella, Brucella melitensis, Proteus mirabilis, and Proteus.

[0109] In certain embodiments, the pathogen antigen is a viral antigen present in cytomegalovirus (CMV), a viral antigen present in Epstein-Barr virus (EBV), a viral antigen present in human immunodeficiency virus (HIV), or a viral antigen present in influenza virus.

[0110] 2.1.2. Chimeric Antigen Receptors (CARs) CARs are engineered receptors that transfer or confer a desired specificity onto immune effector cells. CARs can be used to transfer the specificity of monoclonal antibodies into T cells, and the transfer of their coding sequences is facilitated by retroviral vectors.

[0111] There are three generations of CARs. "First generation" CARs are typically composed of an extracellular antigen-binding domain (e.g., scFv) that binds to the target antigen and an intracellular signaling domain. In certain embodiments, the CAR further comprises a transmembrane domain. "First generation" CARs provide novel antigen recognition and signal transduction via the CD3 zeta chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation. + and CD8 +"Second generation" CARs include those that provide both costimulatory (e.g., CD28 or 4-1BB) and activation (CD3ζ) signals. "Third generation" CARs include those that provide multiple costimulatory (e.g., CD28 and 4-1BB) and activation (CD3ζ) signals.

[0112] In certain embodiments, the first antigen-recognizing receptor is a CAR comprising an extracellular antigen-binding domain that binds to the first antigen and an intracellular signaling domain. In certain embodiments, the CAR further comprises a transmembrane domain. In certain embodiments, the CAR further comprises a hinge / spacer region.

[0113] 2.1.2.1. Extracellular Antigen-Binding Domain In certain embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv) is about 5×10 -7 M or less, approximately 1×10 -7 M or less, about 5 x 10 -8 M or less, approximately 1×10 -8 M or less, about 5 x 10 -9 M or less, or about 1 x 10 -9 M or less, or about 1 x 10 -10 The dissociation constant (K D In certain embodiments, the extracellular antigen-binding domain (e.g., scFv) of the CAR binds to the first antigen at about 1 x 10 -8 K below M D binds to the first antigen.

[0114] The binding of the extracellular antigen-binding domain (e.g., in scFv) can be confirmed, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western blot assay. Each of these assays generally detects the presence of a particular protein-antibody complex of interest by using a labeled reagent (e.g., antibody or scFv) specific to the complex of interest. For example, scFv can be radioactively labeled and used in a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, incorporated herein by reference). Radioisotopes can be detected by means such as the use of a gamma counter or scintillation counter, or autoradioactivity testing. In certain embodiments, the extracellular antigen-binding domain of the CAR is labeled with a fluorescent marker. Non-limiting examples of fluorescent markers include green fluorescent protein (GFP), blue fluorescent protein (e.g., EBFP, EBFP2, Azurite, and mKalama1), cyan fluorescent protein (e.g., ECFP, Cerulean, and CyPet), and yellow fluorescent protein (e.g., YFP, Citrine, Venus, and YPet).

[0115] The extracellular antigen-binding domain may comprise or be an scFv, Fab (optionally cross-linked), or F(ab)2. In certain embodiments, any of the above molecules may be included in a fusion protein with a heterologous sequence to form the extracellular antigen-binding domain. In certain embodiments, the extracellular antigen-binding domain comprises or is an scFv. In certain embodiments, the scFv is a human scFv. In certain embodiments, the scFv is a humanized scFv. In certain embodiments, the scFv is a mouse scFv.

[0116] 2.1.2.1.1. Exemplary Extracellular Antigen-Binding Domains In certain embodiments, the first antigen-recognizing receptor is a CAR comprising an extracellular antigen-binding domain that binds to CD312. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises or consists of the amino acid sequence set forth in SEQ ID NO: 83 and specifically binds to CD312, e.g., human CD312 polypeptide. SEQ ID NO: 83 is provided in Table 1 below.

[0117] In certain embodiments, the extracellular antigen binding domain of the CAR comprises a heavy chain variable region (V) comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 73 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 74 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 75 or a conservative modification thereof. H In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V domain comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 73, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 74, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 75. H SEQ ID NOs: 73-75 are provided in Table 1 below.

[0118] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a light chain variable region (V) comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 76 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 77 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 78 or a conservative modification thereof. L In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V domain comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 76, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 77, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 78. L SEQ ID NOs: 76-78 are provided in Table 1 below.

[0119] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 73, or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 74, or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 75, or a conservative modification thereof. H , a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 76 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 77 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 78 or a conservative modification thereof. L In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V domain comprising a CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 73, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 74, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 75. H and V comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 76, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 77, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 78. L and,

[0120] In certain embodiments, the extracellular antigen-binding domain of the CAR has the amino acid sequence set forth in SEQ ID NO: 79. H V comprising CDR1, CDR2, and CDR3 of H In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V having the amino acid sequence set forth in SEQ ID NO: 81. L V comprising CDR1, CDR2, and CDR3 of L In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V having the amino acid sequence set forth in SEQ ID NO: 79. H V comprising CDR1, CDR2, and CDR3 of H and V having the amino acid sequence set forth in SEQ ID NO: 81. L V comprising CDR1, CDR2, and CDR3 of L and,

[0121] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 79. H For example, the extracellular antigen-binding domain of the first antigen-recognizing receptor comprises a V that comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO:79. H In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V H SEQ ID NO: 79 is provided in Table 1 below.

[0122] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 81. L For example, the extracellular antigen-binding domain of the CAR comprises a V that comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 81. L In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V L SEQ ID NO: 81 is provided in Table 1 below.

[0123] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 79. Hand V comprising an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 81. L and,

[0124] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises the amino acid sequence set forth in SEQ ID NO: 79. H In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V L In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V H and V comprising the amino acid sequence set forth in SEQ ID NO: 81. L and,

[0125] In certain embodiments, the extracellular antigen-binding domain of the CAR is an scFv comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 83. SEQ ID NOs: 73-83 are provided in Table 1 below. In certain embodiments, V H and V L are linked via a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

[0126] In certain embodiments, the CDR regions / sequences disclosed herein are delineated using the Kabat system (Swindells et al., J Mol Biol. 2017 Feb 3;429(3):356-364). [Table 1-1] [Table 1-2]

[0127] In certain embodiments, the first antigen-recognizing receptor is a CAR comprising an extracellular antigen-binding domain that binds to CD276. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises or consists of the amino acid sequence set forth in SEQ ID NO: 95 and specifically binds to CD276, e.g., human CD276 polypeptide. SEQ ID NO: 95 is provided in Table 2 below.

[0128] In certain embodiments, the extracellular antigen binding domain of the CAR comprises a heavy chain variable region (V) comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 85 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 86 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 87 or a conservative modification thereof. H In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V domain comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 85, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 86, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 87. H SEQ ID NOs: 85-87 are provided in Table 2 below.

[0129] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a light chain variable region (V) comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 88 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 89 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 90 or a conservative modification thereof. L In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V domain comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 88, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 89, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 90. L SEQ ID NOs: 88-90 are provided in Table 2 below.

[0130] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 85 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 86 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 87 or a conservative modification thereof. H , CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 88 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 89 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 90 or a conservative modification thereof. L In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V domain comprising a CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 85, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 86, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 87. H and V comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 88, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 89, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 90. L and,

[0131] In certain embodiments, the extracellular antigen-binding domain of the CAR has the amino acid sequence set forth in SEQ ID NO: 91. H V comprising CDR1, CDR2, and CDR3 of H In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V having the amino acid sequence set forth in SEQ ID NO: 93. L V comprising CDR1, CDR2, and CDR3 of L In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V having the amino acid sequence set forth in SEQ ID NO: 91. H V comprising CDR1, CDR2, and CDR3 of H and V having the amino acid sequence set forth in SEQ ID NO: 93 L V comprising CDR1, CDR2, and CDR3 of L and,

[0132] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 91. H For example, the extracellular antigen-binding domain of the first antigen-recognizing receptor comprises a V comprising an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO:91. H In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V H SEQ ID NO: 91 is provided in Table 2 below.

[0133] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 93. L For example, the extracellular antigen-binding domain of the CAR comprises a V that comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO:93. L In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V L SEQ ID NO: 93 is provided in Table 2 below.

[0134] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 91. Hand V comprising an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:93. L and,

[0135] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises the V H In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V L In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V H and V comprising the amino acid sequence set forth in SEQ ID NO: 93 L and,

[0136] In certain embodiments, the extracellular antigen-binding domain of the CAR is an scFv comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 95. SEQ ID NOs: 85-95 are provided in Table 2 below. In certain embodiments, V H and V L are linked via a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

[0137] In certain embodiments, the CDR regions / sequences disclosed herein are delineated using the Kabat system (Swindells et al., J Mol Biol. 2017 Feb 3;429(3):356-364). [Table 2-1] [Table 2-2]

[0138] In certain embodiments, the first antigen-recognizing receptor is a CAR that comprises an extracellular antigen-binding domain that binds to CD22. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises or consists of the amino acid sequence set forth in SEQ ID NO: 107 or SEQ ID NO: 109 and specifically binds to CD22, e.g., human CD22 polypeptide. SEQ ID NO: 107 and SEQ ID NO: 109 are provided in Table 3 below.

[0139] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a heavy chain variable region (V) comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 97 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 98 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 99 or a conservative modification thereof. H In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V domain comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 97, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 98, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 99. H SEQ ID NOs: 97-99 are provided in Table 3 below.

[0140] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a light chain variable region (V) comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 100 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 101 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 102 or a conservative modification thereof. L In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V domain comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 100, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 101, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 102. L SEQ ID NOs: 100-102 are provided in Table 3 below.

[0141] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 97, or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 98, or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 99, or a conservative modification thereof. H , a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 100 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 101 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 102 or a conservative modification thereof. L In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V domain comprising a CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 97, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 98, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 99. H and V comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 100, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 101, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 102. L and,

[0142] In certain embodiments, the extracellular antigen-binding domain of the CAR has the amino acid sequence set forth in SEQ ID NO: 103. H V comprising CDR1, CDR2, and CDR3 of H In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V having the amino acid sequence set forth in SEQ ID NO: 105. L V comprising CDR1, CDR2, and CDR3 of L In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V having the amino acid sequence set forth in SEQ ID NO: 103. H V comprising CDR1, CDR2, and CDR3 of H and V having the amino acid sequence set forth in SEQ ID NO: 105 L V comprising CDR1, CDR2, and CDR3 of L and,

[0143] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 103. H For example, the extracellular antigen-binding domain of the first antigen-recognizing receptor comprises a V that comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 103. H In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V H SEQ ID NO: 103 is provided in Table 3 below.

[0144] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 105. L For example, the extracellular antigen-binding domain of the CAR comprises a V that comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 105. L In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V L SEQ ID NO: 105 is provided in Table 3 below.

[0145] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 103. Hand V comprising an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 105. L and,

[0146] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises the amino acid sequence set forth in SEQ ID NO: 103. H In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V L In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V H and V comprising the amino acid sequence set forth in SEQ ID NO: 105 L and,

[0147] In certain embodiments, the extracellular antigen-binding domain of the CAR is an scFv comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 107 or SEQ ID NO: 109. SEQ ID NOs: 97-109 are provided in Table 3 below. In certain embodiments, V H and V L are linked via a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

[0148] In certain embodiments, the CDR regions / sequences disclosed herein are delineated using the Kabat system (Swindells et al., J Mol Biol. 2017 Feb 3;429(3):356-364). [Table 3-1] [Table 3-2] [Table 3-3]

[0149] In certain embodiments, the first antigen-recognizing receptor is a CAR that comprises an extracellular antigen-binding domain that binds to CD22. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises or consists of the amino acid sequence set forth in SEQ ID NO: 115 or SEQ ID NO: 117 and specifically binds to CD22, e.g., human CD22 polypeptide. SEQ ID NO: 115 and SEQ ID NO: 117 are provided in Table 4 below.

[0150] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a heavy chain variable region (V) comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 97 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 153 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 99 or a conservative modification thereof. H In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V domain comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 97, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 153, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 99. H SEQ ID NOs: 97, 99, and 153 are provided in Table 4 below.

[0151] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a light chain variable region (V) comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 100 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 101 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 102 or a conservative modification thereof. L In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V domain comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 100, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 101, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 102. L SEQ ID NOs: 100-102 are provided in Table 4 below.

[0152] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 97, or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 153, or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 99, or a conservative modification thereof. H , a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 100 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 101 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 102 or a conservative modification thereof. L In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V domain comprising a CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 97, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 153, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 99. H and V comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 100, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 101, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 102. L and,

[0153] In certain embodiments, the extracellular antigen-binding domain of the CAR has the amino acid sequence set forth in SEQ ID NO: 111. H V comprising CDR1, CDR2, and CDR3 of H In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V having the amino acid sequence set forth in SEQ ID NO: 113. L V comprising CDR1, CDR2, and CDR3 of L In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V having the amino acid sequence set forth in SEQ ID NO: 111. H V comprising CDR1, CDR2, and CDR3 of H and V having the amino acid sequence set forth in SEQ ID NO: 113 L V comprising CDR1, CDR2, and CDR3 of L and,

[0154] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 111. H For example, the extracellular antigen-binding domain of the first antigen-recognizing receptor comprises a V that comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO:111. H In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V H SEQ ID NO: 111 is provided in Table 4 below.

[0155] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 113. L For example, the extracellular antigen-binding domain of the CAR comprises a V that comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 113. L In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V L SEQ ID NO: 113 is provided in Table 4 below.

[0156] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 111. Hand V comprising an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO:113. L and,

[0157] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises the amino acid sequence set forth in SEQ ID NO: 111. H In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V L In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V H and V comprising the amino acid sequence set forth in SEQ ID NO: 113. L and,

[0158] In certain embodiments, the extracellular antigen-binding domain of the CAR is an scFv comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 115 or SEQ ID NO: 117. SEQ ID NOs: 99-102 and 111-117 are provided in Table 4 below. In certain embodiments, V H and V L are linked via a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

[0159] In certain embodiments, the CDR regions / sequences disclosed herein are delineated using the Kabat system (Swindells et al., J Mol Biol. 2017 Feb 3;429(3):356-364). [Table 4-1] [Table 4-2]

[0160] In certain embodiments, the first antigen-recognizing receptor is a CAR that comprises an extracellular antigen-binding domain that binds to CD22. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises or consists of the amino acid sequence set forth in SEQ ID NO: 129 or SEQ ID NO: 131 and specifically binds to CD22, e.g., a human CD22 polypeptide. SEQ ID NO: 129 and SEQ ID NO: 131 are provided in Table 5 below.

[0161] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a heavy chain variable region (V) comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 119 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 120 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 121 or a conservative modification thereof. H In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a VDR1 comprising the amino acid sequence set forth in SEQ ID NO: 119, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 120, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 121. H SEQ ID NOs: 119-121 are provided in Table 5 below.

[0162] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a light chain variable region (V) comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 122 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 123 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 124 or a conservative modification thereof. L In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a VDR1 comprising the amino acid sequence set forth in SEQ ID NO: 122, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 123, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 124. L SEQ ID NOs: 122-124 are provided in Table 5 below.

[0163] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 119 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 120 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 121 or a conservative modification thereof. H , CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 122 or a conservative modification thereof, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 123 or a conservative modification thereof, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 124 or a conservative modification thereof. L In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V domain comprising a CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 119, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 120, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 121. H and V comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 122, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 123, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 124. L and,

[0164] In certain embodiments, the extracellular antigen-binding domain of the CAR has the amino acid sequence set forth in SEQ ID NO: 125. H V comprising CDR1, CDR2, and CDR3 of H In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V having the amino acid sequence set forth in SEQ ID NO: 127. L V comprising CDR1, CDR2, and CDR3 of L In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V having the amino acid sequence set forth in SEQ ID NO: 125. H V comprising CDR1, CDR2, and CDR3 of H and V having the amino acid sequence set forth in SEQ ID NO: 127 L V comprising CDR1, CDR2, and CDR3 of L and,

[0165] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 125. H For example, the extracellular antigen-binding domain of the first antigen-recognizing receptor comprises a V that comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 125. H In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V H SEQ ID NO: 125 is provided in Table 5 below.

[0166] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 127. L For example, the extracellular antigen-binding domain of the CAR comprises a V that comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 127. L In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V L SEQ ID NO: 127 is provided in Table 5 below.

[0167] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 125. Hand V comprising an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 127. L and,

[0168] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises the amino acid sequence set forth in SEQ ID NO: 125. H In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V L In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V H and V comprising the amino acid sequence set forth in SEQ ID NO: 127 L and,

[0169] In certain embodiments, the extracellular antigen-binding domain of the CAR is an scFv comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 129 or SEQ ID NO: 131. SEQ ID NOs: 119-132 are provided in Table 5 below. In certain embodiments, V H and V L are linked via a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

[0170] In certain embodiments, the CDR regions / sequences disclosed herein are delineated using the Kabat system (Swindells et al., J Mol Biol. 2017 Feb 3;429(3):356-364). [Table 5-1] [Table 5-2]

[0171] In certain embodiments, the first antigen-recognizing receptor is a CAR that comprises an extracellular antigen-binding domain that binds to CD22. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises or consists of the amino acid sequence set forth in SEQ ID NO: 157 or SEQ ID NO: 158 and specifically binds to CD22, e.g., human CD22 polypeptide. SEQ ID NO: 157 and SEQ ID NO: 158 are provided in Table 6 below.

[0172] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a heavy chain variable region (V) comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 97 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 154 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 99 or a conservative modification thereof. H In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V domain comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 97, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 154, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 99. H SEQ ID NOs: 97, 99, and 154 are provided in Table 6 below.

[0173] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a light chain variable region (V) comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 100 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 101 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 102 or a conservative modification thereof. L In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V domain comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 100, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 101, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 102. L SEQ ID NOs: 100-102 are provided in Table 6 below.

[0174] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 97, or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 154, or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 99, or a conservative modification thereof. H , a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 100 or a conservative modification thereof, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 101 or a conservative modification thereof, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 102 or a conservative modification thereof. L In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V domain comprising a CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 97, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 154, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 99. H and V comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 100, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 101, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 102. L and,

[0175] In certain embodiments, the extracellular antigen-binding domain of the CAR has the amino acid sequence set forth in SEQ ID NO: 155. H V comprising CDR1, CDR2, and CDR3 of H In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V having the amino acid sequence set forth in SEQ ID NO: 105. L V comprising CDR1, CDR2, and CDR3 of L In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V having the amino acid sequence set forth in SEQ ID NO: 155. H V comprising CDR1, CDR2, and CDR3 of H and V having the amino acid sequence set forth in SEQ ID NO: 105 L V comprising CDR1, CDR2, and CDR3 of L and,

[0176] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 155. H For example, the extracellular antigen-binding domain of the first antigen-recognizing receptor comprises a V that comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 155. H In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V H SEQ ID NO: 155 is provided in Table 6 below.

[0177] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 105. L For example, the extracellular antigen-binding domain of the CAR comprises a V that comprises an amino acid sequence that is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 113. L In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V L SEQ ID NO: 105 is provided in Table 6 below.

[0178] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 155. Hand V comprising an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) homologous or identical to the amino acid sequence set forth in SEQ ID NO: 105. L and,

[0179] In certain embodiments, the extracellular antigen-binding domain of the CAR comprises the amino acid sequence set forth in SEQ ID NO: 155. H In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V L In certain embodiments, the extracellular antigen-binding domain of the CAR comprises a V H and V comprising the amino acid sequence set forth in SEQ ID NO: 105 L and,

[0180] In certain embodiments, the extracellular antigen-binding domain of the CAR is an scFv comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 157 or SEQ ID NO: 158. SEQ ID NOs: 154-160 are provided in Table 6 below. In certain embodiments, V H and V L are linked via a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.

[0181] In certain embodiments, the CDR regions / sequences disclosed herein are delineated using the Kabat system (Swindells et al., J Mol Biol. 2017 Feb 3;429(3):356-364). [Table 6-1] [Table 6-2] [Table 6-3]

[0182] V having at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homology or identity to a particular sequence (e.g., SEQ ID NOs: 79, 81, 91, 93, 103, 105, 111, 113, 125, 127, and 155). H and / or V L The amino acid sequence may contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the designated sequence(s) but retain the ability to bind to the target antigen (e.g., CD312, CD276, CD22). In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted within a particular sequence (e.g., SEQ ID NOs: 79, 81, 91, 93, 103, 105, 111, 113, 125, 127, and 155). In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the CDRs (e.g., within the FRs) of the extracellular antigen-binding domain. In certain embodiments, the extracellular antigen-binding domain of the CAR is selected from SEQ ID NOs: 79, 81, 91, 93, 103, 105, 111, 113, 125, 127, and 155, including post-translational modifications of the sequences thereof (SEQ ID NOs: 79, 81, 91, 93, 103, 105, 111, 113, 125, 127, and 155). H and / or V L Contains arrays.

[0183] In addition, the extracellular antigen-binding domain of the CAR can contain a leader or signal peptide that directs the nascent protein into the endoplasmic reticulum. A signal peptide or leader may be essential if the CAR is glycosylated and anchored in the cell membrane. The signal sequence or leader can be a peptide sequence (about 5, about 10, about 15, about 20, about 25, or about 30 amino acids in length) present at the N-terminus of a newly synthesized protein that directs entry into the secretory pathway. In certain embodiments, the signal peptide is covalently linked to the 5' end (N-terminus) of the extracellular antigen-binding domain of the CAR. Exemplary leader sequences include, but are not limited to, a human IL-2 signal sequence (e.g., a human IL-2 signal sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 32), a mouse IL-2 signal sequence (e.g., a mouse IL-2 signal sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 33), a human kappa leader sequence (e.g., a human kappa leader sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 34), a mouse kappa leader sequence (e.g., a mouse kappa leader sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 35), a human CD8 leader sequence (e.g., a human CD8 leader sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 36), a truncated human CD8 signal peptide (e.g., a truncated human CD8 signal peptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 37), a human albumin signal sequence (e.g., a human albumin signal sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 38), and a human prolactin signal sequence (e.g., a human prolactin signal sequence comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 39). SEQ ID NOs: 32-39 are provided below. MYRMQLLSCIALSLALVTNS [SEQ ID NO: 32] MYSMQLASCVTLTLVLLVNS [SEQ ID NO: 33] METPAQLLFLLLLWLPDTTG [SEQ ID NO: 34] METDTLLLWVLLLWVPGSTG [SEQ ID NO: 35] MALPVTALLLPLALLLHAARP [SEQ ID NO: 36] MALPVTALLLPLALLLHA [SEQ ID NO: 37] MKWVTFISLLFSSAYS [SEQ ID NO: 38] MDSKGSSQKGSRLLLLLVVSNLLLCQGVVS [SEQ ID NO: 39]

[0184] In certain embodiments, the signal peptide comprises a CD8 polypeptide, e.g., the CAR comprises a truncated CD8 signal peptide. In certain embodiments, the signal peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 37.

[0185] 2.1.2.2. Transmembrane domain and hinge / spacer region In certain embodiments, the first antigen-recognizing receptor is a CAR that includes a transmembrane domain. Different transmembrane domains result in different receptor stability. After antigen recognition, the receptors cluster and transmit signals to cells. According to the subject matter disclosed herein, the transmembrane domain of the first antigen-recognizing receptor can include the natural or modified transmembrane domain of a CD8 polypeptide, a CD28 polypeptide, a CD3ζ polypeptide, a CD40 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, a CD84 polypeptide, a CD166 polypeptide, a CD8a polypeptide, a CD8b polypeptide, an ICOS polypeptide, an ICAM-1 polypeptide, a CTLA-4 polypeptide, a CD27 polypeptide, a CD40 polypeptide, an NKG2D polypeptide, a synthetic polypeptide (not based on a protein related to immune response), or a combination thereof.

[0186] In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide (e.g., the transmembrane domain of CD28 or a portion thereof). In certain embodiments, the transmembrane domain of the CAR comprises the transmembrane domain of human CD28 or a portion thereof. In certain embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of the amino acid sequence having NCBI Reference Number: NP_006130 (SEQ ID NO:7) that is at least about 20, or at least about 25, or at least about 30, and / or up to about 220 amino acids in length. In certain embodiments, the CD28 polypeptide comprises or consists of the amino acid sequence of amino acids 1-220, 1-50, 50-100, 100-150, 114-220, 150-200, 153-179, or 200-220 of SEQ ID NO:7. In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide comprising or consisting of amino acids 153-179 of SEQ ID NO: 7. SEQ ID NO: 7 is provided below. MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS [SEQ ID NO: 7]

[0187] In certain embodiments, the first antigen-recognizing receptor is a CAR that further comprises a hinge / spacer region that links the extracellular antigen-binding domain to the transmembrane domain. The hinge / spacer region may be flexible enough to allow the antigen-binding domain to orient in different directions to facilitate antigen recognition. In certain embodiments, the hinge / spacer region of the CAR may comprise a natural or modified hinge region of a CD8 polypeptide, a CD28 polypeptide, a CD3ζ polypeptide, a CD40 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, a CD84 polypeptide, a CD166 polypeptide, a CD8a polypeptide, a CD8b polypeptide, an ICOS polypeptide, an ICAM-1 polypeptide, a CTLA-4 polypeptide, a CD27 polypeptide, a CD40 polypeptide, an NKG2D polypeptide, a synthetic polypeptide (not based on a protein associated with an immune response), or a combination thereof. The hinge / spacer region can be a hinge region from IgG1, or an immunoglobulin CH2CH3 region, and a portion of CD3, a portion of a CD28 polypeptide (eg, a portion of SEQ ID NO: 7), a portion of a CD8 polypeptide, or a synthetic spacer sequence.

[0188] In certain embodiments, the first antigen-recognizing receptor is a CAR, further comprising a hinge / spacer region comprising a native or modified hinge region of a CD28 polypeptide. In certain embodiments, the hinge / spacer region of the first antigen-recognizing receptor (e.g., a CAR) comprises a CD28 polypeptide comprising or consisting of amino acids 114 to 152 of SEQ ID NO:7.

[0189] In certain embodiments, the hinge / spacer region is located between the extracellular antigen-binding domain and the transmembrane domain. In certain embodiments, the hinge / spacer region comprises a CD8 polypeptide, a CD28 polypeptide, a CD3ζ polypeptide, a CD4 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, a CD166 polypeptide, a CD8a polypeptide, a CD8b polypeptide, an ICOS polypeptide, an ICAM-1 polypeptide, a CTLA-4 polypeptide, a CD27 polypeptide, a CD40 polypeptide, an NKG2D polypeptide, a synthetic polypeptide (not based on a protein associated with an immune response), or a combination thereof. In certain embodiments, the transmembrane domain comprises a CD8 polypeptide, a CD28 polypeptide, a CD3ζ polypeptide, a CD4 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, a CD166 polypeptide, a CD8a polypeptide, a CD8b polypeptide, an ICOS polypeptide, an ICAM-1 polypeptide, a CTLA-4 polypeptide, a CD27 polypeptide, a CD40 polypeptide, an NKG2D polypeptide, a synthetic polypeptide (not based on a protein associated with an immune response), or a combination thereof.

[0190] In certain embodiments, the transmembrane domain and hinge / spacer region are derived from the same molecule. In certain embodiments, the transmembrane domain and hinge / spacer region are derived from different molecules. In certain embodiments, the hinge / spacer region comprises a CD28 polypeptide, and the transmembrane domain comprises a CD28 polypeptide. In certain embodiments, the hinge / spacer region comprises a CD28 polypeptide, and the transmembrane domain comprises a CD28 polypeptide. In certain embodiments, the hinge / spacer region comprises a CD84 polypeptide, and the transmembrane domain comprises a CD84 polypeptide. In certain embodiments, the hinge / spacer region comprises a CD166 polypeptide, and the transmembrane domain comprises a CD166 polypeptide. In certain embodiments, the hinge / spacer region comprises a CD8a polypeptide, and the transmembrane domain comprises a CD8a polypeptide. In certain embodiments, the hinge / spacer region comprises a CD8b polypeptide, and the transmembrane domain comprises a CD8b polypeptide. In certain embodiments, the hinge / spacer region comprises a CD28 polypeptide and the transmembrane domain comprises an ICOS polypeptide.

[0191] 2.1.2.3. Intracellular signaling domains In certain embodiments, the first antigen-recognizing receptor is a CAR comprising an intracellular signaling domain. In certain embodiments, the intracellular signaling domain of the CAR comprises a CD3ζ polypeptide. CD3ζ can activate or stimulate cells (e.g., lymphoid cells, e.g., T cells). Wild-type ("native") CD3ζ comprises three functional immunoreceptor tyrosine-based activation motifs (ITAMs) and three functional basic-rich extension (BRS) regions (BRS1, BRS2, ​​and BRS3). CD3ζ transmits activation signals to cells (e.g., lymphoid cells, e.g., T cells) after antigen binding. The intracellular signaling domain of the CD3ζ chain is the primary transmitter of signals from endogenous TCRs.

[0192] In certain embodiments, the intracellular signaling domain of the CAR comprises native CD3ζ. In certain embodiments, native CD3ζ comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical or homologous to the amino acid sequence having NCBI Reference Number: NP_932170 (SEQ ID NO: 8), or a fragment thereof, and / or may optionally contain up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, the CD3ζ polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 12 that is at least about 20, or at least about 30, or at least about 40, or at least about 50, and up to about 164 amino acids in length. In certain embodiments, native CD3ζ comprises or consists of the amino acid sequence of amino acids 1-164, 1-50, 50-100, 52-164, 100-150, or 150-164 of SEQ ID NO: 8. In certain embodiments, the intracellular signaling domain of the CAR comprises native CD3ζ comprising or consisting of the amino acid sequence of amino acids 52-164 of SEQ ID NO: 8. SEQ ID NO: 8 is provided below. MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR [SEQ ID NO: 8]

[0193] In certain embodiments, native CD3ζ comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical or homologous to the amino acid sequence set forth in SEQ ID NO: 9. SEQ ID NO: 9 is provided below. RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR [SEQ ID NO: 9]

[0194] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide. In certain embodiments, the modified CD3ζ polypeptide comprises one, two, or three ITAMs. In certain embodiments, the modified CD3ζ polypeptide comprises a native ITAM1. In certain embodiments, the native ITAM1 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 10, as provided below. QNQLYNELNLGRREEYDVLDKR [SEQ ID NO: 10]

[0195] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:10 is set forth in SEQ ID NO:11, provided below. CAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGA [SEQ ID NO: 11]

[0196] In certain embodiments, the modified CD3ζ polypeptide comprises an ITAM1 variant comprising one or more loss-of-function mutations. In certain embodiments, the ITAM1 variant comprises or consists of two loss-of-function mutations. In certain embodiments, each of the one or more (e.g., two) loss-of-function mutations comprises a mutation of a tyrosine residue in ITAM1. In certain embodiments, the ITAM1 variant consists of two loss-of-function mutations. In certain embodiments, the ITAM1 variant comprises or consists of the amino acid sequence set forth in SEQ ID NO: 12, provided below. QNQLFNELNLGRREEFDVLDKR [SEQ ID NO: 12]

[0197] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:12 is set forth in SEQ ID NO:13, provided below. CAGAACCAGCTCTTTAACGAGCTCAATCTAGGACGAAGAGAGGAGTTCGATGTTTTGGACAAGAGA [SEQ ID NO: 13]

[0198] In certain embodiments, the modified CD3ζ polypeptide comprises a native ITAM2, which comprises or consists of the amino acid sequence set forth in SEQ ID NO: 14, provided below. QEGLYNELQKDKMAEAYSEIGMK [SEQ ID NO: 14]

[0199] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:14 is set forth in SEQ ID NO:15, provided below. CAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAA [SEQ ID NO: 15]

[0200] In certain embodiments, the modified CD3ζ polypeptide comprises an ITAM2 variant. In certain embodiments, the ITAM2 variant comprises or consists of one or more loss-of-function mutations. In certain embodiments, the ITAM2 variant comprises or consists of two loss-of-function mutations. In certain embodiments, each of the one or more (e.g., two) loss-of-function mutations comprises a mutation of a tyrosine residue in ITAM2. In certain embodiments, the ITAM1 variant consists of two loss-of-function mutations. In certain embodiments, the ITAM2 variant comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16, provided below. QEGLFNELQKDKMAEAFSEIGMK [SEQ ID NO: 16]

[0201] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:16 is set forth in SEQ ID NO:17, provided below. CAGGAAGGCCTGTTCAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTTCAGTGAGATTGGGATGAAA [SEQ ID NO: 17]

[0202] In certain embodiments, the modified CD3ζ polypeptide comprises a native ITAM3, which comprises or consists of the amino acid sequence set forth in SEQ ID NO: 18, provided below. HDGLYQGLSTATKDTYDALHMQ [SEQ ID NO: 18]

[0203] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:18 is set forth in SEQ ID NO:19, provided below. CACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAG [SEQ ID NO: 19]

[0204] In certain embodiments, the modified CD3ζ polypeptide comprises an ITAM3 variant. In certain embodiments, the ITAM3 variant comprises or consists of two loss-of-function mutations. In certain embodiments, each of the one or more (e.g., two) loss-of-function mutations comprises a mutation of a tyrosine residue in ITAM3. In certain embodiments, the ITAM3 variant comprises or consists of two loss-of-function mutations. In certain embodiments, the ITAM3 variant comprises or consists of the amino acid sequence set forth in SEQ ID NO: 20, provided below. HDGLFQGLSTATKDTFDALHMQ [SEQ ID NO: 20]

[0205] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:20 is set forth in SEQ ID NO:21, provided below. CACGATGGCCTTTTCCAGGGGCTCAGTACAGCCACCAAGGACACCTTCGACGCCCTTCACATGCAG[SEQ ID NO: 21]

[0206] Various modified CD3ζ polypeptides and CARs comprising modified CD3ζ polypeptides are disclosed in International Patent Application Publication No. 2019 / 133969, the entire contents of which are incorporated herein by reference.

[0207] In certain embodiments, the intracellular signaling domain of a CAR comprises a modified CD3ζ polypeptide comprising a native ITAM1, an ITAM2 variant comprising or consisting of one or more (e.g., two) loss-of-function mutations, and an ITAM3 variant comprising or consisting of one or more (e.g., two) loss-of-function mutations. In certain embodiments, the intracellular signaling domain of a CAR comprises a modified CD3ζ polypeptide comprising a native ITAM1, an ITAM2 variant consisting of two loss-of-function mutations, and an ITAM3 variant consisting of two loss-of-function mutations. In certain embodiments, the intracellular signaling domain of a CAR comprises a modified CD3ζ polypeptide comprising a native ITAM1 consisting of the amino acid sequence set forth in SEQ ID NO: 10, an ITAM2 variant consisting of the amino acid sequence set forth in SEQ ID NO: 16, and an ITAM3 variant consisting of the amino acid sequence set forth in SEQ ID NO: 20. In certain embodiments, a CAR is referred to as "1XX." In certain embodiments, the modified CD3ζ polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22. SEQ ID NO: 22 is provided below. RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR [SEQ ID NO: 22]

[0208] In certain embodiments, the intracellular signaling domain of the CAR comprises a modified CD3ζ polypeptide comprising, or consisting of, an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% identical to SEQ ID NO: 22, or a fragment thereof, and / or optionally comprises up to one, or up to two, or up to three conservative amino acid substitutions.

[0209] An exemplary nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO:22 is set forth in SEQ ID NO:23, provided below. AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCC TGTTCAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTTCAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTTCCAGGGGCTCAGTACAGCCACCAAGGACACCTTCGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC [SEQ ID NO: 23]

[0210] In certain embodiments, the intracellular signaling domain of the CAR further comprises at least one costimulatory signaling region. In certain embodiments, the at least one costimulatory region comprises a costimulatory molecule or a portion thereof. In certain embodiments, the at least one costimulatory region comprises at least the intracellular domain of at least one costimulatory molecule or a portion thereof. Non-limiting examples of costimulatory molecules include CD28, 4-1BB, OX40, CD27, CD40, CD154, CD97, CD11a / CD18, ICOS, DAP-10, CD2, CD150, CD226, and NKG2D.

[0211] In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising the intracellular domain of a CD28 polypeptide, e.g., CD28, or a portion thereof. In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising the intracellular domain of human CD28, or a portion thereof.

[0212] In certain embodiments, the CD28 polypeptide comprised in the costimulatory signaling region of the first antigen recognizing receptor comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% identical or homologous to the amino acid sequence set forth in SEQ ID NO: 7, or a fragment thereof, and / or may optionally comprise up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the CD28 polypeptide comprised in the costimulatory signaling region of the CAR comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 7 that is at least about 20, or at least about 30, or at least about 40, or at least about 50, and up to about 220 amino acids in length. Alternatively or additionally, in certain embodiments, the CD28 polypeptide comprised in the costimulatory signaling region of the CAR comprises or consists of amino acids 1-220, 1-50, 50-100, 100-150, 114-220, 150-200, 180-220, or 200-220 of SEQ ID NO: 7. In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising a CD28 polypeptide comprising or consisting of amino acids 180-220 of SEQ ID NO: 7.

[0213] An exemplary nucleic acid sequence encoding the amino acid sequence of amino acids 180-220 of SEQ ID NO:7 is set forth in SEQ ID NO:24, provided below. AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC [SEQ ID NO: 24]

[0214] In certain embodiments, the intracellular signaling domain of the first antigen-recognizing receptor comprises a costimulatory signaling region comprising the intracellular domain of mouse CD28 or a portion thereof. In certain embodiments, the CD28 polypeptide comprised in the costimulatory signaling region comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% identical or homologous to the amino acid sequence having NCBI Reference Number: NP_031668.3 (or SEQ ID NO: 25) or a fragment thereof, and / or may optionally comprise up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, the CD28 polypeptide comprised in the costimulatory signaling region of the CAR comprises or consists of an amino acid sequence that is at least about 20, or at least about 30, or at least about 40, or at least about 50, and up to 218 amino acids in length. In certain embodiments, the CD28 polypeptide comprised in the costimulatory signaling region of the CAR comprises or consists of the amino acid sequence of amino acids 1-218, 1-50, 50-100, 100-150, 150-218, 178-218, or 200-218 of SEQ ID NO: 25. In certain embodiments, the costimulatory signaling region of the CAR comprises a CD28 polypeptide comprising or consisting of amino acids 178-218 of SEQ ID NO: 25. SEQ ID NO: 25 is provided below. MTLRLLFLALNFFSVQVTENKILVKQSPLLVVDSNEVSLSCRYSYNLLAKEFRASLYKGVNSDVEVCVGNGNFTYQPQFRSNAEFNCDGDFDNETVTFRLWNLHVNHTDIYFCKIEFMYPPPYLDNERSNGTIIHIKEKHLCHTQSSPKLFWALVVVAGVLFCYGLLVTVALCVIWTNSRRNRLLQSDYMNMTPRRPGLTRKPYQPYAPARDFAAYRP [SEQ ID NO: 25]

[0215] In certain embodiments, the intracellular signaling domain of the CAR comprises a costimulatory signaling region comprising a 4-1BB polypeptide, e.g., the intracellular domain of 4-1BB, or a portion thereof. In certain embodiments, the costimulatory signaling region comprises the intracellular domain of human 4-1BB, or a portion thereof. In certain embodiments, the 4-1BB comprised in the costimulatory signaling region of the CAR comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% homologous or identical to the amino acid sequence having NCBI Reference Number: NP_001552 (SEQ ID NO: 26), or a fragment thereof, and / or may optionally comprise up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, the 4-1BB included in the costimulatory signaling region of the CAR comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO:26 that is at least about 20, or at least about 30, or at least about 40, or at least about 50, and / or up to about 50, up to about 60, up to about 70, up to about 80, up to about 90, up to about 100, up to about 200, or up to about 255 amino acids in length. In certain embodiments, the costimulatory signaling region of the CAR comprises a 4-1BB polypeptide that comprises or consists of the amino acid sequence of amino acids 1-255, 1-50, 50-100, 100-150, 150-200, or 200-255 of SEQ ID NO:26. In certain embodiments, the costimulatory signaling region of the CAR comprises a 4-1BB polypeptide that comprises or consists of the amino acid sequence of amino acids 214-255 of SEQ ID NO:26. SEQ ID NO:26 is provided below. MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL [SEQ ID NO: 26]

[0216] In certain embodiments, the intracellular signaling domain of the CAR comprises two costimulatory signaling regions, wherein the first costimulatory signaling region comprises the intracellular domain of a first costimulatory molecule or a portion thereof, and the second costimulatory signaling region comprises the intracellular domain of a second costimulatory molecule or a portion thereof. The first and second costimulatory molecules are independently selected from the group consisting of CD28, 4-1BB, OX40, CD27, CD40, CD154, CD97, CD11a / CD18, ICOS, DAP-10, CD2, CD150, CD226, and NKG2D. In certain embodiments, the intracellular signaling domain of the CAR comprises two costimulatory signaling regions, wherein the first costimulatory signaling region comprises the intracellular domain of CD28 or a portion thereof, and the second costimulatory signaling region comprises the intracellular domain of 4-1BB or a portion thereof.

[0217] In certain embodiments, the first antigen-recognizing receptor is a CAR comprising: i) an extracellular antigen-binding domain; ii) a transmembrane domain comprising a CD28 polypeptide (e.g., a human CD28 polypeptide, e.g., the transmembrane domain of CD28 (e.g., human CD28) or a portion thereof); iii) a hinge / spacer region derived from a CD28 polypeptide (e.g., a human CD28 polypeptide); and iv) an intracellular signaling domain comprising: a) a native CD3ζ polypeptide; and b) a costimulatory signaling region comprising a CD28 polypeptide (e.g., a human CD28 polypeptide, e.g., the intracellular domain of CD28 (e.g., human CD28) or a portion thereof). In certain embodiments, the transmembrane domain comprises a CD28 polypeptide comprising or consisting of amino acids 153-179 of SEQ ID NO:7. In certain embodiments, the hinge / spacer region comprises a CD28 polypeptide comprising or consisting of amino acids 114-152 of SEQ ID NO:7. In certain embodiments, the intracellular signaling domain comprises a costimulatory signaling region comprising a native CD3ζ polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:9, and a CD28 polypeptide comprising or consisting of amino acids 180-220 of SEQ ID NO:7. In certain embodiments, the CAR is referred to as "28z." In certain embodiments, the CAR (e.g., 28z) is encoded by a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% identical to the nucleotide sequence set forth in SEQ ID NO:27. In certain embodiments, the CAR (e.g., 28z) comprises the nucleotide sequence set forth in SEQ ID NO:27. SEQ ID NO:27 is provided below. [query number 27]

[0218] In certain embodiments, the first antigen-recognizing receptor is a CAR comprising: i) an extracellular antigen-binding domain; ii) a transmembrane domain comprising a CD28 polypeptide (e.g., a human CD28 polypeptide, e.g., the transmembrane domain of CD28 (e.g., human CD28) or a portion thereof); iii) a hinge / spacer region derived from a CD28 polypeptide (e.g., a human CD28 polypeptide); iv) an intracellular signaling domain comprising: a) a modified CD3ζ polypeptide (e.g., a modified human CD3ζ polypeptide) comprising a native ITAM1, an ITAM2 variant consisting of two loss-of-function mutations, and an ITAM3 variant consisting of two loss-of-function mutations; and b) a costimulatory signaling region comprising a CD28 polypeptide (e.g., a human CD28 polypeptide, e.g., the intracellular domain of CD28 (e.g., human CD28) or a portion thereof). In certain embodiments, the transmembrane domain comprises a CD28 polypeptide comprising or consisting of amino acids 153-179 of SEQ ID NO:7. In certain embodiments, the hinge / spacer region comprises a CD28 polypeptide comprising or consisting of amino acids 114-152 of SEQ ID NO:7. In certain embodiments, the intracellular signaling domain comprises a modified CD3ζ polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:22, and a costimulatory signaling region comprising a CD28 polypeptide comprising or consisting of amino acids 180-220 of SEQ ID NO:7. In certain embodiments, a CAR is referred to as "28z1xx." In certain embodiments, a CAR (e.g., 28z1xx) is encoded by a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% identical to the nucleotide sequence set forth in SEQ ID NO:28 or SEQ ID NO:29. In certain embodiments, a CAR (e.g., 28z1xx) comprises the nucleotide sequence set forth in SEQ ID NO:28 or SEQ ID NO:29. SEQ ID NO:28 and SEQ ID NO:29 are provided below. ATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTTCAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTTCAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTTCCAGGGTCTCAGTACAGCCACCAAGGACACCTTCGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC [SEQ ID NO: 28] ATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTTCAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTTCAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTTCCAGGGTCTCAGTACAGCCACCAAGGACACCTTCGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC [SEQ ID NO: 29]

[0219] In certain embodiments, the first antigen-recognizing receptor is a CAR comprising i) an extracellular antigen-binding domain, ii) a transmembrane domain, iii) a hinge / spacer region, and iv) a costimulatory signaling region comprising a) a native CD3ζ polypeptide, and b) a 4-1BB polypeptide (e.g., a human 4-1BB polypeptide, e.g., the intracellular domain of 4-1BB (e.g., human 4-1BB) or a portion thereof). In certain embodiments, the intracellular signaling domain comprises a native CD3ζ polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 9, and a costimulatory signaling region comprising a 4-1BB polypeptide comprising or consisting of amino acids 214 to 255 of SEQ ID NO: 26. In certain embodiments, the CAR is designated "BBz." In certain embodiments, a CAR (e.g., BBz) is encoded by a nucleotide sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% identical to the nucleotide sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 31. In certain embodiments, a CAR (e.g., BBz) comprises the nucleotide sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 31. SEQ ID NO: 30 and SEQ ID NO: 31 are provided below. [query number 30] [SEQ ID NO: 31]

[0220] 2.1.3. Chimeric Ligand Receptors In certain embodiments, the first antigen-recognizing receptor is a chimeric ligand receptor comprising a ligand or a portion thereof that binds to the first antigen, hi certain embodiments, the chimeric ligand receptor further comprises a transmembrane domain and an intracellular signaling domain.

[0221] In certain embodiments, the transmembrane domain is fused to a ligand or portion thereof. In certain embodiments, the transmembrane domain is fused to an intracellular signaling domain. In certain embodiments, the transmembrane domain is located between the ligand or portion thereof and the intracellular signaling domain. In certain embodiments, the transmembrane domain of the chimeric ligand receptor is a transmembrane domain disclosed in Section 2.1.2.2. In certain embodiments, the intracellular signaling domain of the chimeric ligand receptor comprises a CD3ζ polypeptide (e.g., as disclosed in Section 2.1.2.3).

[0222] Further information regarding the chimeric ligand receptors disclosed herein can be found in Sauer et al., Blood (2021) 138(4):318-330, the contents of which are incorporated by reference in their entirety.

[0223] 2.1.4. Delivery of the First Antigen-Recognizing Receptor In certain embodiments, the first antigen-recognizing receptor is delivered to the cell by a viral method. In certain embodiments, the viral method includes a viral vector. In certain embodiments, the viral vector is a retroviral vector (e.g., a gammaretroviral vector or a lentiviral vector). Other viral vectors include adenoviral vectors, adeno-associated viral vectors, vaccinia virus, bovine papillomavirus, and herpesviruses (e.g., Epstein-Barr virus).

[0224] In certain embodiments, the first antigen-recognizing receptor is delivered to cells by a non-viral method. Any targeted genome editing method can also be used to deliver the first antigen-recognizing receptor to cells. In certain embodiments, the first antigen-recognizing receptor is delivered to cells by a method comprising homologous recombination, zinc finger nuclease, meganuclease, transcription activator-like effector nuclease (TALEN), clustered regularly interspaced short palindromic repeats (CRISPR) system, or a combination thereof. In certain embodiments, the CRISPR system is used to deliver the first antigen-recognizing receptor to cells.

[0225] In certain embodiments, the cell is a T cell, and the first antigen recognition receptor is integrated into a locus within the genome of the T cell. Non-limiting examples of loci include the TRAC locus, the TRBC locus, the TRDC locus, and the TRGC locus. In certain embodiments, the locus is the TRAC locus or the TRBC locus. In certain embodiments, the cell is a T cell, and the first antigen recognition receptor is integrated into the TRAC locus. Methods of targeting a CAR to a site within the genome of a T cell are disclosed in WO2017 / 180989 and Eyquem et al., Nature. (2017 Mar 2); 543(7643): 113-117, both of which are incorporated by reference in their entireties. In certain embodiments, the cell is a T cell, and the first antigen recognition receptor is a CAR, and the first antigen recognition receptor is integrated into the TRAC locus. In certain embodiments, the cell further comprises a genetic disruption of the TRBC locus. In certain embodiments, the gene disruption of the TRBC locus results in a knockout of the TRBC locus.

[0226] 2.2. Second antigen-recognition receptor The second antigen-recognizing receptor targets a second antigen. The second antigen can be a tumor antigen or a pathogen antigen. In certain embodiments, the second antigen-recognizing receptor is a TCR-like fusion molecule.

[0227] 2.2.1. Second Antigen In certain embodiments, the second antigen is a tumor antigen, such as those disclosed in Section 2.1.1. In certain embodiments, the tumor antigen is an antigen with low antigen density. In certain embodiments, the tumor antigen is expressed on cells with low tumor cell frequency.

[0228] In certain embodiments, the second antigen is selected from the group consisting of CD19, CD70, IL1RAP, ABCG2, AChR, ACKR6, ADAMTS13, ADGRE2, ADGRE2(EMR2), ADORA3, ADRA1D, AGER, ALS2, an antigen of a cytomegalovirus (CMV)-infected cell (e.g., a cell surface antigen), ANO9, AQP2, ASIC3, ASPRV1, ATP6V0A4, B3GNT4, B7-H3, BCMA, BEST4, C3orf35, CADM3, CAIX, CAPN3, CCDC155, CCR1, CD10, CD117, CD1 23, CD133, CD135(FLT3), CD138, CD20, CD22, CD244(2B4), CD25, CD26, CD276, CD30, CD300LF, CD312, CD32, CD321, CD33, CD34, CD36, CD38, CD41, CD44, C D44V6, CD47, CD49f, CD56, CD7, CD71, CD74, CD8, CD82, CD96, CD98, CD99, CDH13, CDHR1, CEA, CEACAM6, CHST3, CLEC12A, CLEC1A, CLL1, CNIH2, COL15A1, C OLEC12, CPM, CR1, CX3CR1, CXCR4, CYP4F11, DAGLB, DARC, DFNB31, DGKI, EGF1R, EGFR-VIII, EGP-2, EGP-40, ELOVL6, EMB, EMC10, EMR2, ENG, EpCAM, EphA2 , EPHA4, ERBB, ERBB2, Erb-B3, Erb-B4, E-selectin, EXOC3L4, EXTL3, FAM186B, FBP, FCGR1A, FKBP1B, FLRT1, folate receptor-a, FOLR2, FRMD5, GABRB2, GAS2, GD2, GD3 , GDPD3, GNA14, GNAZ, GPR153, GPR56, GYPA, HEPHL1, HER-2, hERT, HILPDA, HLA-DR, HOOK1, hTERT, HTR2A, ICAM1, IGFBP3, IL10RB, IL20RB, IL23R, ILDR1, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), ITFG3, ITGA4, ITGA5, ITGA8, ITGAX, ITGB5, ITGB8, JAM3, KCND1, KCNJ5, KCNK13, KCNN4, KCNV2, KDR, KIF19,KIF26B, kappa light chain, L1CAM, LAX1, LEPR, Lewis Y (CD174), Lewis Y (LeY), LILRA2, LILRA6, LILRB2, LILRB3, LILRB4, LOXL4, LPAR2, LRRC37A3, LRRC8E, LRRN2, LRRTM2, LTB4R, MAGE-A1, MAGEA3, MANSC1, MART1, GP100, MBOAT1, MBOAT7, melanoma antigen family A, mesothelin (MSLN), MFAP3L, MMP25, MRP1, MT-ND1, mucin 1 (MUC1), mucin 16 (MUC16), MYADM, MYADML2, NGFR, NKCS1, NK G2D ligand, NLGN3, NPAS2, NY-ESO-1, oncofetal antigen (h5T4), OTOA, P2RY13, p53, PDE3A, PEAR1, PIEZO1, PLXNA4, PLXNC1, PNPLA3, PPFIA4, PPP2R5B, PRAME, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), proteinase 3 (PR1), PSD2, PTPRJ, RDH16, receptor tyrosine-protein kinase Erb-B2, RHBDL3, RNF173, RNF183, ROR1, RYR2, SCIN, SCN11A, SCN2A, SCNN1D, SEC31B, SEMA4A, SH3PXD2A, SIGLEC11, SIRPB1, SLC16A6, SLC19A1, SLC22A5, SLC25A36, SLC25A41, SLC30A1 , SLC34A3, SLC43A3, SLC44A1, SLC44A3, SLC45A3, SLC6A16, SLC6A6, SLC8A3, SLC9A1, SLCO2B1, SPAG17, STC1, STON2, SUN3, Survivin, SUSD2, SYNC, T ACSTD2, TAS1R3, TEX29, TFR2, TIM-3 (HAVCR2), TLR2, TMEFF2, TMEM145, TMEM27, TMEM40, TMEM59L, TMEM89, TMPRSS5, TNFRSF14, TNFRSF1B, TRIM55, TSPEAR, TTYH3, tumor-associated glycoprotein 72 (TAG-72), tyrosinase, vascular endothelial growth factor R2 (VEGF-R2), VLA-4, Wilms tumor protein (WT-1), WNT4, WT1, and ZDHHC11.

[0229] In certain embodiments, the second antigen is selected from the group consisting of CD70, SIGLEC-6, IL1RAP, CLEC12A, GRP78, TIM3, CD19, CD20, CD22, BCMA, GPRC5D, SLAMF7, CD276, and CAIX. In certain embodiments, the second antigen is CD70.

[0230] In certain embodiments, the first antigen and the second antigen are different. In certain embodiments, the second antigen is CD70. In certain embodiments, the first antigen is CD312 and the second antigen is CD70. In certain embodiments, the first antigen is CD276 and the second antigen is CD70.

[0231] In certain embodiments, the second antigen is CD 19. In certain embodiments, the first antigen is CD22 and the second antigen is CD19.

[0232] In certain embodiments, the second antigen is a pathogen antigen, such as those disclosed in Section 2.1.1.

[0233] 2.2.2.TCR-like fusion molecules In certain embodiments, the second antigen-recognizing receptor is a TCR-like fusion molecule. Non-limiting examples of TCR fusion molecules include HLA-independent TCR-based chimeric antigen receptors (also known as "HITs" and disclosed, for example, in International Patent Application No. PCT / US19 / 017525, the entire contents of which are incorporated by reference), and T cell receptor fusion constructs (TRuCs) (for example, those disclosed in Baeuerle et al., "Synthetic TRuC receptors engaging the complete T cell receptor for potent anti-tumor response," Nature Communications volume 10, Article number: 2087 (2019), the entire contents of which are incorporated by reference).

[0234] In certain embodiments, the TCR-like fusion molecule is a recombinant T cell receptor (TCR). In certain embodiments, the recombinant TCR comprises at least one antigen-binding chain. In certain embodiments, the antigen-binding domain of the recombinant TCR comprises a ligand for a cell surface receptor, a receptor for a cell surface ligand, an antigen-binding portion of an antibody or fragment thereof, or an antigen-binding portion of a TCR. In certain embodiments, the recombinant TCR comprises two antigen-binding chains, i.e., a first antigen-binding chain and a second antigen-binding chain. In certain embodiments, the first and second antigen-binding chains each comprise a constant domain. In certain embodiments, the recombinant TCR binds to an antigen (e.g., a first antigen or a second antigen) in an HLA-independent manner. Thus, in certain embodiments, the recombinant TCR is an HLA-independent (or non-HLA-restricted) TCR (referred to as "HIT").

[0235] In certain embodiments, the first antigen-binding chain comprises a heavy chain variable region (V H In certain embodiments, the second antigen-binding chain comprises an antigen-binding fragment of the light chain variable region (V L In certain embodiments, the first antigen-binding chain comprises an antigen-binding fragment of the V Hand the second antigen-binding chain comprises an antigen-binding fragment of the V L and antigen-binding fragments of the

[0236] In certain embodiments, the constant domain comprises a TCR constant region selected from the group consisting of a native or modified TRAC polypeptide, a native or modified TRBC polypeptide, a native or modified TRDC polypeptide, a native or modified TRGC polypeptide, and any variant or functional fragment thereof. In certain embodiments, the constant domain comprises a native or modified TRAC polypeptide. In certain embodiments, the constant domain comprises a native or modified TRBC polypeptide. In certain embodiments, the first antigen binding chain comprises a TRAC polypeptide and the second antigen binding chain comprises a TRBC polypeptide. In certain embodiments, the first antigen binding chain comprises a TRBC polypeptide and the second antigen binding chain comprises a TRAC polypeptide.

[0237] In certain embodiments, the first antigen-binding chain is H and a TRAC polypeptide, and the second antigen-binding chain comprises the V L and TRBC polypeptides.

[0238] In certain embodiments, the first antigen-binding chain is H and a TRBC polypeptide, wherein the second antigen-binding chain comprises the V L and TRAC polypeptides.

[0239] In certain embodiments, at least one of the TRAC polypeptide and the TRBC polypeptide is endogenous. In certain embodiments, the TRAC polypeptide is endogenous. In certain embodiments, the TRBC polypeptide is endogenous. In certain embodiments, both the TRAC polypeptide and the TRBC polypeptide are endogenous.

[0240] In certain embodiments, the antigen binding chain can associate with a CD3ζ polypeptide. In certain embodiments, when the antigen binding chain binds to an antigen, it can activate the CD3ζ polypeptide associated with the antigen binding chain. In certain embodiments, activation of the CD3ζ polypeptide can activate immunoresponsive cells. In certain embodiments, the TCR-like fusion molecule can incorporate into the CD3 complex and provide HLA-independent antigen recognition. In certain embodiments, the TCR-like fusion molecule replaces the endogenous TCR in the CD3 / TCR complex.

[0241] In certain embodiments, the first and second antigen binding chains are about 2 x 10 -7 The dissociation constant (K D ) binds to the antigen. In certain embodiments, the first and second antigen binding chains bind to the antigen with high binding affinity. In certain embodiments, the K D is about 2 x 10 -7 M or less, approximately 1×10 -7 M or less, approximately 9 x 10 -8 M or less, approximately 1×10 -8 M or less, approximately 9 x 10 -9 M or less, about 5 x 10 -9 M or less, approximately 4 x 10 -9 M or less, about 3 x 10 -9 Below, approximately 2×10 -9 M or less, or about 1 x 10 -9 In certain embodiments, K D is approximately 1 x 10 -8 In certain embodiments, K D is about 3 x 10 -9 In certain embodiments, K D is about 5 x 10 -9 In certain embodiments, K D is approximately 1 x 10 -9 M ~ approx. 1×10 -8 M. In certain embodiments, K D is approximately 1.5 x 10 -9 M ~ approx. 1×10 -8 M. In certain embodiments, K Dis about 5 x 10 -9 M ~ approx. 1×10 -8 It's M.

[0242] In certain embodiments, the constant domain comprises a TCR constant region, e.g., a T cell receptor alpha constant region (TRAC), a T cell receptor beta constant region (TRBC, e.g., TRBC1 or TRBC2), a T cell receptor gamma constant region (TRGC, e.g., TRGC1 or TRGC2), a T cell receptor delta constant region (TRDC), or any variant or functional fragment thereof.

[0243] In certain embodiments, the first antigen-binding chain or the second antigen-binding chain comprises a constant domain comprising a native or modified TRAC polypeptide. In certain embodiments, the TRAC polypeptide comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 40 or a fragment thereof, and / or may optionally contain up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, the TRAC polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 40. SEQ ID NO: 40 is provided below. IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS [SEQ ID NO: 40]

[0244] An exemplary nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:40 is set forth in SEQ ID NO:41, provided below. ATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCAT GTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC [SEQ ID NO: 41]

[0245] In certain embodiments, a TRAC polypeptide comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 42, or a fragment thereof, and / or may optionally contain up to one, or up to two, or up to three conservative amino acid substitutions. In certain embodiments, a TRAC polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 42. SEQ ID NO: 42 is provided below. IPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS [SEQ ID NO: 42]

[0246] An exemplary nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:42 is set forth in SEQ ID NO:43, provided below. ATTCCCAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTT TGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC [SEQ ID NO: 43]

[0247] In certain embodiments, a TRAC polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to an amino acid sequence encoded by a transcript expressed by the gene of NCBI Genbank ID: 28755, NG_001332.3, range 925603-930229 (SEQ ID NO: 44), or a fragment thereof, and / or may optionally contain up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, a TRAC polypeptide comprises or consists of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 44. SEQ ID NO: 44 is provided below.

[0248] In certain embodiments, the first antigen-binding chain or the second antigen-binding chain comprises a constant domain comprising a native or modified TRBC polypeptide. In certain embodiments, the TRBC polypeptide is a TRBC2 polypeptide. In certain embodiments, the TRBC2 polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 45 or a fragment thereof, and / or may optionally contain up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, the TRBC2 polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 45. SEQ ID NO: 45 is provided below. DLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG [SEQ ID NO: 45]

[0249] An exemplary nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:45 is set forth in SEQ ID NO:46, provided below. [SEQ ID NO: 46]

[0250] In certain embodiments, the TRBC2 polypeptide is a TRBC2 polypeptide. In certain embodiments, the TRBC2 polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 47 or a fragment thereof, and / or may optionally contain up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, the TRBC2 polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 47. SEQ ID NO: 47 is provided below. LEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG [SEQ ID NO: 47]

[0251] An exemplary nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:47 is set forth in SEQ ID NO:48, provided below. [SEQ ID NO: 48]

[0252] In certain embodiments, the TRBC polypeptide is a TRBC1 polypeptide. In certain embodiments, the TRBC1 polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 49 or a fragment thereof, and / or may optionally contain up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, the TRBC1 polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 49. SEQ ID NO: 49 is provided below. LNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF [SEQ ID NO: 49]

[0253] In certain embodiments, the TRBC1 polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 50 or a fragment thereof, and / or may optionally contain up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, the TRBC1 polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 50. SEQ ID NO: 50 is provided below. DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF [SEQ ID NO: 50]

[0254] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:50 is set forth in SEQ ID NO:51, provided below. [SEQ ID NO: 51]

[0255] In certain embodiments, the TRBC polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence or fragment thereof encoded by a transcript expressed by the gene of NCBI Genbank ID: 28639, NG_001333.2, range 645749-647196 (TRBC1, SEQ ID NO: 52), NCBI Genbank ID: 28638, NG_001333.2, range 655095-656583 (TRBC2, SEQ ID NO: 53), and / or optionally contains up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, the TRBC polypeptide comprises or consists of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 52. In certain embodiments, the TRBC polypeptide comprises or consists of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 53. SEQ ID NOs: 52 and 53 are provided below.

[0256] In certain embodiments, the first antigen-binding chain or the second antigen-binding chain comprises a constant domain comprising a native or modified TRGC polypeptide. In certain embodiments, the TRGC polypeptide is a native or modified TRGC1 polypeptide. In certain embodiments, the TRGC1 polypeptide comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 54 provided below. In certain embodiments, the TRGC1 polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 54. DKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGNTMKTNDTYMKFSWLTVPEKSLDKEHRCIVRHENNKNGVDQEIIFPPIKTDVITMDPKDNCSKDANDTLLLQLTNTSAYYMYLLLLLKSVVYFAIITCCLLRRTAFCCNGEKS [SEQ ID NO: 54]

[0257] In certain embodiments, the TRGC polypeptide is a native or modified TRGC2 polypeptide. In certain embodiments, the TRGC2 polypeptide comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 55, provided below. In certain embodiments, the TRGC2 polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 55. DKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDIIKIHWQEKKSNTILGSQEGNTMKTNDTYMKFSWLTVPEESLDKEHRCIVRHENNKNGIDQEIIFPPIKTDVTTVDPKYNYSKDANDVITMDPKDNWSKDANDTLLLQLTNTSAYYTYLLLLLKSVVYFAIITCCLLRRTAFCCNGEKS [SEQ ID NO: 55]

[0258] In certain embodiments, the TRGC polypeptide comprises or consists of an amino acid sequence that is at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% homologous or identical to the amino acid sequence or fragment thereof encoded by a transcript expressed by the gene of NCBI Genbank ID: 6966, NG_001336.2, range 108270-113860 (TRGC1, SEQ ID NO: 56), NCBI Genbank ID: 6967, NG_001336.2, range 124376-133924 (TRGC2, SEQ ID NO: 57), and / or may optionally contain up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, the TRGC polypeptide comprises or consists of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 56. In certain embodiments, the TRGC polypeptide comprises or consists of the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 57. SEQ ID NOs: 56 and 57 are provided below. TGTCTGCTTAGAAGAACGGCTTTCTGCTGCAATGGAGAGAAATCATAA [SEQ ID NO: 57]

[0259] In certain embodiments, the first antigen-binding chain or the second antigen-binding chain comprises a constant domain comprising a native or modified TRDC polypeptide. In certain embodiments, the TRDC polypeptide comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 58, as provided below. In certain embodiments, the TRDC polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58. SQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKLGKYEDSNSVTCSVQHDNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTVAVNFLLTAKLFFL [SEQ ID NO: 58]

[0260] In certain embodiments, the TCR-like fusion molecule comprises a hinge / spacer region linking the first antigen-binding chain to the constant domain. In certain embodiments, the TCR-like fusion molecule comprises a hinge / spacer region linking the second antigen-binding chain to the constant domain. The hinge / spacer region may be sufficiently flexible to allow the antigen-binding chain to orient in different directions to facilitate antigen recognition. In certain embodiments, the hinge / spacer region may be a hinge region from IgG1, an immunoglobulin CH2CH3 region, a portion of CD3, a portion of a TCRα polypeptide, a portion of a TCRβ polypeptide, a portion of a CD28 polypeptide, a portion of a CD8 polypeptide, or a synthetic spacer sequence. In certain embodiments, the hinge / spacer region comprises a portion of a TCRα polypeptide. In certain embodiments, the hinge / spacer region comprises a portion of a variable region (TRAV), a portion of a diversity region (TRAD), a portion of a joining region (TRAJ), a portion of a constant region (TRAC), or a combination thereof. In certain embodiments, the hinge / spacer region comprises a portion of the TRAJ region and a portion of the TRAC region of a TCR alpha polypeptide. In certain embodiments, the hinge / spacer region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 59. In certain embodiments, the hinge / spacer region comprises or consists of amino acids 1-3 of the sequence set forth in SEQ ID NO: 59. An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 59 is set forth in SEQ ID NO: 60. SEQ ID NOs: 59 and 60 are provided below. IPNIQNPDPA [SEQ ID NO: 59] ATTCCCAATATCCAGAACCCTGACCCTGCC [SEQ ID NO: 60]

[0261] In certain embodiments, the hinge / spacer region comprises a portion of a TCR β polypeptide. In certain embodiments, the hinge / spacer region comprises a portion of the variable region (TRBV), a portion of the diversity region (TRBD), a portion of the joining region (TRBJ), a portion of the constant region (TRBC), or a combination thereof. In certain embodiments, the hinge / spacer region comprises a portion of the TRBJ region and a portion of the TRAC region (C) of a TCR β polypeptide. In certain embodiments, the hinge / spacer region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 61. In certain embodiments, the hinge / spacer region comprises or consists of amino acids 1-2 of the sequence set forth in SEQ ID NO: 61. An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 61 is set forth in SEQ ID NO: 62. SEQ ID NOs: 61 and 62 are provided below. LEDLKNVFPPE [SEQ ID NO: 61] CTGGAGGATCTGAAAAACGTGTTCCCTCCTGAA [SEQ ID NO: 62]

[0262] In certain embodiments, the antigen binding chain does not comprise an intracellular domain. In certain embodiments, the antigen binding chain can associate with a CD3ζ polypeptide. In certain embodiments, the antigen binding chain is associated with a CD3ζ polypeptide via a constant domain. In certain embodiments, the CD3ζ polypeptide is endogenous. In certain embodiments, the CD3ζ polypeptide is exogenous. In certain embodiments, binding of the antigen binding chain to a target antigen can activate the CD3ζ polypeptide associated with the antigen binding chain. In certain embodiments, the exogenous CD3ζ polypeptide is fused to or incorporated into a costimulatory molecule disclosed herein.

[0263] In certain embodiments, the TCR-like fusion molecule comprises an antigen-binding chain comprising an intracellular domain. In certain embodiments, the intracellular domain comprises a CD3ζ polypeptide. In certain embodiments, binding of the antigen-binding chain to an antigen can activate the CD3ζ polypeptide of the antigen-binding chain.

[0264] In certain embodiments, CD3ζ polypeptide comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous to the amino acid sequence set forth in SEQ ID NO: 12 or a fragment thereof, and / or may optionally contain up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, CD3ζ polypeptide comprises or consists of an amino acid sequence that is a contiguous portion of SEQ ID NO: 8 that is at least about 20, or at least about 30, or at least about 40, or at least about 50, and up to about 164 amino acids in length. In certain embodiments, CD3ζ comprises or consists of the amino acid sequence of amino acids 1-164, 1-50, 50-100, 52-164, 100-150, or 150-164 of SEQ ID NO: 8. In certain embodiments, the CD3ζ polypeptide comprises or consists of amino acids 52 to 164 of SEQ ID NO:8.

[0265] In certain embodiments, a CD3ζ polypeptide comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to SEQ ID NO:9 or a fragment thereof, and / or may optionally contain up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, a CD3ζ polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:9.

[0266] In certain embodiments, the TCR-like fusion molecule comprises an antigen-binding chain comprising an intracellular domain, wherein the intracellular domain comprises a costimulatory signaling region. In certain embodiments, the intracellular domain comprises a costimulatory signaling region and a CD3ζ polypeptide. In certain embodiments, the intracellular domain comprises a costimulatory signaling region and does not comprise a CD3ζ polypeptide. In certain embodiments, the costimulatory signaling region comprises at least the intracellular domain of a costimulatory molecule disclosed herein.

[0267] In certain embodiments, the TCR-like fusion molecule can associate with the CD3 complex (also known as the "T cell co-receptor"). In certain embodiments, the TCR-like fusion molecule and the CD3 complex form an antigen-recognition receptor complex similar to a natural TCR / CD3 complex. In certain embodiments, the CD3 complex is endogenous. In certain embodiments, the CD3 complex is exogenous. In certain embodiments, the TCR-like fusion molecule replaces a natural and / or endogenous TCR in a CD3 / TCR complex. In certain embodiments, the CD3 complex comprises a CD3γ chain, a CD3δ chain, and two CD3ε chains.

[0268] In certain embodiments, the CD3 gamma chain comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to the amino acid sequence having NCBI Reference Number: NP_000064.1 (SEQ ID NO: 63), or a fragment thereof, and / or may optionally contain up to one, or up to two, or up to three conservative amino acid substitutions. SEQ ID NO: 63 is provided below. MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN [SEQ ID NO: 63]

[0269] In certain embodiments, the CD3 delta chain comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to the amino acid sequence having NCBI Reference Number: NP_000723.1 (SEQ ID NO: 64), or a fragment thereof, or the amino acid sequence having NCBI Reference Number: NP_001035741.1 (SEQ ID NO: 65), or a fragment thereof, and / or may optionally contain up to one, up to two, or up to three conservative amino acid substitutions. SEQ ID NOs: 64 and 65 are provided below. MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNK [SEQ ID NO: 64] MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRTADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNK [SEQ ID NO: 65]

[0270] In certain embodiments, the CD3 epsilon chain comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% homologous or identical to the amino acid sequence having NCBI Reference Number: NP_000724.1 (SEQ ID NO: 66), or a fragment thereof, and / or may optionally contain up to one, or up to two, or up to three conservative amino acid substitutions. SEQ ID NO: 66 is provided below. MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI [SEQ ID NO: 66]

[0271] In certain embodiments, TCR-like fusion molecules exhibit higher antigen sensitivity than CARs targeting the same antigen. In certain embodiments, TCR-like fusion molecules can induce an immune response when bound to an antigen with low antigen density on the surface of tumor cells. In certain embodiments, cells containing TCR-like fusion molecules can be used to treat subjects with tumor cells that have low levels of surface antigen expression, for example, to prevent disease recurrence, and the subject has undergone treatment that results in residual tumor cells. In certain embodiments, tumor cells have a low antigen density of target molecules on the surface of the tumor cells. In certain embodiments, target molecules with low antigen density on the cell surface have a density of less than about 5,000 molecules per cell, less than about 4,000 molecules per cell, less than about 3,000 molecules per cell, less than about 2,000 molecules per cell, less than about 1,500 molecules per cell, less than about 1,000 molecules per cell, less than about 500 molecules per cell, less than about 200 molecules per cell, or less than about 100 molecules per cell. In certain embodiments, target molecules having a low antigen density on the cell surface have a density of less than about 2,000 molecules per cell. In certain embodiments, target molecules having a low antigen density on the cell surface have a density of less than about 1,500 molecules per cell. In certain embodiments, target molecules having a low antigen density on the cell surface have a density of less than about 1,000 molecules per cell. In certain embodiments, target molecules having a low antigen density on the cell surface have a density of about 4,000 molecules per cell to about 2,000 molecules per cell, about 2,000 molecules per cell to about 1,000 molecules per cell, about 1,500 molecules per cell to about 1,000 molecules per cell, about 2,000 molecules per cell to about 500 molecules per cell, about 1,000 molecules per cell to about 200 molecules per cell, or about 1,000 molecules per cell to about 100 molecules per cell.

[0272] In certain embodiments, TCR-like fusion molecules can induce an immune response when binding to an antigen expressed on the surface of tumor cells with a low tumor cell frequency. In certain embodiments, cells containing TCR-like fusion molecules can be used to treat subjects with tumor cells with a low tumor cell frequency, for example, to treat disease recurrence, where the subject has undergone treatment that results in residual tumor cells. In certain embodiments, tumors with a low tumor cell frequency have a frequency of less than about 40% per tumor, less than about 30% per tumor, less than about 20% per tumor, less than about 15% per tumor, less than about 10% per tumor, less than about 5% per tumor, less than about 2% per tumor, or less than about 1% per tumor. In certain embodiments, the low tumor cell frequency is less than about 2% per tumor. In certain embodiments, the low tumor cell frequency is less than about 1.5% per tumor. In certain embodiments, the low tumor cell frequency is less than about 1% per tumor. In certain embodiments, low tumor cell frequency is between about 40% per tumor and about 20% per tumor, between about 20% per tumor and about 10% per tumor, between about 15% per tumor and about 10% per tumor, between about 20% per tumor and about 5% per tumor, between about 10% per tumor and about 2% per tumor, or between about 10% per tumor and about 1% per tumor.

[0273] In certain embodiments, the second antigen-recognizing receptor is a V H and a first antigen-binding chain comprising a constant domain comprising a TRBC polypeptide; and a V L and a second antigen binding chain comprising a constant domain comprising a TRAC polypeptide. In certain embodiments, the first antigen binding chain is a TCR-like fusion molecule comprising a "V H In certain embodiments, the second antigen-binding chain is designated "V-TRBC chain." L In certain embodiments, the first antigen-binding chain is designated as a V-TRAC chain. H and the TRBC polypeptide. In certain embodiments, the hinge region comprises or consists of the amino acid sequence set forth in SEQ ID NO: 59 or SEQ ID NO: 61. In certain embodiments, the second antigen-binding chain comprises a V Land the TRAC polypeptide. In certain embodiments, the hinge region comprises or consists of the amino acid sequence set forth in SEQ ID NO:59 or SEQ ID NO:61.

[0274] In certain embodiments, the second antigen-recognizing receptor is a V H and a first antigen-binding chain comprising a constant domain comprising a TRAC polypeptide; and a V L and a second antigen binding chain comprising a constant domain comprising a TRBC polypeptide. In certain embodiments, the first antigen binding chain is a TCR-like fusion molecule comprising a "V H In certain embodiments, the second antigen-binding chain is designated as "V-TRAC chain." L In certain embodiments, the first antigen-binding chain is designated as a V-TRBC chain. H and the TRAC polypeptide. In certain embodiments, the second antigen-binding chain comprises a hinge region between V L and the TRBC polypeptide. In certain embodiments, the first antigen-binding chain and the second antigen-binding chain bind to a second antigen (e.g., human CD70).

[0275] In certain embodiments, the second antigen-recognizing receptor is a V H and a first antigen-binding chain comprising a constant domain comprising a TRBC polypeptide; and a V L and a second antigen binding chain comprising a constant domain comprising a TRAC polypeptide. In certain embodiments, the first antigen binding chain is a TCR-like fusion molecule comprising a "V H In certain embodiments, the second antigen-binding chain is designated "V-TRBC chain." L In certain embodiments, the first antigen-binding chain is designated as a V-TRAC chain. H and the TRAC polypeptide. In certain embodiments, the second antigen-binding chain comprises a hinge region between V Land the TRBC polypeptide. In certain embodiments, the first antigen-binding chain and the second antigen-binding chain bind to a second antigen (e.g., human CD70).

[0276] 2.2.2.1. Exemplary TCR-Like Fusion Molecules In certain embodiments, the second antigen-recognizing receptor is a TCR-like fusion molecule that binds to CD70 (e.g., human CD70) and comprises two antigen-binding chains, e.g., a V that can dimerize and bind to CD70. H and a first antigen-binding chain ("V") comprising a TRBC polypeptide. H -TRBC chain") and V L and a second antigen-binding chain ("V") comprising a TRBC polypeptide. L In certain embodiments, V H comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 133, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 134, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 135. H comprises the amino acid sequence set forth in SEQ ID NO: 139. In certain embodiments, V L comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 136, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 137, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 138. L comprises the amino acid sequence set forth in SEQ ID NO: 141. In certain embodiments, the TRAC polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 42. In certain embodiments, the TRBC polypeptide is a TRBC2 polypeptide. In certain embodiments, the TRBC2 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 47. In certain embodiments, the TCR-like fusion molecule is designated "70-HIT" or "70H". SEQ ID NOs: 133-142 are provided in Table 7 below.

[0277] In certain embodiments, the CDR regions / sequences disclosed herein are delineated using the Kabat system (Swindells et al., J Mol Biol. 2017 Feb 3;429(3):356-364). [Table 7-1] [Table 7-2]

[0278] In certain embodiments, the second antigen-recognizing receptor is a TCR-like fusion molecule that binds to CD70 (e.g., human CD70) and comprises two antigen-binding chains, e.g., a V that can dimerize and bind to CD70. H and a first antigen-binding chain ("V") comprising a TRBC polypeptide. H -TRBC chain") and V L and a second antigen-binding chain ("V") comprising a TRBC polypeptide. L In certain embodiments, V H is a V of the anti-CD70 antibody disclosed in International Patent Publication No. WO 2007 / 038637, which is incorporated by reference in its entirety. H In certain embodiments, the V H is the V of the anti-CD70 antibody 2H5 disclosed in International Patent Publication No. 2007 / 038637. H In certain embodiments, the V L is the V of the anti-CD70 antibody disclosed in International Patent Publication No. 2007 / 038637. H In certain embodiments, the V L is the V of the anti-CD70 antibody 2H5 disclosed in International Patent Publication No. 2007 / 038637. H It includes the sequences CDR1, CDR2, and CDR3.

[0279] In certain embodiments, the second antigen-recognizing receptor is a TCR-like fusion molecule that binds to CD19 (e.g., human CD19) and comprises two antigen-binding chains, e.g., a V that can dimerize and bind to CD19. H and a first antigen-binding chain ("V") comprising a TRBC polypeptide. H -TRBC chain") and V L and a second antigen-binding chain ("V") comprising a TRBC polypeptide. L In certain embodiments, V H comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 143, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 144, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 145. H comprises the amino acid sequence set forth in SEQ ID NO: 149. In certain embodiments, V L comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 146, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 147, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 148. L comprises the amino acid sequence set forth in SEQ ID NO: 151. In certain embodiments, the TRAC polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 42. In certain embodiments, the TRBC polypeptide is a TRBC2 polypeptide. In certain embodiments, the TRBC2 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 47. In certain embodiments, the TCR-like fusion molecule is designated "19-HIT" or "19H." SEQ ID NOs: 143-152 are provided in Table 8 below.

[0280] In certain embodiments, the CDR regions / sequences disclosed herein are delineated using the Kabat system (Swindells et al., J Mol Biol. 2017 Feb 3;429(3):356-364). [Table 11]

[0281] Various TCR-like fusion molecules are disclosed in International Patent Application Publication No. WO 2019 / 133969, which is incorporated by reference in its entirety.

[0282] 2.2.3. Delivery of a Second Antigen-Recognizing Receptor In certain embodiments, the second antigen-recognizing receptor is delivered to the cell by a viral method. In certain embodiments, the viral method includes a viral vector. In certain embodiments, the viral vector is a retroviral vector (e.g., a gamma retroviral vector or a lentiviral vector). Other viral vectors include adenoviral vectors, adeno-associated viral vectors, vaccinia virus, bovine papillomavirus, and herpesviruses (e.g., Epstein-Barr virus).

[0283] In certain embodiments, the second antigen-recognizing receptor is delivered to cells by a non-viral method. Any targeted genome editing method can also be used to deliver the second antigen-recognizing receptor to cells. In certain embodiments, the second antigen-recognizing receptor is delivered to cells by a method comprising homologous recombination, zinc finger nuclease, meganuclease, transcription activator-like effector nuclease (TALEN), clustered regularly interspaced short palindromic repeats (CRISPR) system, or a combination thereof. In certain embodiments, the CRISPR system is used to deliver the second antigen-recognizing receptor to cells.

[0284] In certain embodiments, the cell is a T cell, and the second antigen-recognizing receptor is integrated into a locus within the genome of the T cell. Non-limiting examples of loci include the TRAC locus, the TRBC locus, the TRDC locus, and the TRGC locus. In certain embodiments, the locus is the TRAC locus or the TRBC locus. In certain embodiments, the cell is a T cell, and the second antigen-recognizing receptor is integrated into the TRAC locus.

[0285] 2.3.CCR In certain embodiments, the cells disclosed herein comprising a first antigen-recognizing receptor and a second antigen-recognizing receptor further comprise a CCR. The term "chimeric costimulatory receptor" or "CCR" refers to a chimeric receptor that binds to an antigen and provides a costimulatory signal but does not provide a T cell activation signal to a CCR-containing cell. Various CCRs are described in US2002 / 0018783, the contents of which are incorporated by reference in their entirety. CCRs mimic costimulatory signals but, unlike CARs, do not provide a T cell activation signal. In certain embodiments, the CCR lacks the CD3ζ polypeptide.

[0286] CCRs provide costimulatory signals (e.g., CD28-like or 4-1BB-like signals) in the absence of natural costimulatory ligands on antigen-presenting cells. Combinatorial antigen recognition, i.e., the use of CCRs in combination with CARs, can enhance T cell reactivity against dual-antigen-expressing T cells, thereby improving selective tumor targeting. Kloss et al. have described a strategy that incorporates combinatorial antigen recognition, split signaling, and, importantly, balanced strength of T cell activation and costimulation to generate T cells that eliminate target cells expressing a combination of antigens while sparing cells expressing each antigen individually (Kloss et al., Nature Biotechnology (2013); 31(1):71-75, the contents of which are incorporated by reference in their entirety). In this approach, T cell activation requires CAR-mediated recognition of one antigen, while costimulation is independently mediated by a CCR specific for a second antigen. To achieve tumor selectivity, combinatorial antigen recognition approaches reduce the efficiency of T cell activation to a level that is ineffective without the rescue provided by simultaneous CCR recognition of a second antigen.

[0287] In certain embodiments, the CCR comprises an extracellular antigen-binding domain that binds to a third antigen and an intracellular domain that can deliver a costimulatory signal to a cell but does not alone deliver an activation signal to a cell. In certain embodiments, the CCR further comprises a transmembrane domain. In certain embodiments, the intracellular domain of the CCR comprises at least the intracellular domain of a costimulatory molecule, or a portion thereof. In certain embodiments, the costimulatory molecule is selected from the group consisting of CD28, 4-1BB, OX40, CD27, CD40, CD154, CD97, CD11a / CD18, ICOS, DAP-10, CD2, CD150, CD226, and NKG2D.

[0288] In certain embodiments, the CCR comprises the intracellular domain of CD28 or a portion thereof. In certain embodiments, the CCR comprises the intracellular domain of 4-1BB or a portion thereof. In certain embodiments, the CCR comprises the intracellular domain of CD28 or a portion thereof and the intracellular domain of 4-1BB or a portion thereof.

[0289] In certain embodiments, the third antigen is selected such that expression of both the first / second antigen and the third antigen is restricted to the target cell (e.g., cancer tissue or cancer cell, or LSC, or AML HSPC). Similar to the CAR, the extracellular antigen-binding domain can be an scFv, Fab, F(ab)2, or a fusion protein with heterologous sequences to form the extracellular antigen-binding domain.

[0290] In certain embodiments, cells comprising a first antigen-recognizing receptor, a second antigen-recognizing receptor, and a CCR exhibit a higher degree of cytolytic activity against cells positive for both the first / second antigen and the third antigen compared to cells positive for the first / second antigen alone. In certain embodiments, cells comprising a first antigen-recognizing receptor, a second antigen-recognizing receptor, and a CCR exhibit substantially no or negligible cytolytic activity against cells positive for the first / second antigen alone.

[0291] In certain embodiments, the first antigen-recognizing receptor and / or the second antigen-recognizing receptor has a low binding affinity, e.g., about 1×10 -8 M or more, about 5 x 10 -8 M or more, approximately 1×10 -7 M or more, about 5 x 10 -7 M or more, or approximately 1 x 10 -6 M or more, or approximately 1 x 10 -8 M ~ approx. 1×10 -6 Dissociation constant of M (K D ) to bind to the first antigen and the second antigen. In certain embodiments, the first antigen-recognizing receptor (e.g., a CAR, TCR, or TCR-like fusion molecule) binds to the first antigen with low binding affinity. In certain embodiments, the first antigen-recognizing receptor (e.g., a CAR, TCR, or TCR-like fusion molecule) binds to the first antigen at an epitope with low accessibility. In certain embodiments, the first antigen-recognizing receptor (e.g., a CAR, TCR, or TCR-like fusion molecule) binds to the first antigen with a lower binding affinity compared to the binding affinity with which the second antigen-recognizing receptor (e.g., a CCR) binds to the second antigen. In certain embodiments, the CCR binds to the first antigen with a lower binding affinity than the binding affinity with which the second antigen-recognizing receptor (e.g., a CCR) binds to the second antigen. -9 M ~ approx. 1×10 -7 M, e.g., about 1 x 10 -7 M or less, approximately 1×10 -8 M or less, or about 1 x 10 -9 Binding affinity K below M D and binds to a third antigen.

[0292] 2.4.T cell receptor (TCR) In certain embodiments, the cells disclosed herein comprising a first antigen-recognizing receptor and a second antigen-recognizing receptor further comprise a TCR. A TCR is a disulfide-linked heterodimeric protein consisting of two variable chains expressed as part of a complex with an invariant CD3 chain molecule. TCRs are found on the surface of T cells and are involved in recognizing antigens as peptides bound to major histocompatibility complex (MHC) molecules. In certain embodiments, the TCR comprises an alpha chain and a beta chain (encoded by TRA and TRB, respectively). In certain embodiments, the TCR comprises a gamma chain and a delta chain (encoded by TRG and TRD, respectively).

[0293] Each chain of the TCR is composed of two extracellular domains: a variable (V) region and a constant (C) region. The constant region is proximal to the cell membrane and is followed by a transmembrane region and a short cytoplasmic tail. The variable region binds to the peptide / MHC complex. The variable domains of both chains each have three complementarity-determining regions (CDRs).

[0294] In certain embodiments, a TCR can form a receptor complex with three dimeric signaling modules, CD3δ / ε, CD3γ / ε, and CD247ζ / ζ or ζ / η. Engagement of the TCR complex with its antigen and MHC (peptide / MHC) activates a T cell expressing the TCR complex.

[0295] In certain embodiments, the TCR is an endogenous TCR. In certain embodiments, the TCR is a naturally occurring TCR.

[0296] In certain embodiments, the TCR is an exogenous TCR. In certain embodiments, the TCR is a recombinant TCR. In certain embodiments, the TCR is a non-naturally occurring TCR. In certain embodiments, a non-naturally occurring TCR differs from any naturally occurring TCR by at least one amino acid residue. In certain embodiments, a non-naturally occurring TCR differs from any naturally occurring TCR by at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, or more amino acid residues. In certain embodiments, a non-naturally occurring TCR is modified from a naturally occurring TCR by at least one amino acid residue. In certain embodiments, a non-naturally occurring TCR has at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, or more amino acid residues modified from a naturally occurring TCR.

[0297] 2.5.Co-stimulatory Ligands In certain embodiments, the cells disclosed herein comprising a first antigen-recognizing receptor and a second antigen-recognizing receptor further comprise at least one recombinant or exogenous costimulatory ligand. For example, the cells disclosed herein can be further transduced with at least one costimulatory ligand such that the cells express or are induced to express the first antigen-recognizing receptor, the second antigen-recognizing receptor, and at least one costimulatory ligand. The at least one costimulatory ligand provides a costimulatory signal to the cells.

[0298] Non-limiting examples of costimulatory ligands include, but are not limited to, members of the tumor necrosis factor (TNF) superfamily and immunoglobulin (Ig) superfamily ligands. TNF is a cytokine involved in systemic inflammation and stimulates the acute phase response. Its primary role is in regulating immune cells. Members of the TNF superfamily share several common properties. Most TNF superfamily members are synthesized as type II transmembrane proteins (extracellular C-terminus) containing a short cytoplasmic segment and a relatively long extracellular region. Non-limiting examples of TNF superfamily members include nerve growth factor (NGF), CD40L (also known as "CD154"), 4-1BBL, TNF-α, OX40L, CD70, Fas ligand (FasL), CD30L, tumor necrosis factor beta (TNFβ) / lymphotoxin-alpha (LTα), lymphotoxin-beta (LTβ), CD257 / B cell activating factor (BAFF) / Blys / THANK / Tall-1, glucocorticoid-induced TNF receptor ligand (GITRL), TNF-related apoptosis-inducing ligand (TRAIL), and LIGHT (TNFSF14). The immunoglobulin (Ig) superfamily is a large group of cell surface and soluble proteins involved in cell recognition, binding, or adhesion processes. These proteins share structural features with immunoglobulins and possess immunoglobulin domains (Ig domains). Non-limiting examples of immunoglobulin superfamily ligands include CD80, CD86, and ICOSLG. In certain embodiments, the at least one costimulatory ligand is selected from the group consisting of 4-1BBL, CD80, CD86, CD70, GITRL, CD40L, OX40L, CD30L, TNFRSF14, ICOSLG, TRAIL, and combinations thereof.

[0299] In certain embodiments, the cells further comprise one exogenous costimulatory ligand that is 4-1BBL. In certain embodiments, the costimulatory ligand is human 4-1BBL. In certain embodiments, 4-1BBL comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% homologous or identical to an amino acid sequence having Uniprot Reference Number: P41273-1 (SEQ ID NO: 67) or a fragment thereof, and / or may optionally contain up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, 4-1BBL comprises or consists of an amino acid sequence that is a contiguous portion of the amino acid sequence of SEQ ID NO: 67. SEQ ID NO: 67 is provided below. MEYASDASLDPEAPWPPAPRARACRVLPWALVAGLLLLLLLAAACAVFLACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE [SEQ ID NO: 67]

[0300] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO:67 is set forth in SEQ ID NO:68. [query number 68]

[0301] In certain embodiments, the cells further comprise one exogenous costimulatory ligand that is CD80. In certain embodiments, the costimulatory ligand is human CD80. In certain embodiments, the CD80 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% homologous or identical to the amino acid sequence having NCBI Reference Number: NP_005182 (SEQ ID NO: 69) or a fragment thereof, and / or may optionally contain up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, the CD80 comprises or consists of an amino acid sequence that is a contiguous portion of the amino acid sequence of SEQ ID NO: 69. SEQ ID NO: 69 is provided below. MGHTRRQGTSPSKCPYLNFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV [SEQ ID NO: 69]

[0302] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 69 is set forth in SEQ ID NO: 70. SEQ ID NO: 70 is provided below. ATGGGCCACACACGGAGGCAGGGAACATCACCATCCAAGTGTCCATACCTCAATTTCTTTCAGCTCTTGGTGCTGGCTGGTCTTTCTCACTTCTGTTCAGGTGTTATCCACGTGACCAAGGAAGTGAAAGAAGTGGCAACGCTGTCCTGTGGTCACAATGTTTCTGTTGAAGAGCTGGCACAAACTCGCATCTACTGGCAAAAGGAGAAGAAAATGGTGCTGACTATGATGTCTGGGGACATGAATATATGGCCCGAGTACAAGAACCGGACCATCTTTGATATCACTAATAACCTCTCCATTGTGATCCTGGCTCTGCGCCCATCTGACGAGGGCACATACGAGTGTGTTGTTCTGAAGTATGAAAAAGACGCTTTCAAGCGGGAACACCTGGCTGAAGTGACGTTATCAGTCAAAGCTGACTTCCCTACACCTAGTATATCTGACTTTGAAATTCCAACTTCTAATATTAGAAGGATAATTTGCTCAACCTCTGGAGGTTTTCCAGAGCCTCACCTCTCCTGGTTGGAAAATGGAGAAGAATTAAATGCCATCAACACAACAGTTTCCCAAGATCCTGAAACTGAGCTCTATGCTGTTAGCAGCAAACTGGATTTCAATATGACAACCAACCACAGCTTCATGTGTCTCATCAAGTATGGACATTTAAGAGTGAATCAGACCTTCAACTGGAATACAACCAAGCAAGAGCATTTTCCTGATAACCTGCTCCCATCCTGGGCCATTACCTTAATCTCAGTAAATGGAATTTTTGTGATATGCTGCCTGACCTACTGCTTTGCCCCAAGATGCAGAGAGAGAAGGAGGAATGAGAGATTGAGAAGGGAAAGTGTACGCCCTGTA [SEQ ID NO: 70]

[0303] In certain embodiments, the cells further comprise two exogenous costimulatory ligands, 4-1BBL and CD80. In certain embodiments, the cells further comprise two exogenous costimulatory ligands, 4-1BBL and CD80, wherein 4-1BBL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 67 and CD80 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 69.

[0304] Cells containing a receptor that includes at least one exogenous costimulatory ligand are described in US Pat. No. 8,389,282, which is incorporated by reference in its entirety.

[0305] 2.6. Fusion Polypeptides In certain embodiments, the cells disclosed herein comprising a first antigen-recognizing receptor and a second antigen-recognizing receptor further comprise a fusion polypeptide. For example, the cells disclosed herein can be further transduced with a fusion polypeptide such that the cells express or are induced to express the first antigen-recognizing receptor, the second antigen-recognizing receptor, and the fusion polypeptide. The fusion polypeptide provides a costimulatory signal to the cells. The fusion polypeptide can enhance the activity and / or efficacy of cells comprising the first antigen-recognizing receptor (e.g., a CAR or TCR-like fusion molecule). In certain embodiments, the fusion polypeptide comprises a) the extracellular domain and transmembrane domain of a costimulatory ligand and b) the intracellular domain of a first costimulatory molecule.

[0306] Non-limiting examples of costimulatory ligands include tumor necrosis factor (TNF) family members, immunoglobulin (Ig) superfamily members, and combinations thereof. TNF family members can be selected from the group consisting of 4-1BBL, OX40L, CD70, GITRL, CD40L, and combinations thereof. Ig superfamily members can be selected from the group consisting of CD80, CD86, ICOS ligand (ICOSLG (also known as "CD275"), and combinations thereof. In certain embodiments, the costimulatory ligand is selected from the group consisting of 4-1BBL, OX40L, CD70, GITRL, CD40L, CD80, CD86, ICOSLG, and combinations thereof.

[0307] In certain embodiments, the fusion polypeptide comprises the extracellular domain and transmembrane domain of a costimulatory ligand that is CD80. In certain embodiments, the costimulatory ligand is human CD80. In certain embodiments, the CD80 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 69 or a fragment thereof, and / or may optionally contain up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, the CD80 comprises or consists of an amino acid sequence that is a contiguous portion of the amino acid sequence of SEQ ID NO: 69.

[0308] In certain embodiments, the extracellular domain of CD80 comprises or consists of an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to amino acids 1-242 of SEQ ID NO: 69. In certain embodiments, the extracellular domain of CD80 comprises or consists of amino acids 1-242 of SEQ ID NO: 69, or a functional fragment thereof. A functional fragment can be a contiguous portion of amino acids 1-242 of SEQ ID NO:69 that is at least about 50, at least about 75, at least about 100, at least about 125, at least about 150, at least about 175, or at least about 200, or at least about 220 amino acids in length. In certain embodiments, a functional fragment retains at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% of the primary functions of the extracellular domain of CD80. Non-limiting examples of the primary functions of the extracellular domain of CD80 include binding to / interacting with CD28, binding to / interacting with CTLA-4, binding to / interacting with PD-L1, and contributing to CD80 homodimerization. In certain embodiments, the extracellular domain of CD80 comprises or consists of amino acids 1-242 of SEQ ID NO:69.

[0309] In certain embodiments, the transmembrane domain of CD80 comprises or consists of an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to amino acids 243-263 of SEQ ID NO: 69. In certain embodiments, the transmembrane domain of CD80 comprises or consists of amino acids 243-263 of SEQ ID NO: 69 or a fragment thereof. Such a fragment can be at least about 5, at least about 10, at least about 15, or at least about 20 amino acids in length. In certain embodiments, the transmembrane domain of CD80 comprises or consists of amino acids 243 to 263 of SEQ ID NO:69.

[0310] Non-limiting examples of costimulatory molecules include CD28, 4-1BB, OX40, ICOS, DAP-10, CD27, CD40, NKG2D, CD2, and combinations thereof.

[0311] In certain embodiments, the fusion polypeptide comprises the extracellular domain and transmembrane domain of a costimulatory molecule that is 4-1BB. In certain embodiments, the costimulatory molecule is human 4-1BB. In certain embodiments, 4-1BB comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:26 or a fragment thereof, and / or may optionally contain up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, 4-1BB comprises or consists of an amino acid sequence that is a continuous portion of the amino acid sequence of SEQ ID NO:26. In certain embodiments, the intracellular domain of 4-1BB comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% homologous or identical to amino acids 214-255 of SEQ ID NO: 26, or a fragment thereof, and / or may optionally contain up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, the intracellular domain of 4-1BB comprises or consists of amino acids 214-255 of SEQ ID NO: 26, or a functional fragment thereof. Such a functional fragment can be a contiguous portion of amino acids 214-255 of SEQ ID NO: 26 that is at least about 20, at least about 25, at least about 30, at least about 35, or at least about 40 amino acids in length. In certain embodiments, a functional fragment of amino acids 214-255 of SEQ ID NO: 26 retains at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% of the primary function of the intracellular domain of 4-1BB, non-limiting examples of which include providing costimulatory signaling for the activation and proliferation of immunoresponsive cells (e.g., T cells) and interacting with and activating downstream adaptors (e.g., TRAFs).In certain embodiments, the intracellular domain of 4-1BB comprises or consists of amino acids 214 to 255 of SEQ ID NO:26.

[0312] In certain embodiments, the costimulatory molecule is CD28. In certain embodiments, the costimulatory molecule is human CD28. In certain embodiments, CD28 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO:7 or a fragment thereof, and / or may optionally contain up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, CD28 comprises or consists of an amino acid sequence that is a contiguous portion of the amino acid sequence of SEQ ID NO:7. In certain embodiments, the intracellular domain of CD28 comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% homologous or identical to amino acids 180-219 of SEQ ID NO: 7, or a fragment thereof, and / or may optionally contain up to one, up to two, or up to three conservative amino acid substitutions. In certain embodiments, the intracellular domain of CD28 comprises or consists of amino acids 180-219 of SEQ ID NO: 7, or a functional fragment thereof. A functional fragment of amino acids 180-219 of SEQ ID NO: 7 can be a contiguous portion of amino acids 180-219 of SEQ ID NO: 7 that is at least about 20, at least about 25, at least about 30, or at least about 35 amino acids in length. In certain embodiments, such functional fragments retain at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% of the primary functions of the intracellular domain of CD28, non-limiting examples of which include providing costimulatory signaling for the activation and proliferation of immunoresponsive cells (e.g., T cells) and interacting with protein adaptors (e.g., PI3K, GRB2, and LCK).In certain embodiments, the intracellular domain of CD28 comprises or consists of amino acids 180 to 219 of SEQ ID NO:7.

[0313] In certain embodiments, the fusion polypeptide comprises the intracellular domain of a second costimulatory molecule. In certain embodiments, the fusion polypeptide comprises the intracellular domain of a third costimulatory molecule. In certain embodiments, the fusion polypeptide comprises the intracellular domain of a fourth costimulatory molecule. In certain embodiments, the fusion polypeptide comprises the intracellular domain of a fifth costimulatory molecule. In certain embodiments, the first, second, third, fourth, and fifth costimulatory molecules can be the same or different from one another.

[0314] In certain embodiments, the fusion polypeptide comprises the extracellular domain and transmembrane domain of a costimulatory ligand that is CD80 and the intracellular domain of a costimulatory molecule that is 4-1BB. In certain embodiments, the fusion polypeptide comprises or consists of an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 71. In certain embodiments, the fusion polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 71. SEQ ID NO: 71 is provided below. MGHTRRQGTSPSKCPYLNFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL [SEQ ID NO: 71]

[0315] In certain embodiments, the fusion polypeptide comprises the extracellular domain and transmembrane domain of a costimulatory ligand that is CD80, the intracellular domain of a first costimulatory molecule that is 4-1BB, and the intracellular domain of a second costimulatory molecule that is CD28.

[0316] In certain embodiments, the fusion polypeptide comprises an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 72. In certain embodiments, the fusion polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 72. SEQ ID NO: 72 is provided below. MGHTRRQGTSPSKCPYLNFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFNWNTTKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL [SEQ ID NO: 72]

[0317] Various modified fusion polypeptides are disclosed in International Patent Application No. PCT / US20 / 42753, which is incorporated herein by reference in its entirety.

[0318] 2.7. Gene Disruption and Gene Modification In certain embodiments, the cells disclosed herein comprising a first antigen-recognizing receptor and a second antigen-recognizing receptor further comprise a gene disruption of the CD70 locus. Gene disruption of the CD70 locus can result in a non-functional CD70 protein or knockout of CD70 gene expression. In certain embodiments, gene disruption of the CD70 locus results in knockout of CD70 gene expression.

[0319] Non-limiting examples of gene disruptions include substitutions, deletions, insertions, or combinations thereof. In certain embodiments, the mutation comprises a missense mutation, a nonsense mutation, or a combination thereof. In certain embodiments, the deletion comprises a non-frameshift deletion, a frameshift deletion, or a combination thereof. In certain embodiments, the insertion comprises a non-frameshift insertion, a frameshift insertion, or a combination thereof.

[0320] In certain embodiments, the CD70 locus is a human CD70 locus. A gene disruption of the CD70 locus can be generated by any suitable gene editing method. In certain embodiments, a gene disruption of the CD70 locus (e.g., a knockout of the CD70 locus) is generated using a viral method. In certain embodiments, the viral method comprises a viral vector. In certain embodiments, the viral vector is a retroviral vector (e.g., a gamma retroviral vector or a lentiviral vector). Other viral vectors include adenoviral vectors, adena-associated viral vectors, vaccinia virus, bovine papillomavirus, and herpesviruses (e.g., Epstein-Barr virus).

[0321] In certain embodiments, a genetic disruption of the CD70 locus (e.g., a knockout of the CD70 locus) is generated using non-viral methods. Non-viral approaches can also be used for the genetic modification of cells. For example, nucleic acid molecules can be delivered by lipofection (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology 101:512, 1983), administration of nucleic acids in the presence of asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263:14621, 1988; Wu et al., Journal of Biological Chemistry 264:16985, 1989), or by microinjection under surgical conditions (Wolff et al., Science 247:1465, 1990). Other non-viral means for gene transfer include in vitro transfection using calcium phosphate, DEAE-dextran, electroporation, and protoplast fusion. Liposomes can also potentially be useful for delivering DNA to cells. Transplantation of normal genes into diseased tissues of a subject can also be achieved by transferring normal nucleic acids into ex vivo culturable cell types (e.g., autologous or heterologous primary cells or their progeny), followed by injection of the cells (or their progeny) into the target tissue or systemically injecting them. Recombinant receptors can also be induced or obtained using transposases or targeted nucleases (e.g., zinc finger nucleases, meganucleases, or TALE nucleases, CRISPR). Transient expression can be obtained by RNA electroporation.

[0322] Any targeted genome editing method can be used to generate a gene disruption at the CD70 locus. In certain embodiments, the gene disruption at the CD70 locus is generated by a method including homologous recombination, zinc finger nucleases, meganucleases, transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR) systems, or a combination thereof.

[0323] In certain embodiments, a CRISPR system is used to generate a gene disruption of the CD70 locus.

[0324] The clustered regularly interspaced short palindromic repeats (CRISPR) system is a genome editing tool discovered in prokaryotic cells. When used for genome editing, the CRISPR system includes Cas9 (a protein that can modify DNA using crRNA as a guide), CRISPR RNA (crRNA, which contains an RNA used by Cas9 to guide Cas9 to the correct part of the host DNA, along with a region that binds to tracrRNA (generally in the form of a hairpin loop) that forms an active complex with Cas9), transactivating crRNA (tracrRNA, which binds to crRNA and forms an active complex with Cas9), and an optional portion of a DNA repair template (DNA that guides the cellular repair process, allowing for the insertion of a specific DNA sequence). CRISPR / Cas9 is often transfected into target cells using a plasmid. The crRNA is the sequence used by Cas9 to identify and directly bind to the target DNA in the cell, so it must be designed for each application. The repair template carrying the CAR expression cassette must also be designed for each application, as it must overlap with the sequences on both sides of the cut and encode the insertion sequence. Multiple crRNAs and tracrRNAs can be packaged together to form a single guide RNA (sgRNA). This sgRNA can be spliced ​​together with a Cas9 gene and made into a plasmid for transfection into cells. In certain embodiments, the CRISPR system comprises a base editor. In certain embodiments, the CRISPR system comprises a transposase / recombinase. In certain embodiments, the CRISPR system comprises a prime editor. In certain embodiments, the CRISPR system comprises an epigenetic regulator. In certain embodiments, the CRISPR system comprises a CRISPRoff system. Further details regarding the CRISPR systems of the subject matter disclosed herein can be found in Anzalone et al., Nature biotechnology 38.7(2020):824-844 and Nunez et al., Cell 184.9(2021):2503-2519, the contents of each of which are incorporated by reference in their entirety.

[0325] In certain embodiments, a gRNA molecule is used to disrupt the CD70 locus to knock out expression of CD70. The gRNA molecule can target a coding sequence of a CD70 gene (e.g., a human CD70 gene) or a non-coding sequence of a CD70 gene (e.g., a human CD70 gene). In certain embodiments, the gRNA molecule targets a coding sequence of a CD70 gene (e.g., a human CD70 gene). In certain embodiments, the gRNA molecule targets a target sequence within the human CD70 gene.

[0326] In certain embodiments, zinc finger nucleases are used to generate gene disruptions in the CD70 locus. Zinc finger nucleases (ZFNs) are artificial restriction enzymes generated by combining a zinc finger DNA-binding domain with a DNA cleavage domain. The zinc finger domain can be engineered to target specific DNA sequences, allowing the zinc finger domain to target desired sequences within the genome. The DNA-binding domain of an individual ZFN typically contains multiple individual zinc finger repeats, each capable of recognizing multiple base pairs. The most common method for generating new zinc finger domains is to combine zinc finger "modules" of known smaller specificity. The most common cleavage domain in ZFNs is the non-specific cleavage domain from the type II restriction endonuclease FokI. Using the endogenous homologous recombination (HR) machinery and a homologous DNA template carrying the CAR expression cassette, ZFNs can be used to insert a CAR expression cassette into the genome. When the target sequence is cleaved by the ZFN, the HR machinery searches for homology between the damaged chromosome and the homologous DNA template, and then copies the sequence of the template between the two broken ends of the chromosome, thereby integrating the homologous DNA template into the genome.

[0327] In certain embodiments, a TALEN system is used to generate gene disruptions at the CD70 locus. Transcription activator-like effector nucleases (TALENs) are restriction enzymes that can be engineered to cleave specific sequences in DNA. TALEN systems operate on roughly the same principle as ZFNs. They are generated by combining a transcription activator-like effector DNA-binding domain with a DNA-cleavage domain. Transcription activator-like effectors (TALEs) consist of a 33-34 amino acid repeat motif with two variable positions that strongly recognize specific nucleotides. By assembling an array of these TALEs, the TALE DNA-binding domain can be engineered to bind to the desired DNA sequence, thereby directing the nuclease to cleave at a specific location in the genome. cDNA expression for use in polynucleotide therapy methods can be derived from any suitable promoter (e.g., human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoter) and regulated by any appropriate mammalian regulatory element or intron (e.g., elongation factor 1a enhancer / promoter / intron structure). For example, if desired, enhancers known to preferentially direct gene expression in specific cell types can be used to direct nucleic acid expression. Enhancers used can include, but are not limited to, those characterized as tissue- or cell-specific enhancers. Alternatively, if a genomic clone is used as a therapeutic construct, regulation can be mediated by cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, including any of the promoters or regulatory elements described above.

[0328] The method for delivering the genome editing agent / system can vary depending on the need. In certain embodiments, the components of the selected genome editing method are delivered as a DNA construct in one or more plasmids. In certain embodiments, the components are delivered via a viral vector. Common delivery methods include, but are not limited to, electroporation, microinjection, gene gun, imparefection, hydrostatic pressure, continuous injection, sonication, magnetofection, adeno-associated virus, envelope protein pseudotyped viral vectors, replication-competent vector cis- and trans-acting elements, herpes simplex virus, and chemical vehicles (e.g., oligonucleotides, lipoplexes, polymersomes, polyplexes, dendrimers, inorganic nanoparticles, and cell-penetrating peptides).

[0329] In certain embodiments, the gene disruption of the CD70 locus can be a disruption of the coding region of the CD70 locus and / or a disruption of a non-coding region of the CD70 locus. In certain embodiments, the gene disruption of the CD70 locus comprises a disruption of the coding region of the CD70 locus. In certain embodiments, the gene disruption of the CD70 locus comprises an insertion in the coding region of the CD70 locus. The human CD70 protein comprises three exons: exon 1, exon 2, and exon 3. In certain embodiments, the gene disruption of the CD70 locus comprises a disruption in one or more of exon 1, exon 2, and exon 3 of the CD70 locus. In certain embodiments, the gene disruption of the CD70 locus comprises a disruption in exon 1 of the CD70 locus. In certain embodiments, the gene disruption of the CD70 locus comprises an insertion in exon 1 of the CD70 locus.

[0330] In certain embodiments, the cells disclosed herein comprising a first antigen-recognizing receptor and a second antigen-recognizing receptor further comprise a genetic modification of the CD70 gene. The genetic modification of the CD70 gene can result in a non-functional CD70 protein or knockdown of CD70 gene expression. In certain embodiments, the genetic modification of the CD70 gene results in knockout of CD70 gene expression.

[0331] In certain embodiments, modification of the CD70 gene involves the use of an RNAi agent, including, but not limited to, shRNA, siRNA, LNA, dsRNA, and miRNA. In certain embodiments, the RNAi agent comprises an shRNA. In certain embodiments, the RNAi agent (e.g., shRNA) targets one or more isoforms of the CD70 gene, thereby reducing or eliminating expression of the CD70 gene or CD70 protein. In certain embodiments, the RNAi agent (e.g., shRNA) is expressed from the same construct that expresses a first antigen-recognizing receptor and / or a second antigen-recognizing receptor disclosed herein. In certain embodiments, expression of the RNAi agent (e.g., shRNA), the first antigen-recognizing receptor, and the second antigen-recognizing receptor is driven by the same promoter (e.g., the same promoter). In certain embodiments, expression of the shRNA disclosed herein, the first antigen-recognizing receptor, and the second antigen-recognizing receptor is driven by different promoters.

[0332] In certain embodiments, an RNAi agent (e.g., shRNA) comprises a nucleotide sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% homologous or identical to at least a portion of a CD70 nucleic acid sequence. In certain embodiments, an RNAi agent (e.g., shRNA) comprises a nucleotide sequence complementary to a CD70 gene that is at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, or at least about 30 nucleotides. In certain embodiments, an RNAi agent (e.g., an shRNA) comprises a nucleotide sequence of at most 15 nucleotides, at most 20 nucleotides, at most 25 nucleotides, at most 30 nucleotides, at most 35 nucleotides, at most 40 nucleotides, at most 55 nucleotides, at most 60 nucleotides, at most 65 nucleotides, at most 70 nucleotides, at most 75 nucleotides, at most 80 nucleotides, at most 85 nucleotides, at most 90 nucleotides, at most 95 nucleotides, or at most 100 nucleotides in length. In certain embodiments, an RNAi agent comprises DNA or an atypical or non-naturally occurring residue, such as, but not limited to, a phosphorothioate residue.

[0333] In certain embodiments, the RNAi agent reduces expression (e.g., endogenous expression) of CD70 by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 100%, or any intermediate value or range thereof. In certain embodiments, the RNAi agent reduces expression (e.g., endogenous expression) of CD70 by about 70%.

[0334] In certain embodiments, an RNAi agent targeting the CD70 gene comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 161-175. In certain embodiments, an RNAi agent targeting the CD70 gene comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 162. In certain embodiments, an RNAi agent targeting the CD70 gene comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 167. In certain embodiments, an RNAi agent targeting the CD70 gene comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 172. SEQ ID NOs: 161-175 are provided below. AUGACAGGUUGAAGCAAGUAGAUA [SEQ ID NO: 161] CAAGUUCAAGAGAAAAAGUGUAUA [SEQ ID NO: 162] CGCUGCUGAUUAGGGUUUUUUAUA [SEQ ID NO: 163] CCGUGAUGGCAUCUACAUGGUAUA [SEQ ID NO: 164] AAAGAGAAAAAGUGUACACACAUA [SEQ ID NO: 165] UCUACUUGCUUCAACCUGUCAGUG [SEQ ID NO: 166] UACACUUUUUCUCUUGAACUUAUG [SEQ ID NO: 167] UAAAAAACCCUAAUCAGCAGCAUG [SEQ ID NO: 168] UACCAUGUAGAUGCCAUCACGAUG [SEQ ID NO: 169] UGUGUGUACACUUUUUCUCUUGUG [SEQ ID NO: 170] TGCTGTTGACAGTGAGCGATGACAGGTTGAAGCAAGTAGATAGTGAAGCCACAGATGTATCTACTTGCTTCAACCTGTCAGTGCCTACTGCCTCGGA [SEQ ID NO: 171] TGCTGTTGACAGTGAGCGCAAGTTCAAGAGAAAAAGTGTATAGTGAAGCCACAGATGTATACACTTTTTCTCTTGAACTTATGCCTACTGCCTCGGA [SEQ ID NO: 172] TGCTGTTGACAGTGAGCGCGCTGCTGATTAGGGTTTTTTATAGTGAAGCCACAGATGTATAAAAAACCCTAATCAGCAGCATGCCTACTGCCTCGGA [SEQ ID NO: 173] TGCTGTTGACAGTGAGCGCCGTGATGGCATCTACATGGTATAGTGAAGCCACAGATGTATACCATGTAGATGCCATCACGATGCCTACTGCCTCGGA [SEQ ID NO: 174] TGCTGTTGACAGTGAGCGAAAGAGAAAAAGTGTACACACATAGTGAAGCCACAGATGTATGTGTGTACACTTTTTCTCTTGTGCCTACTGCCTCGGA [SEQ ID NO: 175]

[0335] In certain embodiments, the cells disclosed herein comprising a first antigen-recognizing receptor and a second antigen-recognizing receptor further comprise a genetic disruption of the TRAC locus. In certain embodiments, the genetic disruption of the TRAC locus results in a non-functional TCR. In certain embodiments, the genetic disruption of the TRAC locus results in knockout of TCR gene expression.

[0336] Any of the methods for generating a gene disruption in the CD70 locus disclosed above can be used to generate a gene disruption in the TRAC locus. In certain embodiments, the gene disruption in the TRAC locus is generated by a method comprising a gene editing method comprising homologous recombination, zinc finger nucleases, meganucleases, transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR) systems, or a combination thereof.

[0337] In certain embodiments, the gene disruption at the TRAC locus can be a disruption of the coding region of the TRAC locus and / or a disruption of a non-coding region of the TRAC locus. In certain embodiments, the gene disruption at the TRAC locus comprises a disruption of the coding region of the TRAC locus. In certain embodiments, the gene disruption at the TRAC locus comprises an insertion in the coding region of the TRAC locus. The human TRAC protein comprises four exons: exon 1, exon 2, exon 3, and exon 4. In certain embodiments, the coding region of the TRAC locus comprises exon 1, exon 2, exon 3, and exon 4. In certain embodiments, the gene disruption at the TRAC locus comprises a disruption in one or more of exons 1 through 4 of the TRAC locus. In certain embodiments, the gene disruption at the TRAC locus comprises a disruption in exon 1 of the TRAC locus. In certain embodiments, the gene disruption at the TRAC locus comprises an insertion in exon 1 of the TRAC locus.

[0338] In certain embodiments, the cells disclosed herein comprising a first antigen-recognizing receptor and a second antigen-recognizing receptor further comprise a genetic modification of the TRAC gene. The genetic modification of the TRAC gene can result in a non-functional TCR protein or knockdown of TCR gene expression. In certain embodiments, the genetic modification of the TRAC gene results in knockout of TCR gene expression.

[0339] In certain embodiments, modification of the TRAC gene involves the use of an RNAi agent, including, but not limited to, shRNA, siRNA, LNA, dsRNA, and miRNA. In certain embodiments, the RNAi agent includes an shRNA. In certain embodiments, the RNAi agent (e.g., shRNA) targets one or more isoforms of the TRAC gene, thereby reducing or eliminating expression of the TRAC gene or TCR protein. In certain embodiments, the RNAi agent (e.g., shRNA) is expressed from the same construct that expresses the first antigen-recognizing receptor and / or the second antigen-recognizing receptor disclosed herein. In certain embodiments, the expression of the RNAi agent (e.g., shRNA), the first antigen-recognizing receptor, and the second antigen-recognizing receptor is driven by the same promoter (e.g., the same promoter). In certain embodiments, the expression of the shRNA disclosed herein, the first antigen-recognizing receptor, and the second antigen-recognizing receptor is driven by different promoters.

[0340] In certain embodiments, an RNAi agent (e.g., shRNA) comprises a nucleotide sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% homologous or identical to at least a portion of a TRAC nucleic acid sequence. In certain embodiments, an RNAi agent (e.g., shRNA) comprises a nucleotide sequence complementary to a TRAC gene that is at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, or at least about 30 nucleotides. In certain embodiments, an RNAi agent (e.g., an shRNA) comprises a nucleotide sequence of at most 15 nucleotides, at most 20 nucleotides, at most 25 nucleotides, at most 30 nucleotides, at most 35 nucleotides, at most 40 nucleotides, at most 55 nucleotides, at most 60 nucleotides, at most 65 nucleotides, at most 70 nucleotides, at most 75 nucleotides, at most 80 nucleotides, at most 85 nucleotides, at most 90 nucleotides, at most 95 nucleotides, or at most 100 nucleotides in length. In certain embodiments, an RNAi agent comprises DNA or an atypical or non-naturally occurring residue, such as, but not limited to, a phosphorothioate residue.

[0341] In certain embodiments, the RNAi agent reduces TCR expression (e.g., endogenous expression) by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 100%, or any intermediate value or range thereof. In certain embodiments, the RNAi agent reduces TCR expression (e.g., endogenous expression) by about 20%. In certain embodiments, the RNAi agent reduces TCR expression (e.g., endogenous expression) by about 30%.

[0342] In certain embodiments, an RNAi agent targeting the TRAC gene comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 176-190. In certain embodiments, an RNAi agent targeting the TRAC gene comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 180. In certain embodiments, an RNAi agent targeting the TRAC gene comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 185. In certain embodiments, an RNAi agent targeting the TRAC gene comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 190. SEQ ID NOs: 176-190 are provided below. CUCACCGAUUUUGAUUCUCAAAUA [SEQ ID NO: 176] AUGGUCGAGAAAAGCUUUGAAAUA [SEQ ID NO: 177] ACGAUUUUGAUUCUCAAACAAAUA [SEQ ID NO: 178] CUUCACCGAUUUUGAUUCUCAAUA [SEQ ID NO: 179] AAGAUACGAACCUAAACUUUCAUA [SEQ ID NO: 180] UUUGAGAAUCAAAAUCGGUGAAUG [SEQ ID NO: 181] UUUCAAAGCUUUUCUCGACCAGUG [SEQ ID NO: 182] UUUGUUUGAGAAUCAAAAUCGGUG [SEQ ID NO: 183] UUGAGAAUCAAAAUCGGUGAAUUG [SEQ ID NO: 184] UGAAAGUUUAGGUUCGUAUCUGUG [SEQ ID NO: 185] TGCTGTTGACAGTGAGCGCTCACCGATTTTGATTCTCAAATAGTGAAGCCACAGATGTATTTGAGAATCAAAATCGGTGAATGCCTACTGCCTCGGA [SEQ ID NO: 186] TGCTGTTGACAGTGAGCGATGGTCGAGAAAAGCTTTGAAATAGTGAAGCCACAGATGTATTTCAAAGCTTTTCTCGACCAGTGCCTACTGCCTCGGA [SEQ ID NO: 187] TGCTGTTGACAGTGAGCGACGATTTTGATTCTCAAACAAATAGTGAAGCCACAGATGTATTTGTTTGAGAATCAAAATCGGTGCCTACTGCCTCGGA [SEQ ID NO: 188] TGCTGTTGACAGTGAGCGCTTCACCGATTTTGATTCTCAATAGTGAAGCCACAGATGTATTGAGAATCAAAATCGGTGAATTGCCTACTGCCTCGGA [SEQ ID NO: 189] TGCTGTTGACAGTGAGCGAAGATACGAACCTAAACTTTCATAGTGAAGCCACAGATGTATGAAAGTTTAGGTTCGTATCTGTGCCTACTGCCTCGGA [SEQ ID NO: 190]

[0343] In certain embodiments, the cells disclosed herein comprising a first antigen-recognizing receptor and a second antigen-recognizing receptor further comprise a gene disruption of a TRBC locus (e.g., the TRBC1 locus, the TRBC2 locus). In certain embodiments, the gene disruption of a TRBC locus (e.g., the TRBC1 locus, the TRBC2 locus) results in a non-functional TCR. In certain embodiments, the gene disruption of a TRBC locus (e.g., the TRBC1 locus, the TRBC2 locus) results in knockout of TCR gene expression.

[0344] Any of the methods for generating a gene disruption in the CD70 locus disclosed above can be used to generate a gene disruption in the TRBC locus (e.g., the TRBC1 locus, the TRBC2 locus). In certain embodiments, the gene disruption in the TRBC locus is generated by a method including a gene editing method including homologous recombination, zinc finger nucleases, meganucleases, transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR) system, or a combination thereof.

[0345] In certain embodiments, the gene disruption of the TRBC locus can be a disruption of the coding region of the TRBC1 locus. In certain embodiments, the gene disruption of the TRBC locus comprises a disruption of the coding region of the TRBC1 locus. In certain embodiments, the gene disruption of the TRBC locus comprises an insertion in the coding region of the TRBC1 locus. The human TRBC1 protein comprises four exons: exon 1, exon 2, exon 3, and exon 4. In certain embodiments, the coding region of the TRBC1 locus comprises exon 1, exon 2, exon 3, and exon 4. In certain embodiments, the gene disruption of the TRBC locus comprises a disruption in one or more of exons 1 to 4 of the TRBC1 locus. In certain embodiments, the gene disruption of the TRBC locus comprises a disruption in exon 1 of the TRBC1 locus. In certain embodiments, the gene disruption of the TRBC locus comprises an insertion in exon 1 of the TRBC1 locus.

[0346] In certain embodiments, the gene disruption of the TRBC locus can be a disruption of the coding region of the TRBC2 locus. In certain embodiments, the gene disruption of the TRBC locus comprises a disruption of the coding region of the TRBC2 locus. In certain embodiments, the gene disruption of the TRBC locus comprises an insertion in the coding region of the TRBC2 locus. The human TRBC2 protein comprises four exons: exon 1, exon 2, exon 3, and exon 4. In certain embodiments, the coding region of the TRBC2 locus comprises exon 1, exon 2, exon 3, and exon 4. In certain embodiments, the gene disruption of the TRBC locus comprises a disruption in one or more of exons 1 to 4 of the TRBC2 locus. In certain embodiments, the gene disruption of the TRBC locus comprises a disruption in exon 1 of the TRBC2 locus. In certain embodiments, the gene disruption of the TRBC locus comprises an insertion in exon 1 of the TRBC2 locus.

[0347] In certain embodiments, the cells disclosed herein comprising a first antigen-recognizing receptor and a second antigen-recognizing receptor further comprise a genetic modification of the TRBC gene. The genetic modification of the TRBC gene can result in a non-functional TCR protein or knockdown of TCR gene expression. In certain embodiments, the genetic modification of the TRBC gene results in knockout of TCR gene expression.

[0348] In certain embodiments, modification of the TRBC gene involves the use of an RNAi agent, including, but not limited to, shRNA, siRNA, LNA, dsRNA, and miRNA. In certain embodiments, the RNAi agent includes an shRNA. In certain embodiments, the RNAi agent (e.g., shRNA) targets one or more isoforms of the TRBC gene, thereby reducing or eliminating expression of the TRBC gene or TCR protein. In certain embodiments, the RNAi agent (e.g., shRNA) is expressed from the same construct that expresses the first antigen-recognizing receptor and / or the second antigen-recognizing receptor disclosed herein. In certain embodiments, the expression of the RNAi agent (e.g., shRNA), the first antigen-recognizing receptor, and the second antigen-recognizing receptor is driven by the same promoter (e.g., the same promoter). In certain embodiments, the expression of the shRNA disclosed herein, the first antigen-recognizing receptor, and the second antigen-recognizing receptor is driven by different promoters.

[0349] In certain embodiments, the RNAi agent (e.g., shRNA) comprises a nucleotide sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% homologous or identical to at least a portion of a TRBC nucleic acid sequence. In certain embodiments, the RNAi agent (e.g., shRNA) comprises a nucleotide sequence complementary to a TRBC gene that is at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, or at least about 30 nucleotides. In certain embodiments, an RNAi agent (e.g., an shRNA) comprises a nucleotide sequence of at most 15 nucleotides, at most 20 nucleotides, at most 25 nucleotides, at most 30 nucleotides, at most 35 nucleotides, at most 40 nucleotides, at most 55 nucleotides, at most 60 nucleotides, at most 65 nucleotides, at most 70 nucleotides, at most 75 nucleotides, at most 80 nucleotides, at most 85 nucleotides, at most 90 nucleotides, at most 95 nucleotides, or at most 100 nucleotides in length. In certain embodiments, an RNAi agent comprises DNA or an atypical or non-naturally occurring residue, such as, but not limited to, a phosphorothioate residue.

[0350] In certain embodiments, the RNAi agent reduces expression (e.g., endogenous expression) of the TCR by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 100%, or any intermediate value or range thereof. In certain embodiments, the RNAi agent reduces expression (e.g., endogenous expression) of the TCR by about 60%.

[0351] In certain embodiments, the RNAi agent targeting the TRBC gene comprises or consists of the nucleotide sequence set forth in SEQ ID NOs: 191-208. In certain embodiments, the RNAi agent targeting the TRBC gene comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 191. In certain embodiments, the RNAi agent targeting the TRBC gene comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 197. In certain embodiments, the RNAi agent targeting the TRBC gene comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 203. SEQ ID NOs: 191-208 are provided below. CCCGCUGUCAAGUCCAGUUCUAUA [SEQ ID NO: 191] ACGCCCUCAAUGACUCCAGAUAUA [SEQ ID NO: 192] AACCCGAGGUCGCUGUGUUUGAUA [SEQ ID NO: 193] ACGCAACCACUUCCGCUGUCAAUA [SEQ ID NO: 194] CGCAGAGAUCUCCCACACCCAAUA [SEQ ID NO: 195] CCGCUGUGUUUGAGCCAUCAGAUA [SEQ ID NO: 196] UAGAACUGGACUUGACAGCGGAUG [SEQ ID NO: 197] UAUCUGGAGUCAUUGAGGGCGGUG [SEQ ID NO: 198] UCAAACACAGCGACCUCGGGUGUG [SEQ ID NO: 199] UUGACAGCGGAAGUGGUUGCGGUG [SEQ ID NO: 200] UUGGGUGUGGGAGAUCUCUGCUUG [SEQ ID NO: 201] UCUGAUGGCUCAAACACAGCGAUG [SEQ ID NO: 202] TGCTGTTGACAGTGAGCGCCGCTGTCAAGTCCAGTTCTATAGTGAAGCCACAGATGTATAGAACTGGACTTGACAGCGGATGCCTACTGCCTCGGA [SEQ ID NO: 203] TGCTGTTGACAGTGAGCGACGCCCTCAATGACTCCAGATATAGTGAAGCCACAGATGTATATCTGGAGTCATTGAGGGCGGTGCCTACTGCCTCGGA [SEQ ID NO: 204] TGCTGTTGACAGTGAGCGAACCCGAGGTCGCTGTGTTTGATAGTGAAGCCACAGATGTATCAAACACAGCGACCTCGGGTGTGCCTACTGCCTCGGA [SEQ ID NO: 205] TGCTGTTGACAGTGAGCGACGCAACCACTTCCGCTGTCAATAGTGAAGCCACAGATGTATTGACAGCGGAAGTGGTTGCGGTGCCTACTGCCTCGGA [SEQ ID NO: 206] TGCTGTTGACAGTGAGCGCGCAGAGATCTCCCACACCCAATAGTGAAGCCACAGATGTATTGGGTGTGGGAGATCTCTGCTTGCTACTGCCTCGGA [SEQ ID NO: 207] TGCTGTTGACAGTGAGCGCCGCTGTGTTTGAGCCATCAGATAGTGAAGCCACAGATGTATCTGATGGCTCAAACACAGCGATGCCTACTGCCTCGGA [SEQ ID NO: 208]

[0352] The cells disclosed herein can be isolated and activated using a CD3 / CD28 antibody prior to generating the gene disruption. In certain embodiments, the cells disclosed herein comprising a first antigen-recognizing receptor and a second antigen-recognizing receptor further comprise a gene disruption at the TRAC locus, the TRBC locus, and / or the CD70 locus. In certain embodiments, the cells disclosed herein comprising a first antigen-recognizing receptor and a second antigen-recognizing receptor further comprise a genetic modification at the TRAC locus, the TRBC locus, and / or the CD70 locus.

[0353] In certain embodiments, the gene disruption of the TRAC locus, the gene disruption of the TRBC locus, and / or the gene disruption of the CD70 locus is generated after isolation and activation of cells (e.g., T cells). In certain embodiments, the gene disruption of the TRAC locus, the gene disruption of the TRBC locus, and / or the gene disruption of the CD70 locus is generated before isolation and activation of cells (e.g., T cells).

[0354] In certain embodiments, the gene disruption of the TRAC locus and / or the TRBC locus is generated prior to isolation and activation of the cells (e.g., T cells), and the gene disruption of the CD70 locus is generated after isolation and activation of the cells (e.g., T cells). In certain embodiments, the gene disruption of the CD70 locus is generated prior to isolation and activation of the cells (e.g., T cells), and the gene disruption of the TRAC locus and / or the TRBC locus is generated after isolation and activation of the cells (e.g., T cells).

[0355] In certain embodiments, the genetic modification of the TRAC locus, the genetic modification of the TRBC locus, and / or the genetic modification of the CD70 locus is generated after isolation and activation of the cells (e.g., T cells). In certain embodiments, the genetic modification of the TRAC locus, the genetic modification of the TRBC locus, and / or the genetic modification of the CD70 locus is generated before isolation and activation of the cells (e.g., T cells).

[0356] In certain embodiments, the genetic modification of the TRAC locus and / or the TRBC locus is generated prior to isolation and activation of the cells (e.g., T cells), and the genetic modification of the CD70 locus is generated after isolation and activation of the cells (e.g., T cells). In certain embodiments, the genetic modification of the CD70 locus is generated prior to isolation and activation of the cells (e.g., T cells), and the genetic modification of the TRAC locus and / or the TRBC locus is generated after isolation and activation of the cells (e.g., T cells).

[0357] 2.7. Exemplary Cells In certain embodiments, the cells disclosed herein are T cells comprising: a) a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain that targets CD312, a transmembrane domain, and an intracellular domain; and b) a TCR-like fusion protein (HIT) that targets CD70, comprising a first antigen-binding chain and a second antigen-binding chain. In certain embodiments, the extracellular antigen-binding domain of the CAR is a V domain comprising CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 73, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 74, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 75. H and V comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 76, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 77, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 78. L and (c) a TRAC polypeptide. In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide comprising or consisting of amino acids 153-179 of SEQ ID NO: 7. In certain embodiments, the intracellular domain of the CAR comprises a CD28 polypeptide and a modified CD3ζ polypeptide. In certain embodiments, the CD28 polypeptide comprises amino acids 180-220 of SEQ ID NO: 7, and the modified CD3ζ polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 22. In certain embodiments, the first antigen-binding chain of the HIT comprises: a) an antigen-binding fragment of an antibody heavy chain variable region (VH) comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 133, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 134, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 135; and b) a TRAC polypeptide. In certain embodiments, the second antigen-binding chain of the HIT comprises: a) an antigen-binding fragment of the light chain variable region (VL) of an antibody comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 136, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 137, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 138; and b) a TRBC polypeptide. In certain embodiments, the CAR and HIT are encoded by exogenous nucleic acids integrated into the TRAC locus. In certain embodiments, the T cells disclosed herein comprise a genetic disruption of the CD70 locus.

[0358] In certain embodiments, the cells disclosed herein are T cells comprising: a) a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain that targets CD312, a transmembrane domain, and an intracellular domain; and b) a TCR-like fusion protein (HIT) that targets CD70, comprising a first antigen-binding chain and a second antigen-binding chain. In certain embodiments, the extracellular antigen-binding domain of the CAR is a V domain comprising CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 73, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 74, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 75. H and V comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 76, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 77, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 78. L and (c) a TRAC polypeptide. In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide comprising or consisting of amino acids 153-179 of SEQ ID NO: 7. In certain embodiments, the intracellular domain of the CAR comprises a CD28 polypeptide and a modified CD3ζ polypeptide. In certain embodiments, the CD28 polypeptide comprises amino acids 180-220 of SEQ ID NO: 7, and the modified CD3ζ polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 22. In certain embodiments, the first antigen-binding chain of the HIT comprises: a) an antigen-binding fragment of an antibody heavy chain variable region (VH) comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 133, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 134, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 135; and b) a TRAC polypeptide. In certain embodiments, the second antigen-binding chain of the HIT comprises: a) an antigen-binding fragment of the light chain variable region (VL) of an antibody comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 136, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 137, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 138; and b) a TRBC polypeptide. In certain embodiments, the CAR and HIT are encoded by exogenous nucleic acids integrated into the TRAC locus. In certain embodiments, the T cells disclosed herein comprise a genetic modification of the CD70 locus.

[0359] In certain embodiments, the cells disclosed herein are T cells comprising: a) a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain that targets CD276, a transmembrane domain, and an intracellular domain; and b) a TCR-like fusion protein (HIT) that targets CD70, comprising a first antigen-binding chain and a second antigen-binding chain. In certain embodiments, the extracellular antigen-binding domain of the CAR is a V domain comprising CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 85, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 86, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 87. H and V comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 88, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 89, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 90. L and (c) a TRAC polypeptide. In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide comprising or consisting of amino acids 153-179 of SEQ ID NO: 7. In certain embodiments, the intracellular domain of the CAR comprises a CD28 polypeptide and a modified CD3ζ polypeptide. In certain embodiments, the CD28 polypeptide comprises amino acids 180-220 of SEQ ID NO: 7, and the modified CD3ζ polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 22. In certain embodiments, the first antigen-binding chain of the HIT comprises: a) an antigen-binding fragment of an antibody heavy chain variable region (VH) comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 133, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 134, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 135; and b) a TRAC polypeptide. In certain embodiments, the second antigen-binding chain of the HIT comprises: a) an antigen-binding fragment of the light chain variable region (VL) of an antibody comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 136, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 137, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 138; and b) a TRBC polypeptide. In certain embodiments, the CAR and HIT are encoded by exogenous nucleic acids integrated into the TRAC locus. In certain embodiments, the T cells disclosed herein comprise a genetic disruption of the CD70 locus.

[0360] In certain embodiments, the cells disclosed herein are T cells comprising: a) a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain that targets CD276, a transmembrane domain, and an intracellular domain; and b) a TCR-like fusion protein (HIT) that targets CD70, comprising a first antigen-binding chain and a second antigen-binding chain. In certain embodiments, the extracellular antigen-binding domain of the CAR is a V domain comprising CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 85, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 86, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 87. H and V comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 88, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 89, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 90. Land (c) a TRAC polypeptide. In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide comprising or consisting of amino acids 153-179 of SEQ ID NO: 7. In certain embodiments, the intracellular domain of the CAR comprises a CD28 polypeptide and a modified CD3ζ polypeptide. In certain embodiments, the CD28 polypeptide comprises amino acids 180-220 of SEQ ID NO: 7, and the modified CD3ζ polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 22. In certain embodiments, the first antigen-binding chain of the HIT comprises: a) an antigen-binding fragment of an antibody heavy chain variable region (VH) comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 133, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 134, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 135; and b) a TRAC polypeptide. In certain embodiments, the second antigen-binding chain of the HIT comprises: a) an antigen-binding fragment of the light chain variable region (VL) of an antibody comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 136, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 137, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 138; and b) a TRBC polypeptide. In certain embodiments, the CAR and HIT are encoded by exogenous nucleic acids integrated into the TRAC locus. In certain embodiments, the T cells disclosed herein comprise a genetic modification of the CD70 locus.

[0361] In certain embodiments, the cells disclosed herein are T cells comprising: a) a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain that targets CD22, a transmembrane domain, and an intracellular domain; and b) a TCR-like fusion protein (HIT) that targets CD19, comprising a first antigen-binding chain and a second antigen-binding chain. In certain embodiments, the extracellular antigen-binding domain of the CAR is a V-type T cell comprising a CDR1 comprising an amino acid sequence set forth in SEQ ID NO: 97, a CDR2 comprising an amino acid sequence set forth in SEQ ID NO: 98, and a CDR3 comprising an amino acid sequence set forth in SEQ ID NO: 99. H and V comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 100, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 101, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 102.L and (c) a TRAC polypeptide. In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide comprising or consisting of amino acids 153-179 of SEQ ID NO: 7. In certain embodiments, the intracellular domain of the CAR comprises a CD28 polypeptide and a modified CD3ζ polypeptide. In certain embodiments, the CD28 polypeptide comprises amino acids 180-220 of SEQ ID NO: 7, and the modified CD3ζ polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 22. In certain embodiments, the first antigen-binding chain of the HIT comprises: a) an antigen-binding fragment of an antibody heavy chain variable region (VH) comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 143, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 144, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 145; and b) a TRAC polypeptide. In certain embodiments, the second antigen-binding chain of the HIT comprises: a) an antigen-binding fragment of the light chain variable region (VL) of an antibody comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 146, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 147, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 148; and b) a TRBC polypeptide. In certain embodiments, the CAR and HIT are encoded by exogenous nucleic acids integrated into the TRAC locus.

[0362] In certain embodiments, the cells disclosed herein are T cells comprising: a) a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain that targets CD22, a transmembrane domain, and an intracellular domain; and b) a TCR-like fusion protein (HIT) that targets CD19, comprising a first antigen-binding chain and a second antigen-binding chain. In certain embodiments, the extracellular antigen-binding domain of the CAR is a V-type T cell comprising a CDR1 comprising an amino acid sequence set forth in SEQ ID NO: 97, a CDR2 comprising an amino acid sequence set forth in SEQ ID NO: 98, and a CDR3 comprising an amino acid sequence set forth in SEQ ID NO: 99. H and V comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 100, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 101, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 102. Land (c) a TRAC polypeptide. In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide comprising or consisting of amino acids 153-179 of SEQ ID NO: 7. In certain embodiments, the intracellular domain of the CAR comprises a CD28 polypeptide and a modified CD3ζ polypeptide. In certain embodiments, the CD28 polypeptide comprises amino acids 180-220 of SEQ ID NO: 7, and the modified CD3ζ polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 22. In certain embodiments, the first antigen-binding chain of the HIT comprises: a) an antigen-binding fragment of an antibody heavy chain variable region (VH) comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 143, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 144, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 145; and b) a TRAC polypeptide. In certain embodiments, the second antigen-binding chain of the HIT comprises: a) an antigen-binding fragment of the light chain variable region (VL) of an antibody comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 146, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 147, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 148; and b) a TRBC polypeptide. In certain embodiments, the CAR and HIT are encoded by exogenous nucleic acids integrated into the TRAC locus.

[0363] In certain embodiments, the cells disclosed herein are T cells comprising: a) a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain that targets CD22, a transmembrane domain, and an intracellular domain; and b) a TCR-like fusion protein (HIT) that targets CD19, comprising a first antigen-binding chain and a second antigen-binding chain. In certain embodiments, the extracellular antigen-binding domain of the CAR is a V fusion protein comprising a CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 97, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 153, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 99. H and V comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 100, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 101, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 102. Land (c) a TRAC polypeptide. In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide comprising or consisting of amino acids 153-179 of SEQ ID NO: 7. In certain embodiments, the intracellular domain of the CAR comprises a CD28 polypeptide and a modified CD3ζ polypeptide. In certain embodiments, the CD28 polypeptide comprises amino acids 180-220 of SEQ ID NO: 7, and the modified CD3ζ polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 22. In certain embodiments, the first antigen-binding chain of the HIT comprises: a) an antigen-binding fragment of an antibody heavy chain variable region (VH) comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 143, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 144, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 145; and b) a TRAC polypeptide. In certain embodiments, the second antigen-binding chain of the HIT comprises: a) an antigen-binding fragment of the light chain variable region (VL) of an antibody comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 146, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 147, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 148; and b) a TRBC polypeptide. In certain embodiments, the CAR and HIT are encoded by exogenous nucleic acids integrated into the TRAC locus.

[0364] In certain embodiments, the cells disclosed herein are T cells comprising: a) a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain that targets CD22, a transmembrane domain, and an intracellular domain; and b) a TCR-like fusion protein (HIT) that targets CD19, comprising a first antigen-binding chain and a second antigen-binding chain. In certain embodiments, the extracellular antigen-binding domain of the CAR is a V-type T cell comprising CDR1 comprising an amino acid sequence set forth in SEQ ID NO: 119, CDR2 comprising an amino acid sequence set forth in SEQ ID NO: 120, and CDR3 comprising an amino acid sequence set forth in SEQ ID NO: 121. H and V comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 122, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 123, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 124. Land (c) a TRAC polypeptide. In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide comprising or consisting of amino acids 153-179 of SEQ ID NO: 7. In certain embodiments, the intracellular domain of the CAR comprises a CD28 polypeptide and a modified CD3ζ polypeptide. In certain embodiments, the CD28 polypeptide comprises amino acids 180-220 of SEQ ID NO: 7, and the modified CD3ζ polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 22. In certain embodiments, the first antigen-binding chain of the HIT comprises: a) an antigen-binding fragment of an antibody heavy chain variable region (VH) comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 143, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 144, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 145; and b) a TRAC polypeptide. In certain embodiments, the second antigen-binding chain of the HIT comprises: a) an antigen-binding fragment of the light chain variable region (VL) of an antibody comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 146, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 147, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 148; and b) a TRBC polypeptide. In certain embodiments, the CAR and HIT are encoded by exogenous nucleic acids integrated into the TRAC locus.

[0365] In certain embodiments, the cells disclosed herein are T cells comprising: a) a chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain that targets CD22, a transmembrane domain, and an intracellular domain; and b) a TCR-like fusion protein (HIT) that targets CD19, comprising a first antigen-binding chain and a second antigen-binding chain. In certain embodiments, the extracellular antigen-binding domain of the CAR is a V-type T cell comprising a CDR1 comprising an amino acid sequence set forth in SEQ ID NO: 97, a CDR2 comprising an amino acid sequence set forth in SEQ ID NO: 154, and a CDR3 comprising an amino acid sequence set forth in SEQ ID NO: 99. H and V comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 100, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 101, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 102. Land (c) a TRAC polypeptide. In certain embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide comprising or consisting of amino acids 153-179 of SEQ ID NO: 7. In certain embodiments, the intracellular domain of the CAR comprises a CD28 polypeptide and a modified CD3ζ polypeptide. In certain embodiments, the CD28 polypeptide comprises amino acids 180-220 of SEQ ID NO: 7, and the modified CD3ζ polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 22. In certain embodiments, the first antigen-binding chain of the HIT comprises: a) an antigen-binding fragment of an antibody heavy chain variable region (VH) comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 143, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 144, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 145; and b) a TRAC polypeptide. In certain embodiments, the second antigen-binding chain of the HIT comprises: a) an antigen-binding fragment of the light chain variable region (VL) of an antibody comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 146, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 147, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 148; and b) a TRBC polypeptide. In certain embodiments, the CAR and HIT are encoded by exogenous nucleic acids integrated into the TRAC locus.

[0366] 3. Nucleic Acid Compositions and Vectors The presently disclosed subject matter provides a nucleic acid composition comprising a first polynucleotide encoding a first antigen-recognizing receptor disclosed herein (e.g., as disclosed in Section 2.1) and a second polynucleotide encoding a second antigen-recognizing receptor disclosed herein (e.g., as disclosed in Section 2.2). Cells comprising such nucleic acid compositions are also provided. In certain embodiments, the nucleic acid composition further comprises a first promoter operably linked to the first antigen-recognizing receptor. In certain embodiments, the nucleic acid composition further comprises a second promoter operably linked to the second antigen-recognizing receptor.

[0367] Additionally, the presently disclosed subject matter provides a nucleic acid composition comprising a first polynucleotide encoding a first antigen-recognizing receptor disclosed herein (e.g., as disclosed in Section 2.1) and a second polynucleotide encoding a fusion polypeptide disclosed herein (e.g., as disclosed in Section 2.2). Also provided are cells comprising such nucleic acid compositions. In certain embodiments, the nucleic acid composition further comprises a first promoter operably linked to the fusion polypeptide...

Claims

1. A cell, a) a chimeric antigen receptor (CAR) that targets a first antigen; and b) a TCR-like fusion molecule that targets a second antigen.

2. 2. The cell of claim 1, wherein the CAR comprises an extracellular antigen-binding domain that binds to the first antigen and an intracellular signaling domain that can deliver an activating signal to the cell.

3. The cell of claim 2, wherein the intracellular signaling domain of the CAR comprises a CD3ζ polypeptide.

4. The cell described in claim 3, wherein the CD3ζ polypeptide is a natural CD3ζ polypeptide or a modified CD3ζ polypeptide.

5. The cell of claim 4, wherein the modified CD3ζ polypeptide comprises a native ITAM1, an ITAM2 variant consisting of two loss-of-function mutations, and an ITAM3 variant consisting of two loss-of-function mutations.

6. The cell described in claim 4, wherein the modified CD3ζ polypeptide comprises the amino acid sequence set forth in SEQ ID NO:

22.

7. 7. The cell of claim 6, wherein the intracellular signaling domain of the CAR further comprises at least one costimulatory signaling region.

8. The cell of claim 7 , wherein the at least one costimulatory signaling region comprises at least the intracellular domain of a costimulatory molecule or a portion thereof.

9. 9. The cell of claim 8, wherein the costimulatory molecule is selected from the group consisting of CD28, 4-1BB, OX40, CD27, CD40, CD154, CD97, CD11a / CD18, ICOS, DAP-10, CD2, CD150, CD226, and NKG2D.

10. The cell of claim 9, wherein the costimulatory molecule comprises amino acids 180 to 220 of SEQ ID NO:

7.

11. The cell of claim 6, wherein the CAR comprises a transmembrane domain.

12. 7. The cell of claim 6, wherein the TCR-like fusion molecule comprises i) a first antigen-binding chain comprising an antigen-binding fragment of the heavy chain variable region (VH) of an antibody, and ii) a second antigen-binding chain comprising an antigen-binding fragment of the light chain variable region (VL) of the antibody, wherein the first and second antigen-binding chains a) each comprise a TRAC polypeptide or a TRBC polypeptide, and b) bind to the second antigen, and the TCR-like fusion molecule binds to the second antigen in an HLA-independent manner.

13. 13. The cell of claim 12, wherein at least one of the TRAC polypeptide and the TRBC polypeptide is endogenous.

14. The first and second antigen-binding chains are about 1 x 10 －８ The cell of claim 12, which binds to the second antigen with a dissociation constant (KD) of M or less.

15. The first and second antigen-binding chains are about 5 x 10 －９ The cell of claim 14, which binds to the second antigen with a dissociation constant (KD) of M or less.

16. 13. The cell of claim 12, wherein the first antigen binding chain comprises an antigen binding fragment of a VH of an antibody and a TRBC polypeptide, and the second antigen binding chain comprises an antigen binding fragment of a VL of the antibody and a TRAC polypeptide.

17. 13. The cell of claim 12, wherein the first antigen binding chain comprises an antigen binding fragment of a VH of an antibody and a TRAC polypeptide, and the second antigen binding chain comprises an antigen binding fragment of a VL of the antibody and a TRBC polypeptide.

18. The cell of claim 12 , wherein the first and second antigen-binding chains are capable of associating with a CD3ζ polypeptide.

19. The cell of claim 18, wherein the first and second antigen-binding chains are capable of activating the CD3ζ polypeptide when bound to the second antigen.

20. The cell of claim 19, wherein the activation of the CD3ζ polypeptide can activate the cell.

21. 13. The cell of claim 12, wherein the cell further comprises a genetic disruption of the TRAC locus and / or the TRBC locus.

22. The cell of claim 12 , wherein the cell further comprises a gene disruption of the CD70 locus.

23. 13. The cell of claim 12, wherein the cell further comprises a gene disruption of the TRAC locus, the TRBC locus, and / or the CD70 locus.

24. 13. The cell of claim 12, wherein the cell further comprises a genetic modification of a TRAC gene and / or a TRBC gene.

25. The cell of claim 12, wherein the cell further comprises a genetic modification of the CD70 gene.

26. 13. The cell of claim 12, wherein the cell further comprises a genetic modification of a TRAC gene, a TRBC gene, and / or a CD70 gene.

27. The cell of claim 12 , wherein the cell is a lymphoid or myeloid lineage cell.

28. 28. The cell of claim 27, wherein the cell of the lymphoid lineage is selected from the group consisting of a T cell, a B cell, a natural killer (NK) cell, and a dendritic cell.

29. 29. The cell of claim 28, wherein the cell is a T cell.

30. 30. The cell of claim 29, wherein the T cell is derived from an induced pluripotent stem cell.

31. The T cell is CD8 ＋ 30. The cell of claim 29, which is a T cell.

32. The CD8 ＋ The cell of claim 31 , wherein the T cell is CD4-independent.

33. 30. The cell of claim 29, wherein the T cell is selected from the group consisting of a cytotoxic T lymphocyte (CTL), a γδ T cell, a tumor-infiltrating lymphocyte (TIL), a regulatory T cell, and a natural killer T (NKT) cell.

34. The T cells are CD62L ＋ 30. The cell of claim 29, wherein

35. The T cells are ＋ 30. The cell of claim 29, wherein

36. The T cells are ＋ and CD62L ＋ 30. The cell of claim 29, wherein

37. 13. The cell of claim 12, wherein the CAR and / or the TCR-like fusion molecule is integrated into a locus within the genome of the T cell.

38. 38. The cell of claim 37, wherein the locus is selected from the group consisting of the TRAC locus, the TRBC locus, the TRDC locus, and the TRGC locus.

39. 38. The cell of claim 37, wherein the locus is the TRAC locus or the TRBC locus.

40. 40. The cell of claim 39, wherein the locus is the TRAC locus.

41. The cell of claim 12 , wherein the first antigen and / or the second antigen is a tumor antigen or a pathogen antigen.

42. The tumor antigen is selected from the group consisting of CD19, CD70, IL1RAP, ABCG2, AChR, ACKR6, ADAMTS13, ADGRE2, ADGRE2 (EMR2), ADORA3, ADRA1D, AGER, ALS2, antigens of cytomegalovirus (CMV)-infected cells (e.g., cell surface antigens), ANO9, AQP2, ASIC3, ASPRV1, ATP6V0A4, B3GNT4, B7-H3, BCMA, BEST4, C3orf35, CADM3, CAIX, CAPN3, CCDC155, CCR1, CD10, CD117, CD123, CD133, and CD1 35 (FLT3), CD138, CD20, CD22, CD244 (2B4), CD25, CD26, CD30, CD300LF, CD3 12, CD32, CD321, CD33, CD34, CD36, CD38, CD41, CD44, CD44V6, CD47, CD49f, C D56, CD7, CD71, CD74, CD8, CD82, CD96, CD98, CD99, CDH13, CDHR1, CEA, CEAC AM6, CHST3, CLEC12A, CLEC1A, CLL1, CNIH2, COL15A1, COLEC12, CPM, CR1, CX3 CR1, CXCR4, CYP4F11, DAGLB, DARC, DFNB31, DGKI, EGF1R, EGFR-VIII, EGP-2 , EGP-40, ELOVL6, EMB, EMC10, EMR2, ENG, EpCAM, EphA2, EPHA4, ERBB, ERBB2, Erb-B3, Erb-B4, E-selectin, EXOC3L4, EXTL3, FAM186B, FBP, FCGR1A, FKBP1B, FLRT1, folate receptor-α, FOLR2, FRMD5, GABRB2, GAS2, GD2, GD3, GDPD3, GNA14, GNAZ, GPR153, GPR56, GYPA, HEPHL1, HER-2, hERT, HILPDA, HLA-DR, HOOK1, hTERT, HTR2A, ICAM1, IGFBP3, IL10RB, IL20RB, IL23R, ILDR1, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), ITFG3, ITGA4, ITGA5, ITGA8, ITGAX, ITGB5, ITGB8, JAM3, KCND1, KCNJ5, KCNK13, KCNN4, KCNV2, KDR, KIF19, KIF26B, κ-light chain, L1CAM,LAX1, LEPR, Lewis Y (CD174), Lewis Y (LeY), LILRA2, LILRA6, LILRB2, LILRB3, LILRB4, LOXL4, LPAR2, LRRC37A3, LRRC8E, LRRN2, LRRTM2, LTB4R, MAGE-A1, MAGEA3, MANSC1, MART1, GP100, MBOAT1, MBOAT7, melanoma antigen family A, mesothelin (MSLN), MFAP3L, MMP25, MRP1, MT-ND1, mucin 1 (MUC1), mucin 16 (MUC16), MYADM, MYADML2, NGFR, NKCS1, NKG2D Ligands, NLGN3, NPAS2, NY-ESO-1, oncofetal antigen (h5T4), OTOA, P2RY13, p53, PDE3A, PEAR1, PIEZO1, PLXNA4, PLXNC1, PNPLA3, PPFIA4, PPP2R5B, PRAME, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), proteinase 3 (PR1), PSD2, PTPRJ, RDH16, receptor tyrosine-protein kinase Erb-B2, RHBDL3, RNF173, RNF183, ROR1, RYR2, S CIN, SCN11A, SCN2A, SCNN1D, SEC31B, SEMA4A, SH3PXD2A, SIGLEC11, SIRP B1, SLC16A6, SLC19A1, SLC22A5, SLC25A36, SLC25A41, SLC30A1, SLC34A3 , SLC43A3, SLC44A1, SLC44A3, SLC45A3, SLC6A16, SLC6A6, SLC8A3, SLC9A 1, SLCO2B1, SPAG17, STC1, STON2, SUN3, Survivin, SUSD2, SYNC, TACSTD2, TA The cell of claim 41, wherein the cell is selected from the group consisting of S1R3, TEX29, TFR2, TIM-3 (HAVCR2), TLR2, TMEFF2, TMEM145, TMEM27, TMEM40, TMEM59L, TMEM89, TMPRSS5, TNFRSF14, TNFRSF1B, TRIM55, TSPEAR, TTYH3, tumor-associated glycoprotein 72 (TAG-72), tyrosinase, vascular endothelial growth factor R2 (VEGF-R2), VLA-4, Wilms' tumor protein (WT-1), WNT4, WT1, and ZDHHC11.

43. 42. The cell of claim 41, wherein the first antigen is selected from the group consisting of CD312, CD19, CD20, CD22, CD276, and CAIX.

44. 44. The cell of claim 43, wherein the second antigen is selected from the group consisting of CD70, CD19, CD20, and CD22.

45. The cell of claim 41 , wherein the first antigen and the second antigen are CD312 and CD70.

46. The extracellular antigen-binding domain of the CAR targeting CD312 comprises a CDR1 comprising an amino acid sequence set forth in SEQ ID NO: 73, a CDR2 comprising an amino acid sequence set forth in SEQ ID NO: 74, and a CDR3 comprising an amino acid sequence set forth in SEQ ID NO:

75. Ｈ and V comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 76, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 77, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

78. Ｌ and the cell of claim 45.

47. 47. The cell of claim 46, wherein the TCR-like fusion molecule targeting CD70 comprises a first antigen-binding chain comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 133, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 134, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 135, and a second antigen-binding chain comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 136, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 137, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

138.

48. The cell of claim 38, wherein the first antigen and the second antigen are CD276 and CD70.

49. The extracellular antigen-binding domain of the CAR targeting CD276 comprises a CDR1 comprising an amino acid sequence set forth in SEQ ID NO: 85, a CDR2 comprising an amino acid sequence set forth in SEQ ID NO: 86, and a CDR3 comprising an amino acid sequence set forth in SEQ ID NO:

87. Ｈ and V comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 88, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 89, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

90. Ｌ and the cell of claim 48.

50. 50. The cell of claim 49, wherein the TCR-like fusion molecule targeting CD70 comprises a first antigen-binding chain comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 133, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 134, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 135, and a second antigen-binding chain comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 136, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 137, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

138.

51. The cell of claim 41 , wherein the first antigen and the second antigen are CAIX and CD70.

52. 42. The cell of claim 41, wherein the first antigen and the second antigen are CD19 and CD22.

53. 42. The cell of claim 41, wherein the first antigen and the second antigen are CD19 and CD20.

54. 42. The cell of claim 41, wherein the first antigen and the second antigen are CD20 and CD22.

55. 42. The cell of claim 41, wherein the first antigen and the second antigen are CD20 and CD19.

56. 42. The cell of claim 41, wherein the first antigen and the second antigen are CD22 and CD20.

57. 42. The cell of claim 41, wherein the first antigen and the second antigen are CD22 and CD19.

58. the extracellular antigen-binding domain of the CAR targeting CD22 is a) a V comprising a CDR1 comprising an amino acid sequence set forth in SEQ ID NO: 97, a CDR2 comprising an amino acid sequence set forth in SEQ ID NO: 98, and a CDR3 comprising an amino acid sequence set forth in SEQ ID NO: 99; Ｈ and a V comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 100, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 101, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

102. Ｌ , b) a V comprising a CDR1 comprising an amino acid sequence set forth in SEQ ID NO: 97, a CDR2 comprising an amino acid sequence set forth in SEQ ID NO: 153, and a CDR3 comprising an amino acid sequence set forth in SEQ ID NO: 99; Ｈ and a V comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 100, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 101, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

102. Ｌ , c) V comprising a CDR1 having an amino acid sequence set forth in SEQ ID NO: 119, a CDR2 having an amino acid sequence set forth in SEQ ID NO: 120, and a CDR3 having an amino acid sequence set forth in SEQ ID NO:

121. Ｈ and a V comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 122, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 123, and a CDR3 having the amino acid sequence set forth in SEQ ID NO:

124. Ｌ , or d) V comprising a CDR1 having an amino acid sequence set forth in SEQ ID NO: 97, a CDR2 having an amino acid sequence set forth in SEQ ID NO: 154, and a CDR3 having an amino acid sequence set forth in SEQ ID NO:

99. Ｈ and a V comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 100, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 101, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

102. Ｌ 58. The cell of claim 57, comprising:

59. The cell of claim 58, wherein the TCR-like fusion molecule targeting CD19 comprises a first antigen-binding chain comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 143, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 144, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 145, and a second antigen-binding chain comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 146, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 147, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

148.

60. 42. The cell of claim 41 , wherein the first antigen and the second antigen are selected from Table 8.

61. The cell of claim 12, further comprising a chimeric costimulatory receptor (CCR).

62. 62. The cell of claim 61 , wherein the CCR comprises an extracellular antigen-binding domain that binds to a third antigen and an intracellular domain that can deliver a costimulatory signal to the cell but does not alone deliver an activating signal to the cell.

63. The cell of claim 62, wherein the intracellular domain of the CCR comprises at least the intracellular domain of a costimulatory molecule or a portion thereof.

64. 64. The cell of claim 63, wherein the costimulatory molecule is selected from the group consisting of CD28, 4-1BB, OX40, CD27, CD40, CD154, CD97, CD11a / CD18, ICOS, DAP-10, CD2, CD150, CD226, and NKG2D.

65. The cell of claim 12 , wherein the cell further comprises at least one exogenous costimulatory ligand.

66. 66. The cell of claim 65, wherein the at least one exogenous costimulatory ligand is selected from the group consisting of tumor necrosis factor (TNF) family members, immunoglobulin (Ig) superfamily members, and combinations thereof.

67. The cell of claim 66, wherein the TNF family member is selected from the group consisting of 4-1BBL, OX40L, CD70, FasL, GITRL, TNF-related apoptosis-inducing ligand (TRAIL), CD30L, LIGHT (TNFSF14), and CD40L.

68. 67. The cell of claim 66, wherein the Ig superfamily member is selected from the group consisting of CD80, CD86, ICOSLG, and combinations thereof.

69. 66. The cell of claim 65, wherein the at least one exogenous costimulatory ligand comprises CD80.

70. The cell of claim 65, wherein the at least one exogenous costimulatory ligand comprises 4-1BBL.

71. 66. The cell of claim 65, wherein the cell comprises two exogenous costimulatory ligands.

72. The cell of claim 71, wherein the at least two exogenous costimulatory ligands comprise CD80 and 4-1BBL.

73. The cell of claim 72, wherein the at least two exogenous costimulatory ligands comprise the amino acid sequence set forth in SEQ ID NO: 67 and / or the amino acid sequence set forth in SEQ ID NO:

69.

74. 13. The cell of claim 12, wherein the cell further comprises a fusion polypeptide comprising: a) the extracellular domain and transmembrane domain of a costimulatory ligand; and b) the intracellular domain of a first costimulatory molecule.

75. 75. The cell of claim 74, wherein the costimulatory ligand is selected from the group consisting of tumor necrosis factor (TNF) family members, immunoglobulin (Ig) superfamily members, and combinations thereof.

76. 76. The cell of claim 75, wherein the TNF family member is selected from the group consisting of 4-1BBL, OX40L, CD70, GITRL, CD40L, and combinations thereof.

77. 76. The cell of claim 75, wherein the Ig superfamily member is selected from the group consisting of CD80, CD86, ICOSLG, and combinations thereof.

78. The cell of claim 74, wherein the costimulatory ligand is CD80.

79. 75. The cell of claim 74, wherein the first costimulatory molecule is selected from the group consisting of CD28, 4-1BB, OX40, ICOS, DAP-10, CD27, CD40, NKG2D, CD2, and combinations thereof.

80. The cell of claim 79, wherein the first costimulatory molecule is 4-1BB.

81. The cell of claim 74, wherein the costimulatory ligand is CD80 and the first costimulatory molecule is 4-1BB.

82. 82. The cell of claim 81, wherein the fusion polypeptide comprises the amino acid sequence set forth in SEQ ID NO:

71.

83. 75. The cell of claim 74, wherein the fusion polypeptide further comprises the intracellular domain of a second costimulatory molecule.

84. 84. The cell of claim 83, wherein the second costimulatory molecule is selected from the group consisting of CD28, 4-1BB, OX40, ICOS, DAP-10, CD27, CD40, NKG2D, CD2, and combinations thereof.

85. The cell of claim 83, wherein the second costimulatory molecule is CD28.

86. The cell of claim 74, wherein the costimulatory ligand is CD80, the first costimulatory molecule is 4-1BB, and the second costimulatory molecule is CD28.

87. 87. The cell of claim 86, wherein the fusion polypeptide comprises the amino acid sequence set forth in SEQ ID NO:

72.

88. The cell of any one of claims 1 to 87, wherein the cell is autologous.

89. The cell of any one of claims 1 to 87, wherein the cell is allogeneic.

90. A composition comprising a cell according to any one of claims 1 to 89.

91. 91. The composition of claim 90, which is a pharmaceutical composition further comprising a pharmaceutically acceptable excipient.

92. 1. A nucleic acid composition comprising: a first polynucleotide encoding a chimeric antigen receptor (CAR) that targets a first antigen; and a second polynucleotide encoding a TCR-like fusion molecule that targets a second antigen.

93. 1. A vector comprising: a first polynucleotide encoding a chimeric antigen receptor (CAR) that targets a first antigen; and a second polynucleotide encoding a TCR-like fusion molecule that targets a second antigen.

94. 94. The vector of claim 93, wherein the vector is a lentiviral vector.

95. The vector of claim 93 or 94, wherein the vector is a gamma-retroviral vector.

96. A lipid nanoparticle comprising a first polynucleotide encoding a chimeric antigen receptor (CAR) that targets a first antigen, and a second polynucleotide encoding a TCR-like fusion molecule that targets a second antigen.

97. A polynucleotide encoding a chimeric antigen receptor (CAR) that targets a first antigen and a TCR-like fusion molecule that targets a second antigen.

98. A vector comprising the polynucleotide of claim 97.

99. 99. The vector of claim 98, wherein the vector is a lentiviral vector.

100. 100. The vector of claim 98 or 99, wherein the vector is a gamma-retroviral vector.

101. A lipid nanoparticle comprising the polynucleotide of claim 97.

102. A composition comprising a polynucleotide according to claim 97, a vector according to any one of claims 93 to 95 or 98 to 100, or a lipid nanoparticle according to claim 96 or 101.

103. 103. The composition of claim 102, which is a pharmaceutical composition further comprising a pharmaceutically acceptable excipient.

104. 103. A method for producing a cell according to any one of claims 1 to 89, said method comprising introducing into said cell a nucleic acid composition according to claim 92, a vector according to any one of claims 93 to 95 or 98 to 100, a polynucleotide according to claim 97, a lipid nanoparticle according to claim 96 or 101, or a composition according to claim 102 or 103.

105. 105. The method of claim 104, further comprising generating a gene disruption in the CD70 locus.

106. 106. The method of claim 104 or 105, further comprising generating a gene disruption at the TRAC locus.

107. 107. The method of claim 105 or 106, wherein the gene disruption comprises a substitution, deletion, insertion, mutation, or a combination thereof.

108. 108. The method of claim 107, wherein the mutation comprises a missense mutation, a nonsense mutation, or a combination thereof.

109. 108. The method of claim 107, wherein the deletion comprises a non-frameshift deletion, a frameshift deletion, or a combination thereof.

110. 108. The method of claim 107, wherein the insertion comprises a non-frameshift insertion, a frameshift insertion, or a combination thereof.

111. 111. The method of any one of claims 104 to 110, wherein said genetic disruption of the CD70 locus results in a non-functional CD70 protein or results in knockout of CD70 gene expression.

112. 112. The method of any one of claims 104-111, wherein said genetic disruption of the TRAC locus results in a non-functional TRAC protein or results in knockout of TRAC gene expression.

113. 113. The method of any one of Claims 104-112, wherein generating the gene disruption comprises a gene editing method comprising homologous recombination, zinc finger nucleases, meganucleases, transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR) systems, or a combination thereof.

114. 114. The method of any one of claims 104 to 113, wherein said gene disruption at the CD70 locus or the TRAC locus is generated prior to activation of the cell.

115. 115. The method of any one of claims 104 to 114, wherein said gene disruption of the CD70 locus or the TRAC locus is generated following activation of a cell.

116. 115. The method of any one of claims 104-114, wherein a) the gene disruption at the CD70 locus is generated prior to activation of the cell, and b) the gene disruption at the TRAC locus is generated after activation of the cell.

117. 115. The method of any one of claims 104-114, wherein a) the gene disruption at the TRAC locus is generated prior to activation of the cell, and b) the gene disruption at the CD70 locus is generated after activation of the cell.

118. 105. The method of claim 104, further comprising generating a genetic modification of the CD70 gene.

119. 119. The method of claim 104 or 118, further comprising generating a genetic modification of the TRAC gene and / or the TRBC gene.

120. 120. The method of claim 118 or 119, wherein said genetic modification of said CD70 gene results in a non-functional CD70 protein or results in knockdown of said CD70 gene expression.

121. 121. The method of any one of claims 118-120, wherein said genetic modification of said TRAC locus results in a non-functional TRAC protein or results in knockdown of said TRAC gene expression.

122. 122. The method of any one of claims 118 to 121, wherein said genetic modification of the TRBC locus results in a non-functional TRBC protein or results in knockdown of TRBC gene expression.

123. 123. The method of any one of claims 104 to 122, further comprising introducing a chimeric costimulatory receptor (CCR).

124. 124. The method of any one of claims 104 to 123, further comprising introducing at least one exogenous costimulatory ligand.

125. 125. The method of any one of claims 104-124, further comprising introducing a fusion polypeptide comprising a) the extracellular and transmembrane domains of a costimulatory ligand, and b) the intracellular domain of a first costimulatory molecule.

126. A cell produced by the method of any one of claims 104 to 125.

127. 13. A method of reducing tumor burden in a subject, said method comprising administering to said subject an effective amount of a cell of any one of claims 1 to 89 or 126, or a composition of claim 90 or 91.

128. 128. The method of claim 127, wherein the method reduces the number of tumor cells, reduces tumor size, and / or eradicates the tumor in the subject.

129. 13. A method of preventing and / or treating a neoplasm or tumor in a subject, comprising administering to said subject an effective amount of a cell according to any one of claims 1 to 89 or 126, or a composition according to claim 90 or 91.

130. 130. The method of any one of claims 127 to 129, wherein the neoplasm or tumor is cancer.

131. 131. The method of any one of claims 127 to 130, wherein the neoplasm or tumor comprises antigenic heterogeneity of the first antigen and the second antigen.

132. 132. The method of claim 131, wherein the second antigen has a low antigen density.

133. 133. The method of claim 131 or 132, wherein the second antigen is expressed on tumor cells having a low tumor cell frequency.

134. the first antigen and the second antigen are independently selected from CD19, CD70, IL1RAP, ABCG2, AChR, ACKR6, ADAMTS13, ADGRE2, ADGRE2 (EMR2), ADORA3, ADRA1D, AGER, ALS2, an antigen of a cytomegalovirus (CMV)-infected cell, ANO9, AQP2, ASIC3, ASPRV1, ATP6V0A4, B3GNT4, B7-H3, BCMA, BEST4, C3orf35, CADM3, CAIX, CAPN3, CCDC155, CCR1, CD10, CD117, CD123, and CD133; , CD135 (FLT3), CD138, CD20, CD22, CD244 (2B4), CD25, CD26, CD30, CD300LF , CD312, CD32, CD321, CD33, CD34, CD36, CD38, CD41, CD44, CD44V6, CD47, CD 49f, CD56, CD7, CD71, CD74, CD8, CD82, CD96, CD98, CD99, CDH13, CDHR1, CEA , CEACAM6, CHST3, CLEC12A, CLEC1A, CLL1, CNIH2, COL15A1, COLEC12, CPM, CR 1, CX3CR1, CXCR4, CYP4F11, DAGLB, DARC, DFNB31, DGKI, EGF1R, EGFR-VIII, EGP-2, EGP-40, ELOVL6, EMB, EMC10, EMR2, ENG, EpCAM, EphA2, EPHA4, ERBB, ERBB2, Erb-B3, Erb-B4, E-selectin, EXOC3L4, EXTL3, FAM186B, FBP, FCGR1A, F KBP1B, FLRT1, folate receptor-α, FOLR2, FRMD5, GABRB2, GAS2, GD2, GD3, GDPD3, GNA14 , GNAZ, GPR153, GPR56, GYPA, HEPHL1, HER-2, hERT, HILPDA, HLA-DR, HOOK1, hTERT, HTR2A, ICAM1, IGFBP3, IL10RB, IL20RB, IL23R, ILDR1, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), ITFG3, ITGA4, ITGA5, ITGA8, ITGAX, ITGB5, ITGB8, JAM3, KCND1, KCNJ5, KCNK13, KCNN4, KCNV2, KDR, KIF19, KIF26B, κ-light chain,L1CAM, LAX1, LEPR, Lewis Y (CD174), Lewis Y (LeY), LILRA2, LILRA6, LILRB2, LILRB3, LILRB4, LOXL4, LPAR2, LRRC37A3, LRRC8E, LRRN2, LRRTM2, LTB4R, MAGE-A1, MAGEA3, MANSC1, MART1, GP100, MBOAT1, MBOAT7, melanoma antigen family A, mesothelin (MSLN), MFAP3L, MMP25, MRP1, MT-ND1, mucin 1 (MUC1), mucin 16 (MUC16), MYADM, MYADML2, NGFR, NKCS1, NK G2D ligand, NLGN3, NPAS2, NY-ESO-1, oncofetal antigen (h5T4), OTOA, P2RY13, p53, PDE3A, PEAR1, PIEZO1, PLXNA4, PLXNC1, PNPLA3, PPFIA4, PPP2R5B, PRAME, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), proteinase 3 (PR1), PSD2, PTPRJ, RDH16, receptor tyrosine-protein kinase Erb-B2, RHBDL3, RNF173, RNF183, ROR1, RYR2, SCIN, SCN11A, SCN2A, SCNN1D, SEC31B, SEMA4A, SH3PXD2A, SIGLEC 11, SIRPB1, SLC16A6, SLC19A1, SLC22A5, SLC25A36, SLC25A41, SLC30A1 , SLC34A3, SLC43A3, SLC44A1, SLC44A3, SLC45A3, SLC6A16, SLC6A6, SLC 8A3, SLC9A1, SLCO2B1, SPAG17, STC1, STON2, SUN3, Survivin, SUSD2, SYNC, T selected from the group consisting of ACSTD2, TAS1R3, TEX29, TFR2, TIM-3 (HAVCR2), TLR2, TMEFF2, TMEM145, TMEM27, TMEM40, TMEM59L, TMEM89, TMPRSS5, TNFRSF14, TNFRSF1B, TRIM55, TSPEAR, TTYH3, tumor-associated glycoprotein 72 (TAG-72), tyrosinase, vascular endothelial growth factor R2 (VEGF-R2), VLA-4, Wilms' tumor protein (WT-1), WNT4, WT1, and ZDHHC11;The method according to any one of claims 131 to 133.

135. 135. The method of any one of claims 131 to 134, wherein the first antigen is selected from the group consisting of CD312, CD19, CD20, CD22, CD276, and CAIX.

136. 135. The method of any one of claims 131 to 134, wherein the second antigen is selected from the group consisting of CD70, CD19, CD20, and CD22.

137. 135. The method of any one of claims 131 to 134, wherein the first antigen and the second antigen are selected from a) CD312 and CD70, b) CD276 and CD70, c) CAIX and CD70, d) CD19 and CD22, e) CD19 and CD20, f) CD20 and CD22, g) CD20 and CD19, h) CD22 and CD20, or i) CD22 and CD19.

138. 135. The method of any one of claims 131 to 134, wherein the first antigen and the second antigen are selected from Table 8.

139. 139. The method of any one of claims 127 to 138, wherein the neoplasm or tumor is a solid tumor.

140. 140. The method of claim 139, wherein the solid tumor is selected from the group consisting of melanoma, renal cell carcinoma, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, lung neuroendocrine carcinoma, small cell lung cancer, pancreatic cancer, breast cancer, astrocytoma, glioblastoma, laryngeal / pharyngeal cancer, EBV-associated nasopharyngeal carcinoma, and ovarian cancer.

141. 141. The method of claim 140, wherein the solid tumor is melanoma.

142. 139. The method of any one of claims 127 to 138, wherein the neoplasm or tumor is a hematological cancer.

143. 143. The method of any one of claims 127-138 or 142, wherein the neoplasm or tumor is a bone marrow disorder.

144. 144. The method of claim 143, wherein the bone marrow disorder is selected from the group consisting of myelodysplastic syndrome, myeloproliferative neoplasm, chronic myelomonocytic leukemia, or acute myeloid leukemia (AML), blastic plasmacytoid dendritic cell neoplasm, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, chronic myelocytic leukemia, and polycythemia vera.

145. 145. The method of claim 144, wherein the bone marrow disorder is acute myeloid leukemia (AML).

146. 143. The method of any one of claims 127-138 or 142, wherein the neoplasm or tumor is a B-cell malignancy.

147. 147. The method of claim 146, wherein the B-cell malignancy is selected from the group consisting of B-cell non-Hodgkin's lymphoma (NHL), B-cell Hodgkin's lymphoma, B-cell acute lymphocytic leukemia (ALL), B-cell chronic lymphocytic leukemia (CLL), multiple myeloma (MM), CLL with Richter's transformation, and CNS lymphoma.

148. 148. The method of claim 147, wherein the B-cell malignancy is B-cell acute lymphocytic leukemia.

149. 143. The method of any one of claims 127-138 or 142, wherein the neoplasm or tumor is leukemia.

150. 150. The method of claim 149, wherein the leukemia is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute promyelocytic leukemia (APL), mixed phenotype acute leukemia (MLL), hairy cell leukemia, B-cell prolymphocytic leukemia, B-cell precursor acute lymphoblastic leukemia, and T-cell precursor acute lymphoblastic leukemia.

151. 143. The method of any one of claims 127-138 or 142, wherein the neoplasm or tumor is a lymphoma.

152. 152. The method of claim 151, wherein the lymphoma is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell non-Hodgkin's lymphoma, T-cell non-Hodgkin's lymphoma, and T-cell precursor acute lymphoblastic lymphoma.

153. 153. The method of any one of claims 127 to 152, wherein the subject has a recurrence of the neoplasm or tumor.

154. 154. The method of any one of claims 127 to 153, wherein the subject has undergone a treatment that results in residual tumor cells.

155. 13. A method of preventing and / or treating a pathogen infection in a subject, said method comprising administering to said subject an effective amount of a cell according to any one of claims 1 to 89 or 126, or a composition according to claim 90 or 91.

156. 13. A method for preventing and / or treating an autoimmune disease in a subject, said method comprising administering to said subject an effective amount of a cell according to any one of claims 1 to 89 or 126, or a composition according to claim 90 or 91.

157. 13. A method of preventing and / or treating an infectious disease in a subject, said method comprising administering to said subject an effective amount of a cell according to any one of claims 1 to 89 or 126, or a composition according to claim 90 or 91.

158. 127. A cell according to any one of claims 1 to 89 or 126 or a composition according to claim 90 or 91 for use in reducing tumor burden in a subject, treating and / or preventing a neoplasm or tumor, preventing and / or treating a pathogen infection, preventing and / or treating an autoimmune disease, and / or preventing and / or treating an infectious disease.

159. A kit comprising a cell according to any one of claims 1 to 89 or 126, or a composition according to claim 90 or 91.

160. 160. The kit of claim 159, wherein the kit further comprises written instructions for reducing tumor burden, treating and / or preventing a neoplasm or tumor, preventing and / or treating a pathogen infection, preventing and / or treating an autoimmune disease, and / or preventing and / or treating an infectious disease.

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