Targeting cytotoxic cells with chimeric receptors for adoptive immunotherapy

Abstract

To provide compositions and methods for regulating the specificity and activity of immune effector cells for use in immunotherapy.SOLUTION: A composition is provided comprising 1×106 to 5×109±20% cytotoxic cells comprising a nucleic acid comprising a sequence encoding an activating killer cell immunoglobulin-like receptor chimeric antigen receptor (actKIR-CAR), wherein the actKIR-CAR comprises: (i) an extracellular antigen-binding domain comprising an antibody that binds to mesothelin or CD19; (ii) a transmembrane domain of an activating killer cell immunoglobulin-like receptor (actKIR) capable of interacting with a DAP12 polypeptide; and (iii) a cytoplasmic domain.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims priority to PCT Application No. PCT / CN2014 / 086694, filed September 17, 2014, and PCT Application No. PCT / CN2014 / 090578, filed November 7, 2014, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Background of the Invention Using gene transfer technology, T cells can be engineered to stably express antibody-binding domains on their surface, conferring specificity to T cells independent of the constraints imposed by the major histocompatibility complex (MHC). Chimeric antigen receptors (CARs) are synthetic proteins expressed on T cells (CAR cells) that fuse an antigen-recognition fragment of an antibody (e.g., an scFv, or single-chain variable fragment) with the intracellular domain of the CD3 zeta chain. Upon interaction with target cells expressing the scFv's cognate antigen, the CAR expressed on T cells can trigger T cell activation, resulting in target cell killing (also known as target cell lysis). When combined with additional costimulatory signals, such as the intracellular domains of CD137 or CD28, these receptors can also induce proliferation. However, some of this proliferation appears to be antigen-independent, unlike normal T cell receptor (TCR) responses (Milone et al., 2009, Mol Ther 17(8):1453-64). Artificial receptors do not fully recapitulate the intracellular signaling produced by natural TCR binding to antigenic peptides complexed with MHC molecules (Brocker, 2000, Blood 96(5):1999-2001). Signaling defects can limit the long-term survival of adoptively transferred CART cells in the absence of high levels of cytokines such as IL-2 (Lo et al., 2010, Clin Cancer Res 16(10):2769-80). They also have regulatory alterations that may be beneficial in certain anticancer applications (Loskog et al., 2006, Leukemia 20(10):1819-28). However, these regulatory defects also present a potential barrier to controlling their "off-target" activity against normal tissues that express the antigen at even very low levels. These "off-target" effects severely limit CAR-based therapeutics and result in high probability of death during early Phase I evaluation of CAR-modified T cells (Morgan et al., 2010, Mol Ther 18(4):843-51). Summary of the Invention [Problem to be solved by the invention]

[0003] Therefore, there is a need in the art for alternative approaches to CAR construction that overcome the limitations of current CAR-based therapeutics. The present invention addresses this unmet need in the art. [Means for solving the problem]

[0004] summary In a first aspect, the present invention relates to a purified or non-naturally occurring natural killer cell immune function receptor-chimeric antigen receptor (NKR-CAR) polypeptide comprising one, two, or all of a purified or non-naturally occurring extracellular antigen-binding domain, a transmembrane domain, e.g., an NKR transmembrane domain, and a cytoplasmic domain, e.g., an NKR cytoplasmic domain. In some embodiments, the NKR-CAR polypeptide comprises an extracellular antigen-binding domain, and one or both of a transmembrane domain, e.g., an NKR transmembrane domain; or a cytoplasmic domain, e.g., an NKR cytoplasmic domain. In some embodiments, the NKR-CAR polypeptide comprises an extracellular antigen-binding domain; a transmembrane domain, and an NKR cytoplasmic domain. In some embodiments, the NKR-CAR polypeptide comprises an extracellular antigen-binding domain, an NKR transmembrane domain, and a cytoplasmic domain. In some embodiments, the NKR-CAR polypeptide comprises an extracellular antigen-binding domain, an NKR transmembrane domain, and a cytoplasmic domain.

[0005] In some embodiments, the NKR-CAR polypeptide comprises a KIR-CAR, e.g., actKIR-CAR or inhKIR-CAR, an NCR-CAR, e.g., actNCR-CAR, an SLAMF-CAR, e.g., inhSLAMF-CAR, an FcR-CAR, e.g., a CD16-CAR, e.g., actCD16-CAR, or a CD64-CAR, e.g., actCD64-CAR, or a Ly49-CAR, e.g., actLy49-CAR or inhLy49-CAR.

[0006] In some embodiments, the NKR-CAR polypeptide comprises a killer cell immunoglobulin-like receptor chimeric antigen receptor (KIR-CAR), wherein the KIR-CAR comprises one or both of a transmembrane domain from a KIR (KIR transmembrane domain) or a cytoplasmic domain comprising a functional signaling domain from a KIR (KIR cytoplasmic domain). In some embodiments, the KIR transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of KIR2DS2, KIR2DL3, KIR2DL1, KIR2DL2, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DS1, KIR3DL2, KIR3DL3, KIR2DP1, and KIR3DP1. In some embodiments, the KIR cytoplasmic domain comprises a functional signaling domain of a protein selected from the group consisting of KIR2DS2, KIR2DL3, KIR2DL1, KIR2DL2, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DS1, KIR3DL2, KIR3DL3, KIR2DP1 and KIR3DP1. In some embodiments, the KIR-CAR further comprises one or more of a KIR D0 domain, a KIR D1 domain and / or a KIR D2 domain.

[0007] In some embodiments, the NKR CAR polypeptide comprises a natural cytotoxicity receptor-chimeric antigen receptor (NCR-CAR), wherein the NCR-CAR comprises one or both of a transmembrane domain from an NCR (NCR transmembrane domain) or a cytoplasmic domain comprising a functional signaling domain from an NCR (NCR cytoplasmic domain). In some embodiments, the NCR transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of NKp46, NKp30, and NKp44. In some embodiments, the NCR cytoplasmic domain comprises a functional signaling domain of a protein selected from the group consisting of NKp46, NKp30, and NKp44.

[0008] In some embodiments, the NKR CAR polypeptide comprises a signaling lymphocyte activation molecule family chimeric antigen receptor (SLAMF-CAR), wherein the SLAMF-CAR comprises one or both of a transmembrane domain from SLAMF (SLAMF transmembrane domain) or a cytoplasmic domain comprising a functional signaling domain from SLAMF (SLAMF cytoplasmic domain). In some embodiments, the SLAMF transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of SLAM, CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, and CD2F-10. In some embodiments, the SLAMF cytoplasmic domain comprises a functional signaling domain of a protein selected from the group consisting of SLAM, CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, and CD2F-10.

[0009] In one embodiment, the NKR CAR polypeptide comprises an Fc receptor-chimeric antigen receptor (FcR-CAR), wherein the FcR-CAR comprises one or both of a transmembrane domain from an FcR selected from CD16 or CD64 or a cytoplasmic domain comprising a functional signaling domain from an FcR selected from CD16 or CD64.

[0010] In some embodiments, the NKR CAR polypeptide comprises a Ly49 receptor-chimeric antigen receptor (Ly49-CAR), wherein the Ly49-CAR comprises one or both of a transmembrane domain from Ly49 (Ly49 transmembrane domain) or a cytoplasmic domain comprising a functional signaling domain from Ly49 (Ly49 cytoplasmic domain). In some embodiments, the Ly49 transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of Ly49A, Ly49C, Ly49H, and Ly49D. In some embodiments, the Ly49 cytoplasmic domain comprises a functional signaling domain of a protein selected from the group consisting of Ly49A, Ly49C, Ly49H, and Ly49D.

[0011] In some embodiments, the transmembrane domain of the NKR-CAR polypeptide is an NKR transmembrane domain, wherein the NKR transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of KIR2DS2, KIR2DL3, NKp46, KIR, NCR, SLAMF, FcR, and Ly49. In some embodiments, the transmembrane domain comprises i) the amino acid sequence of SEQ ID NO: 357, 358, or 359; ii) an amino acid sequence containing at least one, two, or three modifications, but not more than five modifications, of the amino acid sequence of SEQ ID NO: 357, 358, or 359; or iii) an amino acid sequence with 95-99% sequence identity to SEQ ID NO: 357, 358, or 359.

[0012] In some embodiments, the encoded transmembrane domain of the NKR-CAR polypeptide is a transmembrane domain, e.g., TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FcεRI, CD66d, DAP10, DAP12, the alpha, beta, or zeta chain of the T cell receptor, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, activating NK cell receptors, BTLA, T Ill ligand receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (CD8 alpha or CD8 beta), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD5+, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), B7-H3, CD5+, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGH TR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB 7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSG L1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp,a transmembrane domain of a TCAR as described herein, comprising a transmembrane domain of a protein selected from the group consisting of a ligand that specifically binds to CD19a and CD83, or any combination thereof;

[0013] In some embodiments, the cytoplasmic domain of the NKR-CAR polypeptide is an NKR cytoplasmic domain comprising one or more functional signaling domains of a protein selected from the group consisting of KIR2DS2, KIR2DL3, NKp46, KIR, NCR, SLAMF, FcR, and Ly49. In some embodiments, the cytoplasmic domain comprises i) the amino acid sequence of SEQ ID NO: 360, 361, or 362; ii) an amino acid sequence of SEQ ID NO: 360, 361, or 362 containing at least one, two, or three modifications, but not more than 20, 10, or 5 modifications; or iii) an amino acid sequence with 95-99% sequence identity to SEQ ID NO: 360, 361.

[0014] In some embodiments, the cytoplasmic domain of an NKR-CAR polypeptide comprises an intracellular signaling domain or an adaptor molecule, e.g., DAP12. In some embodiments, the encoded cytoplasmic domain of an NKR-CAR polypeptide comprises an adaptor molecule, e.g., DAP12 or FcεRγ. In some embodiments, the cytoplasmic domain comprises an encoded adaptor molecule comprising: i) the amino acid sequence of amino acids 1-113 of SEQ ID NO: 333 or amino acids 1-86 of SEQ ID NO: 335; ii) an amino acid sequence having at least one, two, or three modifications but not more than 20, 10, or 5 modifications in amino acids 1-113 of SEQ ID NO: 333 or amino acids 1-86 of SEQ ID NO: 335; or iii) an amino acid sequence with 95-99% identity to amino acids 1-113 of SEQ ID NO: 333 or amino acids 1-86 of SEQ ID NO: 335. In some embodiments, the NKR-CAR comprises a cytoplasmic domain comprising an antigen binding domain described herein, a CD8 transmembrane domain, and DAP12.

[0015] In some embodiments, the cytoplasmic domain of the NKR-CAR polypeptide is selected from the group consisting of, for example, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FcεRI, CD66d, DAP10, DAP12, the alpha, beta, or zeta chain of the T cell receptor, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, activating NK cell receptors, BTLA, Toll ligand receptors Body, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (CD8 alpha or CD8 beta), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD5+, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), B7-H3, CD5+, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KI RDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, N KG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1 , CD100(SEMA4D), CD69, SLAMF6(NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME(SLAMF8), SELPLG(CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp,a cytoplasmic domain of a TCAR as described herein, comprising one or more functional signaling domains of a protein selected from the group consisting of a ligand that specifically binds to CD19a and CD83, or any combination thereof.

[0016] In certain embodiments, the NKR-CAR polypeptide further comprises a leader or signal sequence, eg, comprising the amino acid sequence of SEQ ID NO:1.

[0017] In some embodiments, the NKR-CAR comprises a transmembrane domain and an extracellular antigen-binding domain, and further comprises a hinge domain located between the transmembrane domain and the extracellular antigen-binding domain. In some embodiments, the hinge domain is selected from the group consisting of a GS hinge, a CD8 hinge, an IgG4 hinge, an IgD hinge, a KIR2DS2 hinge, a KIR hinge, an NCR hinge, an SLAMF hinge, a CD16 hinge, a CD64 hinge, and an LY49 hinge. In some embodiments, the hinge domain comprises: i) the amino acid sequence of SEQ ID NO: 5, 2, 3, or 4; ii) an amino acid sequence having at least one, two, or three modifications, but not more than five modifications, of the amino acid sequence of SEQ ID NO: 5, 2, 3, or 4; or iii) an amino acid sequence having 95-99% identity to the amino acid sequence of SEQ ID NO: 5, 2, 3, or 4.

[0018] In certain embodiments, the transmembrane domain and cytoplasmic domain collectively comprise: i) the amino acid sequence of amino acids 413 to 487 of SEQ ID NO: 333 or amino acids 386 to 454 of SEQ ID NO: 335 or SEQ ID NO: 371; ii) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications in amino acids 413 to 487 of SEQ ID NO: 333 or amino acids 386 to 454 of SEQ ID NO: 335 or SEQ ID NO: 371; or iii) an amino acid sequence with 95 to 99% identity to amino acids 413 to 487 of SEQ ID NO: 333 or amino acids 386 to 454 of SEQ ID NO: 335 or SEQ ID NO: 371.

[0019] In any of the foregoing embodiments, the NKR-CAR is an activating NKR-CAR and the extracellular antigen-binding domain is an antigen-binding domain described herein, e.g., in Table 4. In any of the foregoing embodiments, the extracellular antigen-binding domain is a non-murine antigen-binding domain that binds to mesothelin. In some embodiments, the non-murine antigen-binding domain that binds to extracellular mesothelin comprises a human or humanized antigen-binding domain that binds to mesothelin. In some embodiments, the human or humanized antigen-binding domain that binds to mesothelin is set forth in Table 4.

[0020] In some embodiments, the human antigen-binding domain that binds to mesothelin comprises heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3) of any of the human anti-mesothelin heavy chain amino acid sequences listed in Table 4; and / or light chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2), and light chain complementarity determining region 3 (LC CDR3) of any of the human anti-mesothelin light chain amino acid sequences listed in Table 4. In certain embodiments, the human antigen-binding domain that binds to mesothelin comprises: i) the amino acid sequence of any of the human antimesothelin heavy chain variable regions listed in Table 4; ii) an amino acid sequence having at least one, two, or three modifications but not more than 30, 20, or 10 modifications of any of the amino acid sequences of any of the human antimesothelin heavy chain variable regions listed in Table 4; or iii) a heavy chain variable region comprising an amino acid sequence that is 95-99% identical to any of the amino acid sequences of any of the human antimesothelin heavy chain variable regions listed in Table 4; and / or i) the amino acid sequence of any of the human antimesothelin light chain variable regions listed in Table 4; ii) an amino acid sequence having at least one, two, or three modifications but not more than 30, 20, or 10 modifications of any of the amino acid sequences of any of the human antimesothelin light chain variable regions listed in Table 4; or iii) a light chain variable region comprising an amino acid sequence that is 95-99% identical to any of the amino acid sequences of any of the human antimesothelin light chain variable regions listed in Table 4. In certain embodiments, the human antigen-binding domain that binds to mesothelin comprises i) an amino acid sequence of any of SEQ ID NOs: 230-253; ii) an amino acid sequence having at least one, two, or three modifications but not more than 30, 20, or 10 modifications of an amino acid sequence of any of SEQ ID NOs: 230-253; or iii) an amino acid sequence having 95-99% identity to an amino acid sequence of any of SEQ ID NOs: 230-253. In such embodiments, the NKR-CAR further comprises a transmembrane domain and a cytoplasmic domain that, for example, collectively comprise the amino acid sequence of SEQ ID NO: 371, an amino acid sequence having at least one, two, or three modifications but not more than 10 or 5 modifications of SEQ ID NO: 371, or an amino acid sequence with at least 95-99% sequence identity to SEQ ID NO: 371.

[0021] In a first aspect, the present invention relates to a purified or non-naturally occurring nucleic acid molecule encoding a natural killer cell immune function receptor-chimeric antigen receptor (NKR-CAR) polypeptide described herein, e.g., comprising one, two, or all of an extracellular antigen binding domain, a transmembrane domain, e.g., an NKR transmembrane domain, and a cytoplasmic domain, e.g., an NKR cytoplasmic domain. In some embodiments, the nucleic acid molecule encoding the NKR-CAR comprises one or both of an extracellular antigen binding domain and a transmembrane domain, e.g., an NKR transmembrane domain; or a cytoplasmic domain, e.g., an NKR cytoplasmic domain. In some embodiments, the nucleic acid molecule encoding the NKR-CAR comprises an extracellular antigen binding domain; a transmembrane domain, and an NKR cytoplasmic domain. In some embodiments, the nucleic acid molecule encoding the NKR-CAR comprises an extracellular antigen binding domain, an NKR transmembrane domain, and a cytoplasmic domain. In some embodiments, the nucleic acid molecule encoding the NKR-CAR comprises an extracellular antigen binding domain, an NKR transmembrane domain, and a cytoplasmic domain.

[0022] In some embodiments, the nucleic acid molecule encodes a KIR-CAR, e.g., an actKIR-CAR or an inhKIR-CAR, an NCR-CAR, e.g., an actNCR-CAR, an SLAMF-CAR, e.g., an inhSLAMF-CAR, an FcR-CAR, e.g., a CD16-CAR, e.g., an actCD16-CAR, or a CD64-CAR, e.g., an actCD64-CAR, or an NKR-CAR, including a Ly49-CAR, e.g., an actLy49-CAR or an inhLy49-CAR.

[0023] In some embodiments, the encoded KIR-CAR comprises one or both of a transmembrane domain from a KIR (KIR transmembrane domain) or a cytoplasmic domain comprising a functional signaling domain from a KIR (KIR cytoplasmic domain). In some embodiments, the KIR transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of KIR2DS2, KIR2DL3, KIR2DL1, KIR2DL2, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DS1, KIR3DL2, KIR3DL3, KIR2DP1 and KIR3DP1. In some embodiments, the KIR cytoplasmic domain comprises a functional signaling domain of a protein selected from the group consisting of KIR2DS2, KIR2DL3, KIR2DL1, KIR2DL2, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DS1, KIR3DL2, KIR3DL3, KIR2DP1 and KIR3DP1. In some embodiments, the KIR-CAR further comprises one or more of a KIR D0 domain, a KIR D1 domain and / or a KIR D2 domain.

[0024] In some embodiments, the encoded NCR-CAR comprises one or both of a transmembrane domain from an NCR (NCR transmembrane domain) or a cytoplasmic domain comprising a functional signaling domain from an NCR (NCR cytoplasmic domain). In some embodiments, the NCR transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of NKp46, NKp30, and NKp44. In some embodiments, the NCR cytoplasmic domain comprises a functional signaling domain of a protein selected from the group consisting of NKp46, NKp30, and NKp44.

[0025]

[0013] In some embodiments, the encoded SLAMF-CAR comprises one or both of a transmembrane domain from SLAMF (SLAMF transmembrane domain) or a cytoplasmic domain comprising a functional signaling domain from SLAMF (SLAMF cytoplasmic domain). In some embodiments, the SLAMF transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of SLAM, CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, and CD2F-10. In some embodiments, the SLAMF cytoplasmic domain comprises a functional signaling domain of a protein selected from the group consisting of SLAM, CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, and CD2F-10.

[0026] In some embodiments, the encoded FcR-CAR comprises one or both of a transmembrane domain from an FcR selected from CD16 or CD64 or a cytoplasmic domain comprising a functional signaling domain from an FcR selected from CD16 or CD64.

[0027] In some embodiments, the encoded Ly49-CAR comprises one or both of a transmembrane domain from Ly49 (Ly49 transmembrane domain) or a cytoplasmic domain comprising a functional signaling domain from Ly49 (Ly49 cytoplasmic domain). In some embodiments, the Ly49 transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of Ly49A, Ly49C, Ly49H, and Ly49D. In some embodiments, the Ly49 cytoplasmic domain comprises a functional signaling domain of a protein selected from the group consisting of Ly49A, Ly49C, Ly49H, and Ly49D.

[0028] In some embodiments, the encoded transmembrane domain of the NKR-CAR is an NKR transmembrane domain, wherein the NKR transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of KIR2DS2, KIR2DL3, NKp46, KIR, NCR, SLAMF, FcR, and Ly49. In some embodiments, the encoded transmembrane domain of the NKR-CAR comprises the amino acid sequence of SEQ ID NO: 357, 358, or 359; an amino acid sequence containing at least one, two, or three modifications but not more than five modifications of the amino acid sequence of SEQ ID NO: 357, 358, or 359; or an amino acid sequence with 95-99% sequence identity to SEQ ID NO: 357, 358, or 359. In some embodiments, the nucleic acid molecule encoding the NKR-CAR comprises a nucleic acid sequence comprising nucleotides 803-875 of SEQ ID NO: 347, or a nucleic acid sequence with 95-99% sequence identity thereto, which encodes the transmembrane domain.

[0029] In some embodiments, the encoded transmembrane domain of the NKR-CAR polypeptide is selected from the group consisting of, for example, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FcεRI, CD66d, DAP10, DAP12, the alpha, beta, or zeta chain of the T cell receptor, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, activating NK cell receptors, BTLA, Toll ligases, and the like. Endonuclear receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (CD8 alpha or CD8 beta), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD5+, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), B7-H3, CD5+, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR) , KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7 , NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL 1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp,a transmembrane domain of a TCAR as described herein, comprising a transmembrane domain of a protein selected from the group consisting of a ligand that specifically binds to CD19a and CD83, or any combination thereof;

[0030] In some embodiments, the encoded cytoplasmic domain of the NKR-CAR is an NKR cytoplasmic domain, wherein the NKR cytoplasmic domain comprises one or more functional signaling domains of a protein selected from the group consisting of KIR2DS2, KIR2DL3, NKp46, KIR, NCR, SLAMF, FcR, and Ly49. In some embodiments, the encoded cytoplasmic domain comprises i) the amino acid sequence of SEQ ID NO: 360, 361, or 362; ii) an amino acid sequence containing at least one, two, or three modifications, but not more than 20, 10, or 5 modifications, of the amino acid sequence of SEQ ID NO: 360, 361, or 362; or iii) an amino acid sequence with 95-99% sequence identity to SEQ ID NO: 360, 361. In some embodiments, the nucleic acid molecule encoding the NKR-CAR comprises a nucleic acid sequence comprising nucleotides 831-947 of SEQ ID NO: 343, nucleotides 833-1060 of SEQ ID NO: 345, or nucleotides 876-949 of SEQ ID NO: 347, or a nucleic acid sequence having 95-99% sequence identity thereof, which encodes a cytoplasmic domain.

[0031] In some embodiments, the encoded cytoplasmic domain of the NKR-CAR polypeptide comprises an intracellular signaling domain or an adapter molecule, e.g., DAP12. In some embodiments, the encoded cytoplasmic domain of the NKR-CAR polypeptide comprises an adapter molecule, e.g., DAP12 or FcεRγ. In some embodiments, the encoded cytoplasmic domain comprises an encoded adapter molecule comprising: i) the amino acid sequence of amino acids 1-113 of SEQ ID NO: 333 or amino acids 1-86 of SEQ ID NO: 335; ii) an amino acid sequence having at least one, two, or three modifications but not more than 20, 10, or 5 modifications in amino acids 1-113 of SEQ ID NO: 333 or amino acids 1-86 of SEQ ID NO: 335; or iii) an amino acid sequence with 95-99% identity to amino acids 1-113 of SEQ ID NO: 333 or amino acids 1-86 of SEQ ID NO: 335. In some embodiments, the nucleic acid molecule encoding the NKR-CAR comprises a nucleic acid sequence comprising nucleotides comprising nucleotides 1-339 of SEQ ID NO: 332 or nucleotides 1-258 of SEQ ID NO: 334, or a nucleic acid sequence comprising 95-99% identity thereof, which encodes an adaptor molecule. In some embodiments, the NKR-CAR comprises an antigen binding domain described herein, a CD8 transmembrane domain, and a cytoplasmic domain comprising DAP12.

[0032] In some embodiments, the encoded cytoplasmic domain of the NKR-CAR polypeptide is selected from a group consisting of, for example, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FcεRI, CD66d, DAP10, DAP12, the alpha, beta, or zeta chain of the T cell receptor, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, activating NK cell receptors, BTLA, Toll ligases, and the like. Endonuclear receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (CD8 alpha or CD8 beta), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD5+, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), B7-H3, CD5+, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR) , KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7 , NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL 1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp,a cytoplasmic domain of a TCAR as described herein, comprising one or more functional signaling domains of a protein selected from the group consisting of a ligand that specifically binds to CD19a and CD83, or any combination thereof.

[0033] In some embodiments, the nucleic acid molecule encoding NKR-CAR further comprises a leader or signal sequence encoding the amino acid sequence of SEQ ID NO:1.

[0034] In some embodiments, the encoded NKR-CAR comprises an extracellular antigen-binding domain linked to a transmembrane domain by a hinge domain. In some embodiments, the hinge domain is selected from the group consisting of a GS hinge, a CD8 hinge, an IgG4 hinge, an IgD hinge, a KIR2DS2 hinge, a KIR hinge, an NCR hinge, an SLAMF hinge, a CD16 hinge, a CD64 hinge, and an LY49 hinge. In some embodiments, the encoded hinge domain comprises i) the amino acid sequence of SEQ ID NO: 5, 2, 3, or 4; ii) an amino acid sequence having at least one, two, or three modifications, but not more than five modifications, of the amino acid sequence of SEQ ID NO: 5, 2, 3, or 4; or iii) an amino acid sequence having 95-99% identity to the amino acid sequence of SEQ ID NO: 5, 2, 3, or 4. In one embodiment, the nucleic acid molecule encoding the NKR-CAR comprises a nucleic acid sequence comprising the nucleic acid sequence of SEQ ID NO: 356, 16, 13, 14, or 15, or a nucleic acid sequence having 95-99% identity thereto, which encodes a hinge domain.

[0035] In some embodiments, the encoded transmembrane domain and the encoded cytoplasmic domain collectively comprise i) the amino acid sequence of amino acids 413-487 of SEQ ID NO:333 or amino acids 386-454 of SEQ ID NO:335 or SEQ ID NO:371; ii) an amino acid sequence having at least one, two, or three modifications but not more than 30, 20, or 10 modifications in amino acids 413-487 of SEQ ID NO:333 or amino acids 386-454 of SEQ ID NO:335 or SEQ ID NO:371; or iii) an amino acid sequence with 95-99% identity to amino acids 413-487 of SEQ ID NO:333 or amino acids 386-454 of SEQ ID NO:335 or SEQ ID NO:371. In some embodiments, a nucleic acid molecule encoding an NKR-CAR comprises a nucleic acid sequence comprising nucleotides 1237-1464 of SEQ ID NO:332 or nucleotides 1156-1365 of SEQ ID NO:334, or a sequence with 95-99% identity thereof, which collectively encodes the transmembrane domain and the cytoplasmic domain.

[0036] In another aspect, the invention pertains to a nucleic acid molecule, e.g., a purified or non-naturally occurring nucleic acid, e.g., a nucleic acid, including DNA or RNA, sequence, e.g., mRNA, comprising a sequence encoding an NKR-CAR described herein.

[0037] In some embodiments, the nucleic acid molecule further comprises a nucleic acid sequence encoding an adapter molecule or an intracellular signaling domain that interacts with said NKR-CAR. In some embodiments, the encoded adapter molecule comprises a functional signaling domain of DAP12 or FcεRγ. In some embodiments, the encoded adapter molecule comprises the amino acid sequence of amino acids 1-113 of SEQ ID NO: 333 or amino acids 1-86 of SEQ ID NO: 335; an amino acid sequence having at least one, two, or three modifications but not more than 20, 10, or 5 modifications in amino acids 1-113 of SEQ ID NO: 333 or amino acids 1-86 of SEQ ID NO: 335; or an amino acid sequence with 95-99% identity to amino acids 1-113 of SEQ ID NO: 333 or amino acids 1-86 of SEQ ID NO: 335. In some embodiments, the nucleic acid molecule encoding an NKR-CAR comprises a nucleic acid sequence comprising nucleotides 1-339 of SEQ ID NO: 332 or nucleotides 1-258 of SEQ ID NO: 334, or a nucleic acid sequence comprising 95-99% identity thereto, which encodes an adapter molecule.

[0038] In some embodiments, the nucleic acid molecule further comprises a nucleic acid sequence encoding a TCAR or a second NKR-CAR. In some embodiments, the encoded TCAR or the encoded second NKR-CAR comprises an antigen-binding domain that binds to a target antigen that is not mesothelin.

[0039] In some embodiments, the nucleic acid molecule encoding NKR-CAR further comprises a nucleic acid sequence encoding a peptide cleavage site selected from the group consisting of T2A, P2A, E2A, and F2A, wherein the nucleic acid encoding the peptide cleavage site links the nucleic acid sequence encoding NKR-CAR to a second nucleic acid sequence, e.g., a nucleic acid sequence encoding an adapter molecule. In some embodiments, the nucleic acid sequence encoding the peptide cleavage site encodes the amino acid sequence of SEQ ID NO: 57, 58, 59, or 60; or an amino acid sequence having 95-99% sequence identity thereto.

[0040] In some embodiments, the nucleic acid encodes an activating NKR-CAR, and the encoded extracellular antigen-binding domain is an antigen-binding domain described herein, e.g., in Table 4. In any of the foregoing embodiments, the encoded extracellular antigen-binding domain is a non-murine antigen-binding domain that binds mesothelin. In some embodiments, the encoded non-murine antigen-binding domain that binds mesothelin comprises a human or humanized antigen-binding domain that binds mesothelin. In some embodiments, the human or humanized antigen-binding domain that binds mesothelin is set forth in Table 4.

[0041] In some embodiments, the encoded human antigen-binding domain that binds mesothelin comprises heavy chain complementarity-determining region 1 (HC CDR1), heavy chain complementarity-determining region 2 (HC CDR2), and heavy chain complementarity-determining region 3 (HC CDR3) of any of the human anti-mesothelin heavy chain amino acid sequences listed in Table 4; and / or light chain complementarity-determining region 1 (LC CDR1), light chain complementarity-determining region 2 (LC CDR2), and light chain complementarity-determining region 3 (LC CDR3) of any of the human anti-mesothelin light chain amino acid sequences listed in Table 4. In certain embodiments, the encoded human antigen-binding domain that binds to mesothelin comprises: i) the amino acid sequence of any of the human antimesothelin heavy chain variable regions listed in Table 4; ii) an amino acid sequence having at least one, two, or three modifications but not more than 30, 20, or 10 modifications of any of the amino acid sequences of any of the human antimesothelin heavy chain variable regions listed in Table 4; or iii) a heavy chain variable region comprising an amino acid sequence that is 95-99% identical to an amino acid sequence of any of the human antimesothelin heavy chain variable regions listed in Table 4; and / or i) the amino acid sequence of any of the human antimesothelin light chain variable regions listed in Table 4; ii) an amino acid sequence having at least one, two, or three modifications but not more than 30, 20, or 10 modifications of any of the amino acid sequences of any of the human antimesothelin light chain variable regions listed in Table 4; or iii) a light chain variable region comprising an amino acid sequence that is 95-99% identical to an amino acid sequence of any of the human antimesothelin light chain variable regions listed in Table 4. In certain embodiments, the encoded human antigen-binding domain that binds to mesothelin comprises i) an amino acid sequence of any of SEQ ID NOs: 230-253; ii) an amino acid sequence having at least one, two, or three modifications but not more than 30, 20, or 10 modifications of an amino acid sequence of any of SEQ ID NOs: 230-253; or iii) an amino acid sequence with 95-99% identity to an amino acid sequence of any of SEQ ID NOs: 230-253. In such embodiments, the NKR-CAR further comprises a transmembrane domain and a cytoplasmic domain that, for example, collectively comprise the amino acid sequence of SEQ ID NO: 371, an amino acid sequence having at least one, two, or three modifications but not more than 10 or 5 modifications of SEQ ID NO: 371, or an amino acid sequence with at least 95-99% sequence identity to SEQ ID NO: 371.

[0042] In another aspect, the present invention relates to a vector comprising a nucleic acid molecule encoding an NKR-CAR described herein. In some embodiments, the vector is a DNA vector or an RNA vector. In some embodiments, the vector is selected from the group consisting of a plasmid, a lentiviral vector, an adenoviral vector, and a retroviral vector. In some embodiments, the vector further comprises a nucleic acid sequence comprising an adapter molecule or an intracellular signaling domain described herein. In some embodiments, the vector further comprises a promoter described herein, e.g., an EF-1 promoter, e.g., comprising the sequence of SEQ ID NO: 11.

[0043] In other aspects, the invention further pertains to a cell, e.g., an immune effector cell, e.g., a cytotoxic cell, e.g., a naturally occurring or non-occurring T cell, NK cell, or cytotoxic T cell or NK cell line, comprising an NKR-CAR described herein, a nucleic acid molecule encoding an NKR-CAR described herein, a vector described herein, or an NKR-CAR complex described herein.

[0044] In some embodiments, the cytotoxic cell further comprises an adaptor molecule or intracellular signaling domain that interacts with said NKR-CAR.

[0045] In another aspect, the invention relates to a method of producing a cell described herein, e.g., an immune effector cell, e.g., a cytotoxic cell, e.g., a naturally occurring or non-occurring T cell, NK cell, or cytotoxic T cell or NK cell line, comprising an NKR-CAR described herein, comprising introducing into a cytotoxic cell a nucleic acid, e.g., mRNA, comprising a sequence encoding an NKR-CAR described herein. In some embodiments, the method further comprises producing an NKR-CAR described herein in the cytotoxic cell. In some embodiments, the method comprises producing a population of cells described herein, e.g., a population of immune effector cells, comprising an NKR-CAR described herein.

[0046] In another aspect, the invention pertains to a method of treating a subject, e.g., a method of providing anti-tumor immunity in a mammal, comprising administering to the mammal an effective amount of a cell described herein or a population of cells described herein, e.g., a cytotoxic cell, e.g., a naturally occurring or non-occurring T cell, NK cell, or cytotoxic T cell or NK cell line, comprising an NKR-CAR described herein.

[0047] In another aspect, the invention relates to a method of treating a subject having a disease associated with expression of a tumor antigen, e.g., a tumor antigen described herein (e.g., a proliferative disease, a precancerous condition, and a non-cancer indication associated with expression of a tumor antigen), comprising administering to the subject an effective amount of cells comprising an NKR-CAR, e.g., as described herein. In some embodiments, the NKR-CAR is an activated NKR-CAR, and the extracellular antigen-binding domain is an antigen-binding domain described herein, e.g., in Table 4. In some embodiments, the method further comprises administration of cells comprising a TCAR, e.g., as described herein.

[0048] In some embodiments, the disease associated with tumor antigen expression is cancer, e.g., a cancer described herein. In some embodiments, the cancer is a solid tumor, e.g., a solid tumor described herein.

[0049] In some embodiments, the disease or disorder is associated with mesothelin expression, e.g., mesothelioma (e.g., malignant pleural mesothelioma), lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, squamous cell lung cancer, or large cell lung cancer), pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), ovarian cancer, colorectal cancer, and bladder cancer, or any combination thereof. In some embodiments, the disease is pancreatic cancer, e.g., metastatic pancreatic ductal adenocarcinoma (PDA), e.g., in a subject that has progressed on at least one prior standard of care. In some embodiments, the disease is mesothelioma (e.g., malignant pleural mesothelioma), e.g., in a subject that has progressed on at least one prior standard of care regimen. In some embodiments, the disease is ovarian cancer, e.g., serous epithelial ovarian cancer, in a subject that has progressed after at least one prior standard of care regimen.

[0050] Further features and embodiments of the compositions and methods include one or more of the following: In another aspect, the invention pertains to a purified or non-naturally occurring KIR-CAR comprising an extracellular antigen-binding domain and a transmembrane domain, e.g., a KIR transmembrane domain or a cytoplasmic domain, e.g., an ITIM-containing cytoplasmic domain or a KIR cytoplasmic domain. In one embodiment, the KIR-CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an ITIM-containing cytoplasmic domain or a KIR cytoplasmic domain.

[0051] In some embodiments, the transmembrane domain can interact with, e.g., bind to, the transmembrane domain of DAP12. In some embodiments, the transmembrane domain comprises an amino acid residue comprising a positively charged group, e.g., a positively charged group, e.g., a side chain. In some embodiments, the transmembrane domain comprises a KIR transmembrane domain.

[0052] In some embodiments, the KIR-CAR is an activating KIR-CAR. In some embodiments, the KIR-CAR comprises a KIR transmembrane domain. In some embodiments, the KIR-CAR is an inhibitory KIR-CAR. In some embodiments, the KIR-CAR comprises a KIR cytoplasmic domain. In some embodiments, the KIR-CAR comprises an extracellular antigen-binding domain and a transmembrane domain, e.g., a transmembrane domain comprising an amino acid residue comprising a positively charged group, e.g., a positively charged group, e.g., a side chain, or a KIR transmembrane domain.

[0053] In some embodiments, the KIR-CAR described herein comprises an antigen-binding domain comprising an scFv. In some embodiments, the antigen-binding domain is a single VH domain, such as a camelid, shark, or lamprey single VH domain, or a single VH domain derived from a human or mouse sequence, or a non-antibody scaffold, such as fibronectin, e.g., a fibronectin type III antibody-like molecule. In some embodiments, the antigen-binding domain comprises a nanobody. In some embodiments, the antigen-binding domain comprises a camelid VHH domain.

[0054] In one embodiment, the KIR-CAR is an activating KIR-CAR and the extracellular antigen-binding domain is an antigen-binding domain described herein, e.g., in Table 4.

[0055] In some embodiments, the KIR-CAR described herein comprises an extracellular hinge domain. In some embodiments, the extracellular hinge domain is other than a KIR hinge domain, e.g., other than a KIR2DS2 hinge domain. In some embodiments, the extracellular hinge domain is derived from a naturally occurring molecule. In some embodiments, the extracellular hinge domain is derived from a naturally occurring molecule other than KIR. In some embodiments, the extracellular hinge domain comprises a non-naturally occurring polypeptide sequence. In some embodiments, the extracellular hinge domain comprises an extracellular hinge from human CD8 alpha. In some embodiments, the extracellular hinge domain comprises a synthetic extracellular hinge. In some embodiments, the extracellular hinge domain is less than 50, 20, or 10 amino acids in length. In some embodiments, the extracellular hinge domain has fewer amino acids than the KIR2DS2 hinge domain.

[0056] In some embodiments, the KIR-CAR described herein is an actKIR-CAR. In some embodiments, the actKIR-CAR comprises a transmembrane domain or an actKIR transmembrane domain comprising a positively charged group, e.g., an amino acid residue comprising a positively charged group, e.g., a positively charged side chain. In some embodiments, the actKIR-CAR can interact with and promote signaling from an ITAM-containing polypeptide or an adaptor molecule. In some embodiments, the actKIR-CAR can interact with and promote signaling from a DAP12 polypeptide. In some embodiments, the actKIR-CAR comprises a KIR D domain. In some embodiments, the actKIR-CAR comprises a KIR D1 domain. In some embodiments, the actKIR-CAR comprises a KIR D2 domain. In some embodiments, the actKIR-CAR does not comprise a KIR D domain. In some embodiments, the actKIR-CAR comprises a KIR2DS2 transmembrane domain. In some embodiments, the actKIR-CAR further comprises a KIR2DS2 cytoplasmic domain. In some embodiments, the actKIR-CAR does not comprise a KIR D domain.

[0057] In some embodiments, the antigen binding domain of a KIR-CAR described herein binds to an antigen presented on a target cell, e.g., a cancer cell. In some embodiments, the antigen binding domain is an antigen that is more highly expressed on a target cell, e.g., a cancer cell, than on a non-target cell, e.g., a non-cancerous cell, e.g., a non-cancerous cell of the same type as the target cell. In some embodiments, the antigen binding domain binds to an antigen described herein, e.g., a tumor antigen described herein. In some embodiments, the tumor antigen is expressed on a solid tumor, e.g., a solid tumor described herein, e.g., mesothelioma (e.g., malignant pleural mesothelioma), lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, squamous cell lung cancer, or large cell lung cancer), pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), ovarian cancer, colorectal cancer, and bladder cancer, or any combination thereof.

[0058] In some embodiments, the KIR-CAR described herein is an inhKIR-CAR. In some embodiments, the inhKIR-CAR comprises an inhKIR transmembrane domain. In some embodiments, the inhKIR-CAR comprises an ITIM-containing cytoplasmic domain, e.g., an inhKIR cytoplasmic domain, e.g., a KIR2DL or KIR3DL cytoplasmic domain. In some embodiments, the inhKIR-CAR comprises a transmembrane domain other than a KIR transmembrane, e.g., a transmembrane domain from PD-1, CTLA4, or an ITIM-containing receptor from a receptor in the ILT (CD85), Siglec, LMIR (CD300), and / or SLAM gene family. In some embodiments, the inhKIR-CAR comprises a cytoplasmic domain from an inhibitory receptor other than KIR, e.g., PD-1, CTLA4, or an ITIM-containing receptor from a receptor in the ILT (CD85), Siglec, LMIR (CD300), and / or SLAM gene family. In some embodiments, the inhKIR-CAR comprises a transmembrane domain and a cytoplasmic domain from an inhibitory receptor other than KIR, e.g., a transmembrane domain and a cytoplasmic domain from PD-1, CTLA4, or an ITIM-containing receptor, independently, e.g., from a receptor in the ILT (CD85), Siglec, LMIR (CD300), and / or SLAM gene family. In some embodiments, the cytoplasmic domain comprises an ITIM. In some embodiments, the inhKIR-CAR comprises a KIR D domain. In some embodiments, the inhKIR-CAR comprises a KIR D0 domain. In some embodiments, the inhKIR-CAR comprises a KIR D1 domain. In some embodiments, the inhKIR-CAR comprises a KIR D2 domain. In some embodiments, the inhKIR-CAR does not comprise a KIR D domain.

[0059] In some embodiments, the antigen-binding domain of an inhKIR-CAR described herein binds to an antigen that is not presented on target cells, e.g., cancer cells. In some embodiments, the antigen-binding domain binds to an antigen that is more highly expressed on non-target cells, e.g., non-cancer cells, than on target cells, e.g., cancerous cells, e.g., cancerous cells of the same type as the target cells. In some embodiments, the antigen-binding domain binds to desmoglein 1 / 3 (DSG1 / 3). In some embodiments, an inhCAR, e.g., an inhTCAR or inhNKR-CAR, e.g., an inhKIR-CAR, and an actCAR, e.g., an actTCAR or actNKR-CAR, e.g., an actKIR-CAR, are provided, wherein the inhCAR comprises an antigen-binding domain targeting desmoglein 1 / 3 (DSG1 / 3), and the actCAR comprises an antigen-binding domain targeting an antigen other than DSG1 / 3, e.g., EGFR. In some embodiments, this pair is used to treat EGFR-expressing cancer, e.g., lung or colon adenocarcinoma. In some embodiments, cancer cells express less DSG1 / 3 than non-cancer cells. In some embodiments, this combination can minimize CAR-mediated invasiveness of skin cells or squamous epithelial cells of the GI tract (i.e., oral mucosa). In some embodiments, the antigen-binding domain binds to an ephrin receptor or a claudin.

[0060] In another aspect, the invention pertains to (a) a nucleic acid, e.g., a purified or non-naturally occurring nucleic acid, e.g., a nucleic acid, comprising a DNA, or RNA, sequence, e.g., mRNA, comprising a sequence encoding a KIR-CAR, e.g., a first KIR-CAR described herein. In one embodiment, the KIR-CAR, e.g., the first KIR-CAR, is an actKIR-CAR, e.g., an actKIR-CAR described herein. In one embodiment, the nucleic acid encodes an actKIR-CAR, and the encoded extracellular antigen-binding domain is an antigen-binding domain described herein, e.g., in Table 4. In one embodiment, the KIR-CAR, e.g., the first KIR-CAR, is an inhKIR-CAR, e.g., an inhKIR-CAR described herein.

[0061] In some embodiments, the nucleic acid comprises a DNA sequence. In some embodiments, the nucleic acid comprises an RNA sequence, e.g., an mRNA sequence.

[0062] In some embodiments, the nucleic acid comprises a sequence encoding an inhibitory molecule comprising a KIR-CAR, e.g., an actKIR-CAR, and an inhKIR cytoplasmic domain; a transmembrane domain, e.g., a KIR transmembrane domain; and an inhibitor cytoplasmic domain, e.g., an ITIM domain, e.g., an inhKIR ITIM domain. In some embodiments, the inhibitory molecule is a naturally occurring inhKIR or a sequence that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% homologous to a naturally occurring inhKIR or that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 residues.

[0063] In some embodiments, the nucleic acid comprises a sequence encoding a KIR-CAR, such as an actKIR-CAR, and a sequence encoding an inhibitory molecule comprising a SLAM family cytoplasmic domain; a transmembrane domain, such as a SLAM family transmembrane domain; and an inhibitor cytoplasmic domain, such as a SLAM family domain, such as a SLAM family ITIM domain. In some embodiments, the inhibitory molecule is a naturally occurring SLAM family member or a naturally occurring sequence that has at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99% homology with a SLAM family member, or does not differ by more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 residues.

[0064] In some embodiments, the nucleic acid described herein further comprises (b) a sequence encoding a second KIR-CAR described herein, e.g., a second KIR-CAR different from the first KIR-CAR. In some embodiments, (a) and (b) are disposed in the same nucleic acid molecule, e.g., the same vector, e.g., the same viral vector, e.g., a lentiviral vector. In some embodiments, one of (a) and (b) is disposed in a first nucleic acid molecule, e.g., a first vector, e.g., a viral vector, e.g., a lentiviral vector, and the other is disposed in a second nucleic acid molecule, e.g., a second vector, e.g., a viral vector, e.g., a lentiviral vector. In some embodiments, the first KIR-CAR and the second KIR-CAR are actKIR-CARs. In some embodiments, binding of either actKIR-CAR alone is insufficient to induce a significant level of activation. In some embodiments, binding of both the first and second actKIR-CARs results in an additive or synergistic level of activation. In some embodiments, the first KIR-CAR and the second KIR-CAR are inhKIR-CARs. In some embodiments, one of the first KIR-CAR and the second KIR-CAR is an actKIR-CAR, and the other is an inhKIR-CAR. In some embodiments, the actKIR-CAR is an actKIR-CAR described herein. In some embodiments, the inhKIR-CAR is an inhKIR-CAR described herein. In some embodiments, the nucleic acid described herein comprises an actKIR-CAR described herein and an inhKIR-CAR described herein.

[0065] In some embodiments, the core further comprises (c) a sequence encoding an intracellular signaling domain, e.g., an adapter molecule, capable of generating an activation signal. In some embodiments, the intracellular signaling domain comprises an ITAM motif. In some embodiments, the sequence encodes a DAP12 polypeptide comprising a DAP12 intracellular signaling domain. In some embodiments, the DAP12 polypeptide further comprises a transmembrane domain. In some embodiments, the DAP12 polypeptide further comprises an extracellular domain. In some embodiments, each of (a), (b), and (c) is represented by the same nucleic acid molecule, e.g., a vector, e.g., a viral vector, e.g., a lentiviral vector. In some embodiments, one of (a), (b), and (c) is encoded by a first nucleic acid molecule, e.g., a vector, e.g., a viral vector, e.g., a lentiviral vector, and the second and third of (a), (b), and (c) are encoded by a second nucleic acid molecule, e.g., a vector, e.g., a viral vector, e.g., a lentiviral vector. In one embodiment, (a) is present in a first nucleic acid molecule, e.g., a vector, e.g., a viral vector, e.g., a lentiviral vector, and (b) and (c) are present in a second nucleic acid molecule, e.g., a vector, e.g., a viral vector, e.g., a lentiviral vector. In another embodiment, (b) is present in a first nucleic acid molecule, e.g., a vector, e.g., a viral vector, e.g., a lentiviral vector, and (a) and (c) are present in a second nucleic acid molecule, e.g., a vector, e.g., a viral vector, e.g., a lentiviral vector. In one embodiment, (c) is present in a first nucleic acid molecule, e.g., a vector, e.g., a viral vector, e.g., a lentiviral vector, and (b) and (a) are present in a second nucleic acid molecule, e.g., a vector, e.g., a viral vector, e.g., a lentiviral vector. In one embodiment, each of (a), (b), and (c) is present in a different nucleic acid molecule, e.g., a different vector, e.g., a viral vector, e.g., a lentiviral vector.

[0066] In certain embodiments, (i) the antigen binding domain of one of the first KIR-CAR and the second KIR-CAR does not comprise a light chain variable domain and a heavy chain variable domain, (ii) the antigen binding domain of one of the first KIR-CAR and the second KIR-CAR is an scFv and the other is other than an scFv, (iii) when presented on a cell surface, the antigen binding domains of the first KIR-CAR and the second KIR-CAR bind to each other less than when both are scFv antigen binding domains, (iv) wherein, when presented on a cell surface, binding of the antigen binding domain of the first KIR-CAR to its cognate antigen is not substantially reduced by the presence of the second KIR-CAR, or (v) the antigen binding domain of one of the first KIR-CAR and the second KIR-CAR is a single VH domain, e.g., a camelid, shark or lamprey single VH domain. (vi) the antigen-binding domain of one of the first KIR-CAR and the second KIR-CAR is an scFv and the other comprises a single VH domain, such as a camelid, shark or lamprey single VH domain, or a single VH domain derived from a human or mouse sequence or a non-antibody scaffold, such as a fibronectin type III antibody-like molecule; (vii) the antigen-binding domain of one of the first KIR-CAR and the second KIR-CAR is an scFv and the other comprises a nanobody; or (viii) the antigen-binding domain of one of the first KIR-CAR and the second KIR-CAR is an scFv and the other comprises a camelid VHH domain.

[0067] In some embodiments, the antigen-binding domain of one of the first KIR-CAR and the second KIR-CAR does not comprise a light chain variable domain and a heavy chain variable domain. In some embodiments, the antigen-binding domain of one of the first KIR-CAR and the second KIR-CAR is an scFv, and the other is other than an scFv.

[0068] In some embodiments, when displayed on a cell surface, the antigen-binding domains of the first KIR-CAR and the second KIR-CAR bind to each other less than when both are scFv antigen-binding domains. In some embodiments, when displayed on a cell surface, binding of the antigen-binding domain of the first KIR-CAR to its cognate antigen is not substantially reduced by the presence of the second KIR-CAR. In some embodiments, the antigen-binding domain of one of the first KIR-CAR and the second KIR-CAR comprises a single VH domain, e.g., a camelid, shark, or lamprey single VH domain, or a single VH domain derived from a human or mouse sequence, or a non-antibody scaffold, e.g., fibronectin, e.g., a fibronectin type III antibody-like molecule. In some embodiments, the antigen-binding domain of one of the first KIR-CAR and the second KIR-CAR is an scFv, and the other comprises a single VH domain, e.g., a camelid, shark, or lamprey single VH domain, or a single VH domain derived from a human or mouse sequence, or a non-antibody scaffold, e.g., fibronectin, e.g., a fibronectin type III antibody-like molecule. In some embodiments, the antigen-binding domain of one of the first KIR-CAR and the second KIR-CAR is an scFv, and the other comprises a nanobody. In some embodiments, the antigen-binding domain of one of the first KIR-CAR and the second KIR-CAR is an scFv, and the other comprises a camelid VHH domain.

[0069] In some embodiments, the nucleic acid comprises a sequence encoding a TCAR.In some embodiments, the TCAR comprises an antigen binding domain and an activation cytoplasmic domain from a T cell receptor complex with CD3, such as CD3 zeta chain, CD3 epsilon chain, CD3 gamma chain, or CD3 delta chain.In some embodiments, the TCAR comprises a costimulatory domain from a costimulatory receptor, such as CD28, CD137, CD27, ICOS, or OX40.

[0070] In certain embodiments, (i) the antigen binding domains of the KIR-CAR and TCAR do not comprise a light chain variable domain and a heavy chain variable domain, (ii) the antigen binding domain of one of the KIR-CAR and TCAR is an scFv and the other is other than an scFv, (iii) when presented on a cell surface, the antigen binding domains of the KIR-CAR and TCAR bind to each other less than when both are scFv antigen binding domains, (iv) when presented on a cell surface, binding of the KIR-CAR to its cognate antigen is not substantially reduced by the presence of the second TCAR, or (v) the antigen binding domain of one of the KIR-CAR and TCAR is a single VH domain, e.g., a camelid, shark, or lamprey single VH domain, or derived from a human or mouse sequence. or a non-antibody scaffold, such as a fibronectin type III antibody-like molecule, (vi) the antigen-binding domain of one of the KIR-CAR and the TCAR is an scFv, and the other comprises a single VH domain, such as a camelid, shark, or lamprey single VH domain, or a single VH domain derived from a human or mouse sequence, or a non-antibody scaffold, such as a fibronectin type III antibody-like molecule, (vii) the antigen-binding domain of one of the KIR-CAR and the TCAR is an scFv, and the other comprises a nanobody, or (viii) wherein the antigen-binding domain of one of the KIR-CAR and the TCAR is an scFv, and the other comprises a camelid VHH domain. In certain embodiments, the antigen-binding domain of one of the KIR-CAR and the TCAR does not comprise a light chain variable domain and a heavy chain variable domain.

[0071] In some embodiments, the antigen-binding domains of the KIR-CAR and the TCAR are scFvs, and the other is other than scFvs. In some embodiments, when presented on the cell surface, the antigen-binding domains of the KIR-CAR and the TCAR bind to each other less than when both are scFv antigen-binding domains. In some embodiments, when presented on the cell surface, the binding of the KIR-CAR to its cognate antigen is not substantially reduced by the presence of the second TCAR. In some embodiments, the antigen-binding domain of one of the KIR-CAR and the TCAR comprises a single VH domain, e.g., a camelid, shark, or lamprey single VH domain, or a single VH domain derived from a human or mouse sequence, or a non-antibody scaffold, e.g., fibronectin, e.g., a fibronectin type III antibody-like molecule. In some embodiments, the antigen-binding domain of one of the KIR-CAR and the TCAR is an scFv, and the other comprises a single VH domain, such as a camelid, shark, or lamprey single VH domain, or a single VH domain derived from a human or mouse sequence, or a non-antibody scaffold, such as fibronectin, for example, a fibronectin type III antibody-like molecule. In some embodiments, the antigen-binding domain of one of the KIR-CAR and the TCAR is an scFv, and the other comprises a nanobody. In some embodiments, the antigen-binding domain of one of the KIR-CAR and the TCAR is an scFv, and the other comprises a camelid VHH domain.

[0072] In some embodiments, the antigen-binding domain of one of the KIR-CAR and the TCAR binds to mesothelin, and the other binds to a different antigen-binding domain, e.g., the same target (mesothelin) or a different target (e.g., a target other than mesothelin on stromal cells, e.g., FAP; a target other than mesothelin on prostate cancer cells, e.g., androgen receptor, OR51E2, PSMA, PSCA, PDGRF-β, TARP, GloboH, MAD-CT-1, or MAD-CT-2; a target other than mesothelin on ovarian cancer cells, e.g., Tn, PRSS21, CD171, Lewis Y, folate receptor alpha, claudin 6, GloboH, or sperm protein 17).

[0073] In another aspect, the invention pertains to a cytotoxic cell, e.g., a naturally occurring or non-occurring T cell, NK cell, or cytotoxic T cell or NK cell line cell, e.g., NK92, comprising (a) a first KIR-CAR described herein. In some embodiments, the cytotoxic cell is a T cell. In some embodiments, the cytotoxic cell is an NK cell. In some embodiments, the cytotoxic cell is from an NK cell line, e.g., an NK92 cell. In some embodiments, the first KIR-CAR is an actKIR-CAR described herein. In some embodiments, the first KIR-CAR is an inhKIR-CAR described herein.

[0074] In some embodiments, the cytotoxic cell comprises an inhibitory molecule comprising a KIR-CAR, e.g., an actKIR-CAR, and an inhKIR cytoplasmic domain; a transmembrane domain, e.g., a KIR transmembrane domain; and an inhibitor cytoplasmic domain, e.g., an ITIM domain, e.g., an inhKIR ITIM domain. In some embodiments, the inhibitory molecule is a naturally occurring inhKIR or a sequence that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% homologous to a naturally occurring inhKIR or that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 residues from a naturally occurring inhKIR.

[0075] In some embodiments, the cytotoxic cell comprises an inhibitory molecule comprising a KIR-CAR (e.g., actKIR-CAR) and a SLAM family cytoplasmic domain; a transmembrane domain (e.g., a SLAM family transmembrane domain); and an inhibitor cytoplasmic domain (e.g., a SLAM family domain, e.g., a SLAM family ITIM domain). In some embodiments, the inhibitory molecule is a naturally occurring SLAM family member or has at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99% homology with a naturally occurring SLAM family member, or a sequence that does not differ by more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 residues.

[0076] In some embodiments, the cytotoxic cell further comprises (b) a second KIR-CAR described herein, e.g., a second KIR-CAR different from the first KIR-CAR. In some embodiments, one of the KIR-CAR and the second KIR-CAR is an actKIR-CAR, and the other is an inhKIR-CAR. In some embodiments, the actKIR-CAR is an actKIR-CAR described herein. In some embodiments, one of the inhKIR-CARs is an inhKIR-CAR described herein. In some embodiments, the cytotoxic cell described herein comprises an actKIR-CAR described herein and an inhKIR-CAR described herein.

[0077] In some embodiments, the cytotoxic cell further comprises an intracellular signaling domain, e.g., an adapter molecule, capable of generating an activation signal, e.g., an activation signal exogenous to the cell. In some embodiments, the intracellular signaling domain comprises an ITAM motif. In some embodiments, the intracellular signaling domain comprises a DAP12 polypeptide comprising a DAP12 intracellular signaling domain. In some embodiments, the DAP12 polypeptide further comprises a transmembrane domain. In some embodiments, the DAP12 polypeptide further comprises an extracellular domain.

[0078] In some embodiments, the cytotoxic cell comprises a first and a second KIR-CAR described herein, wherein (i) the antigen binding domain of one of the first KIR-CAR and the second KIR-CAR does not comprise a light chain variable domain and a heavy chain variable domain, (ii) the antigen binding domain of one of the first KIR-CAR and the second KIR-CAR is an scFv and the other is other than an scFv, (iii) when presented on a cell surface, the antigen binding domains of the first KIR-CAR and the second KIR-CAR bind to each other less than when both are scFv antigen binding domains, (iv) when presented on a cell surface, binding of the antigen binding domain of the first KIR-CAR to its cognate antigen is not substantially reduced by the presence of the second KIR-CAR, or (v) the antigen binding domain of one of the first KIR-CAR and the second KIR-CAR is a single VH domain, e.g., a VH domain derived from a camelid, shark, or other mammal. or a lamprey single VH domain, or a single VH domain derived from a human or mouse sequence or a non-antibody scaffold, such as fibronectin, e.g., a fibronectin type III antibody-like molecule; (vi) the antigen-binding domain of one of the first KIR-CAR and the second KIR-CAR comprises an scFv, and the other comprises a single VH domain, such as a camelid, shark or lamprey single VH domain, or a single VH domain derived from a human or mouse sequence or a non-antibody scaffold, such as fibronectin, e.g., a fibronectin type III antibody-like molecule; (vii) wherein the antigen-binding domain of one of the first KIR-CAR and the second KIR-CAR comprises an scFv, and the other comprises a nanobody; or (viii) the antigen-binding domain of one of the first KIR-CAR and the second KIR-CAR comprises an scFv, and the other comprises a camelid VHH domain.

[0079] In some embodiments, the antigen-binding domain of one of the first KIR-CAR and the second KIR-CAR does not comprise a light chain variable domain and a heavy chain variable domain. In some embodiments, the antigen-binding domain of one of the first KIR-CAR and the second KIR-CAR is an scFv, and the other is other than an scFv. In some embodiments, when displayed on a cell surface, the antigen-binding domains of the first KIR-CAR and the second KIR-CAR bind to each other less than when both are scFv antigen-binding domains. In some embodiments, when displayed on a cell surface, the binding of the antigen-binding domain of the first KIR-CAR to its cognate antigen is not substantially reduced by the presence of the second KIR-CAR. In some embodiments, the antigen-binding domain of one of the first KIR-CAR and the second KIR-CAR comprises a single VH domain, e.g., a camelid, shark, or lamprey single VH domain, or a single VH domain derived from a human or mouse sequence, or a non-antibody scaffold, e.g., fibronectin, e.g., a fibronectin type III antibody-like molecule. In some embodiments, the antigen-binding domain of one of the first KIR-CAR and the second KIR-CAR comprises an scFv, and the other comprises a single VH domain, e.g., a camelid, shark, or lamprey single VH domain, or a single VH domain derived from a human or mouse sequence or a non-antibody scaffold, e.g., fibronectin, e.g., a fibronectin type III antibody-like molecule. In some embodiments, the antigen-binding domain of one of the first KIR-CAR and the second KIR-CAR comprises an scFv, and the other comprises a nanobody. In some embodiments, the antigen-binding domain of one of the first KIR-CAR and the second KIR-CAR comprises an scFv, and the other comprises a camelid VHH domain.

[0080] In some embodiments, one antigen-binding domain of the first KIR-CAR and the second KIR-CAR binds to mesothelin, and the other antigen-binding domain binds to a different antigen, e.g., the same target (mesothelin) or a different target (e.g., a target other than mesothelin on stromal cells, e.g., FAP; a target other than mesothelin on prostate cancer cells, e.g., androgen receptor, OR51E2, PSMA, PSCA, PDGRF-β, TARP, GloboH, MAD-CT-1, or MAD-CT-2; a target other than mesothelin on ovarian cancer cells, e.g., Tn, PRSS21, CD171, Lewis Y, folate receptor alpha, claudin 6, GloboH, or sperm protein 17).

[0081] In some embodiments, the cytotoxic cell comprises a KIR-CAR as described herein and further comprises a TCAR. In some embodiments, the TCAR comprises an antigen-binding domain and a primary stimulatory domain. In some embodiments, the TCAR comprises a costimulatory domain.

[0082] In some embodiments, the cytotoxic cells, such as naturally occurring or non-occurring T cells, NK cells, or cytotoxic T cells or NK cell lines, e.g., NK92 cells, comprise a nucleic acid as described herein; or a KIR-CAR encoded by a nucleic acid as described herein. In some embodiments, the cytotoxic cells are T cells. In some embodiments, the cytotoxic cells are NK cells. In some embodiments, the cytotoxic cells are from NK cell lines, e.g., NK92.

[0083] In another aspect, the invention relates to a method of producing a cell described herein, comprising introducing into a cytotoxic cell a nucleic acid described herein into the cell. In one embodiment, the method comprises forming in the cytotoxic cell a KIR-CAR described herein.

[0084] In another aspect, the invention relates to a method of treating a subject, e.g., a method of providing anti-tumor immunity in a mammal, comprising administering to the mammal an effective amount of the cells described herein. In some embodiments, the cells are autologous. In some embodiments, the cells are allogeneic. In some embodiments, the cells are T cells, e.g., autologous T cells. In some embodiments, the cells are allogeneic T cells. In some embodiments, the cells are NK cells, e.g., autologous NK cells. In some embodiments, the cells are allogeneic NK cells. In some embodiments, the cells are cells from an NK cell line, e.g., NK92. In some embodiments, the mammal is human. In some embodiments, the method comprises assessing side effects of the treatment in the mammal, e.g., human. In some embodiments, the side effects include acute respiratory distress syndrome, febrile neutropenia, hypotension, encephalopathy, hepatic transaminaemia, seizures, or macrophage activation syndrome. In some embodiments, the method further comprises treating the human having the side effect with an agent described herein, e.g., an anti-cytokine agent, e.g., a tumor necrosis factor antagonist, e.g., a TNF-Ig fusion, e.g., etanercept, an IL-6 antagonist, e.g., an IL-6 receptor antagonist, e.g., an anti-IL6 receptor antibody, e.g., tocilizumab, or a corticosteroid. In some embodiments, the treatment comprises administering an anti-IL6 receptor antibody to the human.

[0085] In some embodiments, the disease associated with tumor antigen expression is cancer, e.g., a cancer described herein. In some embodiments, the cancer is a solid tumor, e.g., a solid tumor described herein, e.g., mesothelioma (e.g., malignant pleural mesothelioma), lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, squamous cell lung cancer, or large cell lung cancer), pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), ovarian cancer, colorectal cancer, and bladder cancer, or any combination thereof. In some embodiments, the disease is pancreatic cancer, e.g., metastatic pancreatic ductal adenocarcinoma (PDA), e.g., in a subject that has progressed on at least one prior standard of care. In some embodiments, the disease is mesothelioma (e.g., malignant pleural mesothelioma), e.g., in a subject that has progressed on at least one prior standard of care regimen. In some embodiments, the disease is ovarian cancer, e.g., serous epithelial ovarian cancer, in a subject that has progressed after at least one prior standard of care regimen.

[0086] In some embodiments, the methods include treating a mammal, e.g., a human, having a disease associated with mesothelin or CD19 expression. In some embodiments, the methods include treating a mammal, e.g., a human, having a disorder associated with unwanted cell proliferation, e.g., cancer. In some embodiments, the disorder is pancreatic cancer, mesothelioma, lung cancer, ovarian cancer, leukemia, or lymphoma.

[0087] In another aspect, the invention pertains to a purified or non-naturally occurring NCR-CAR, e.g., an activated NCR-CAR, comprising an extracellular antigen-binding domain, a transmembrane domain, e.g., a transmembrane domain comprising an amino acid residue comprising a positively charged group, e.g., a positively charged group, e.g., a positively charged side chain, or an NCR transmembrane domain, and a cytoplasmic domain, e.g., an NCR cytoplasmic domain.

[0088] In some embodiments, the NCR-CAR comprises a transmembrane domain, e.g., an NCR transmembrane domain, e.g., an NKp30, NKp44, or NKp46 cytoplasmic domain, comprising an amino acid residue comprising a positively charged group, e.g., a positively charged group, e.g., a positively charged side chain. In some embodiments, the NCR-CAR comprises a cytoplasmic domain capable of interacting with an adaptor molecule or an intracellular signaling molecule, e.g., a DAP12, FcRγ, or CD3ζ cytoplasmic domain. In some embodiments, the NCR-CAR, e.g., an NKp30-CAR, comprises a transmembrane domain, e.g., DAP12, capable of interacting with an adaptor molecule or an intracellular signaling molecule. In some embodiments, the NCR-CAR comprises an NKp46-CAR. In some embodiments, the NKp46-CAR comprises a transmembrane domain comprising an amino acid residue comprising a positively charged group, e.g., a positively charged group, e.g., a positively charged side chain, capable of interacting with an adaptor molecule or an intracellular signaling molecule, or e.g., an NCR transmembrane domain, e.g., an FcRγ or CD3ζ cytoplasmic domain. In some embodiments, the NCR-CAR described herein further comprises a hinge domain disposed between the transmembrane domain and the extracellular antigen-binding domain.

[0089] In some embodiments, the NCR-CAR is an activated NCR-CAR and the extracellular antigen-binding domain is an antigen-binding domain described herein, e.g., in Table 4.

[0090] In another aspect, the present invention relates to a nucleic acid, e.g., a purified or non-naturally occurring nucleic acid, e.g., a nucleic acid comprising DNA or RNA, sequence, e.g., mRNA, comprising a sequence encoding an NCR-CAR described herein. In one embodiment, the nucleic acid comprises a sequence encoding an NKp30-CAR and, optionally, an adaptor molecule or intracellular signaling molecule, e.g., DAP12. In one embodiment, the NCR-CAR, e.g., NKp46-CAR, comprises a transmembrane domain or NCR transmembrane domain comprising an amino acid residue comprising a positively charged group, e.g., a positively charged group, e.g., a positively charged side chain, that can interact with an adaptor molecule or intracellular signaling molecule, e.g., an FcRγ or CD3ζ molecule. In one embodiment, the nucleic acid further comprises a sequence encoding an adaptor molecule or intracellular signaling molecule, e.g., DAP12, FcRγ, or CD3ζ.

[0091] In some embodiments, the nucleic acid encodes an actNCR-CAR and the encoded extracellular antigen-binding domain is an antigen-binding domain described herein, e.g., in Table 4.

[0092] In another aspect, the present invention relates to a cytotoxic cell, e.g., a naturally occurring or non-occurring T cell, NK cell, or cytotoxic T cell or NK cell line, comprising an NCR-CAR described herein. In some embodiments, the cytotoxic cell further comprises an adapter molecule or intracellular signaling molecule, e.g., comprising a DAP12, FcRγ, or CD3ζ cytoplasmic domain. In some embodiments, the cytotoxic cell comprises an NKp30-CAR and optionally an adapter molecule or intracellular signaling molecule, e.g., DAP12. In some embodiments, the NKp46-CAR comprises a transmembrane domain capable of interacting with an adapter molecule or intracellular signaling molecule, e.g., an FcRγ or CD3ζ molecule.

[0093] In another aspect, the invention relates to a method of producing a cell described herein, comprising introducing into a cytotoxic cell a nucleic acid comprising a sequence encoding an NCR-CAR described herein, hi some embodiments, the method comprises forming an NCR-CAR described herein into the cytotoxic cell.

[0094] In another aspect, the invention features a method of treating a subject, e.g., a method of providing anti-tumor immunity in a mammal, comprising administering to the mammal an effective amount of a cell described herein, e.g., a cell of the invention described herein, including an NCR-CAR described herein.

[0095] In another aspect, the invention relates to a method of treating a subject having a disease associated with expression of a tumor antigen, e.g., a tumor antigen described herein (e.g., a proliferative disease, a precancerous condition, and a non-cancer indication associated with expression of a tumor antigen), comprising administering to the subject an effective amount of cells comprising an NCR-CAR, e.g., as described herein. In one embodiment, the NCR-CAR is an activated NCR-CAR, and the extracellular antigen-binding domain is an antigen-binding domain described herein, e.g., in Table 4.

[0096] In some embodiments, the disease associated with tumor antigen expression is cancer, e.g., a cancer described herein. In some embodiments, the cancer is a solid tumor, e.g., a solid tumor described herein, e.g., mesothelioma (e.g., malignant pleural mesothelioma), lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, squamous cell lung cancer, or large cell lung cancer), pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), ovarian cancer, colorectal cancer, and bladder cancer, or any combination thereof. In some embodiments, the disease is pancreatic cancer, e.g., metastatic pancreatic ductal adenocarcinoma (PDA), e.g., in a subject that has progressed on at least one prior standard of care. In some embodiments, the disease is mesothelioma (e.g., malignant pleural mesothelioma), e.g., in a subject that has progressed on at least one prior standard of care regimen. In some embodiments, the disease is ovarian cancer, e.g., serous epithelial ovarian cancer, in a subject that has progressed after at least one prior standard of care regimen.

[0097] In another aspect, the invention relates to a purified or non-naturally occurring SLAMF-CAR, e.g., an inhibitory SLAMF-CAR, comprising an extracellular antigen-binding domain, a transmembrane domain, e.g., a transmembrane domain comprising an amino acid residue comprising a positively charged group, e.g., a positively charged group, e.g., a positively charged side chain, e.g., an SLAMF transmembrane domain, and an SLAMF cytoplasmic domain. In some embodiments, the SLAMF-CAR comprises a SLAMF, CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, or CD2F-10 cytoplasmic domain. In some embodiments, the SLAMF-CAR further comprises a hinge domain disposed between the transmembrane domain and the extracellular antigen-binding domain.

[0098] In another aspect, the invention pertains to a nucleic acid, e.g., a purified or non-naturally occurring nucleic acid, e.g., a nucleic acid comprising DNA or RNA, sequence, e.g., mRNA, comprising a sequence encoding a SLAMF-CAR described herein.

[0099] In another aspect, the invention pertains to a cytotoxic cell, e.g., a naturally occurring or non-occurring T cell, NK cell, or cytotoxic T cell or NK cell line, comprising a SLAMF-CAR described herein.

[0100] In another aspect, the invention relates to a method of producing a cytotoxic cell comprising a SLAMF-CAR described herein, comprising introducing into a cytotoxic cell a nucleic acid comprising a sequence encoding a SLAMF-CAR described herein, hi some embodiments, the method comprises forming a SLAMF-CAR described herein into a cytotoxic cell.

[0101] In another aspect, the invention features a method of treating a subject, e.g., a method of providing anti-tumor immunity in a mammal, comprising administering to the mammal an effective amount of cells comprising a SLAMF-CAR described herein.

[0102] In another aspect, the present invention relates to a purified or non-naturally occurring FcR-CAR, e.g., a CD16-CAR, e.g., an activating CD16-CAR or CD64-CAR, e.g., an activating CD64-CAR, comprising an extracellular antigen-binding domain, a transmembrane domain, and a CD16 or CD64 cytoplasmic domain. In some embodiments, the FcR-CAR is a CD16-CAR. In some embodiments, the FcR-CAR is a CD64-CAR. In some embodiments, the FcR-CAR can interact with an adaptor molecule or an intracellular signaling molecule, e.g., an FcRγ or CD3ζ domain, e.g., via a transmembrane domain, e.g., a transmembrane domain comprising an amino acid residue comprising a positively charged group, e.g., a positively charged group, e.g., a positively charged side chain, or e.g., a CD16 or CD64 transmembrane domain. In some embodiments, the FcR-CAR further comprises a hinge domain disposed between the transmembrane domain and the extracellular antigen-binding domain.

[0103] In another aspect, the invention pertains to a purified or non-naturally occurring nucleic acid, e.g., a nucleic acid comprising DNA or RNA, sequence, e.g., mRNA, comprising a sequence encoding an FcR-CAR described herein. In some embodiments, the nucleic acid further comprises an adapter molecule or intracellular signaling molecule comprising a cytoplasmic activation domain, e.g., an FcRγ or CD3ζ cytoplasmic domain. In some embodiments, the FcR-CAR and the cytoplasmic activation domain are disposed in separate nucleic acid molecules, e.g., separate vectors, e.g., separate viral vectors, e.g., separate lentiviral vectors.

[0104] In another aspect, the invention pertains to a cytotoxic cell, e.g., a naturally occurring or non-naturally occurring T cell, NK cell, or cytotoxic T cell or NK cell line, comprising an FcR-CAR described herein. In one embodiment, the cytotoxic cell further comprises a cytoplasmic activation domain, e.g., an FcRγ or CD3ζ cytoplasmic domain.

[0105] In another aspect, the invention relates to a method of producing a cell comprising an FcR-CAR described herein, comprising introducing into a cytotoxic cell a nucleic acid comprising a sequence encoding an FcR-CAR described herein, hi one embodiment, the method comprises forming an FcR-CAR described herein into a cytotoxic cell.

[0106] In another aspect, the invention features a method of treating a subject, e.g., a method of providing anti-tumor immunity in a mammal, comprising administering to the mammal an effective amount of cells comprising an FcR-CAR described herein.

[0107] In another aspect, the present invention relates to a Ly49-CAR comprising a purified or non-naturally occurring extracellular antigen-binding domain and a transmembrane domain, e.g., a Ly49 transmembrane domain or a cytoplasmic domain, e.g., an ITIM-containing cytoplasmic domain, e.g., a Ly49 cytoplasmic domain. In some embodiments, the Ly49-CAR comprises a transmembrane domain and a Ly49 cytoplasmic domain. In some embodiments, the Ly49-CAR is an activating Ly49-CAR, e.g., Ly49D or Ly49H. In some embodiments, the Ly49-CAR comprises a positively charged transmembrane domain, e.g., a positively charged Ly49 transmembrane domain. In some embodiments, the Ly49-CAR can interact with an ITAM-containing cytoplasmic domain, e.g., DAP12. In some embodiments, the Ly49-CAR comprises a Ly49 transmembrane domain. In some embodiments, the KIR-CAR is an inhibitory Ly49-CAR, e.g., Ly49A or Ly49C. In some embodiments, the Ly49-CAR comprises an ITIM-containing cytoplasmic domain, e.g., a Ly49 cytoplasmic domain. In some embodiments, the Ly49-CAR comprises a Ly49 transmembrane domain or a Ly49 cytoplasmic domain, independently selected from Ly49A-Ly49W. In some embodiments, the Ly49-CAR further comprises a hinge domain disposed between the transmembrane domain and the extracellular antigen-binding domain.

[0108] In another aspect, the present invention pertains to a nucleic acid, e.g., a purified or non-naturally occurring nucleic acid, e.g., a nucleic acid comprising DNA or RNA, sequence, e.g., mRNA, comprising a sequence encoding a Ly49-CAR as described herein. In some embodiments, the nucleic acid further comprises a cytoplasmic activation domain, e.g., a DAP12 cytoplasmic domain. In some embodiments, the Ly49-CAR and the cytoplasmic activation domain are disposed in separate nucleic acid molecules, e.g., separate vectors, e.g., separate viral vectors, e.g., separate lentiviral vectors.

[0109] In another aspect, the invention pertains to a cytotoxic cell, e.g., a naturally occurring or non-occurring T cell, NK cell, or cytotoxic T cell or NK cell line, comprising a Ly49-CAR described herein. In one embodiment, the cytotoxic cell further comprises a cytoplasmic activation domain, e.g., a DAP12 cytoplasmic domain.

[0110] In another aspect, the invention features a method of producing a cytotoxic cell comprising a Ly49-CAR described herein, comprising introducing into a cytotoxic cell a nucleic acid comprising a sequence encoding a Ly49-CAR described herein.

[0111] In another aspect, the invention features a method of producing a cell comprising a Ly49-CAR described herein, comprising formulating a Ly49-CAR described herein into a cytotoxic cell.

[0112] In another aspect, the invention features a method of treating a subject, e.g., a method of providing anti-tumor immunity in a mammal, comprising administering to the mammal an effective amount of a cell described herein, e.g., a cell comprising a Ly49-CAR described herein.

[0113] In another aspect, the invention relates to a cell, e.g., a cytotoxic cell, comprising a first non-naturally occurring chimeric membrane-embedded receptor comprising an antigen binding domain and a second non-naturally occurring chimeric membrane-embedded receptor comprising an antigen binding domain, wherein: (i) the antigen binding domain of one of the first and second non-naturally occurring chimeric membrane-embedded receptors does not comprise a light chain variable domain and a heavy chain variable domain; (ii) the antigen binding domain of one of the first and second non-naturally occurring chimeric membrane-embedded receptors comprises an scFv, and the other is other than an scFv; (iii) when displayed on a cell surface, the antigen binding domains of the first and second non-naturally occurring chimeric membrane-embedded receptors bind to each other less than when both are scFv antigen binding domains; (iv) when displayed on a cell surface, binding of the antigen binding domain of the first non-naturally occurring chimeric membrane-embedded receptor to its cognate antigen is not substantially reduced by the presence of the second non-naturally occurring chimeric membrane-embedded receptor; (vi) one antigen-binding domain of the membrane-embedded receptor comprises a single VH domain, e.g., a camelid, shark, or lamprey single VH domain, or a single VH domain derived from a human or mouse sequence or a non-antibody scaffold, e.g., fibronectin, e.g., a fibronectin type III antibody-like molecule; (vi) one antigen-binding domain of the first and second non-naturally occurring chimeric membrane-embedded receptors comprises an scFv, and the other comprises a single VH domain, e.g., a camelid, shark, or lamprey single VH domain; (vii) the antigen-binding domain of one of the first and second non-naturally occurring chimeric membrane-embedded receptors comprises an scFv, and the other comprises a nanobody; and (viii) the antigen-binding domain of one of the first and second non-naturally occurring chimeric membrane-embedded receptors comprises an scFv, and the other comprises a camelid VHH domain. In some embodiments, the cell is a T cell. In some embodiments, the cell is an NK cell. In some embodiments, the cell is derived from an NK cell line, for example, NK92.In some embodiments, one of the first and second non-naturally occurring chimeric membrane-embedded receptors is a TCAR. In some embodiments, both the first and second non-naturally occurring chimeric membrane-embedded receptors are TCARs. In some embodiments, one of the first and second non-naturally occurring chimeric membrane-embedded receptors is an NKR-CAR, e.g., a KIR-CAR. In some embodiments, both the first and second non-naturally occurring chimeric membrane-embedded receptors are NKR-CARs, e.g., KIR-CARs. In some embodiments, the antigen-binding domain of one of the first and second non-naturally occurring chimeric membrane-embedded receptors does not comprise a light chain variable domain and a heavy chain variable domain. In some embodiments, when presented on a cell surface, the antigen-binding domains of the first and second non-naturally occurring chimeric membrane-embedded receptors bind to each other less than when both are scFv antigen-binding domains. In some embodiments, when presented on a cell surface, binding of the antigen-binding domain of the first non-naturally occurring chimeric membrane-embedded receptor to its cognate antigen is not substantially reduced by the presence of the second non-naturally occurring chimeric membrane-embedded receptor. In some embodiments, the antigen-binding domain of one of the first and second non-naturally occurring chimeric membrane-embedded receptors comprises a single VH domain, such as a camelid, shark, or lamprey single VH domain, or a single VH domain derived from a human or mouse sequence or a non-antibody scaffold, such as fibronectin, e.g., a fibronectin type III antibody-like molecule. In some embodiments, the antigen-binding domain of one of the first and second non-naturally occurring chimeric membrane-embedded receptors comprises an scFv, and the other comprises a single VH domain, such as a camelid, shark, or lamprey single VH domain, or a single VH domain derived from a human or mouse sequence or a non-antibody scaffold, such as fibronectin, e.g., a fibronectin type III antibody-like molecule. In some embodiments, the antigen-binding domain of one of the first and second non-naturally occurring chimeric membrane-embedded receptors comprises an scFv, and the other comprises a nanobody. In certain embodiments, the antigen-binding domain of one of said first and second non-naturally occurring chimeric membrane-embedded receptors comprises an scFv and the other comprises a camelid VHH domain.In some embodiments, the invention includes a nucleic acid, e.g., a purified or non-naturally occurring nucleic acid, comprising a sequence encoding a first and a second non-naturally occurring chimeric membrane-embedded receptor comprising an antigen-binding domain described herein. In some embodiments, the invention includes a method of making a cell described herein, comprising introducing a nucleic acid described herein into a cell. In some embodiments, the invention includes a method of making a cell described herein, comprising forming a first and a second non-naturally occurring chimeric membrane-embedded receptor described herein into a cytotoxic cell. In some embodiments, the invention relates to a method of treating a subject, e.g., a method of providing anti-tumor immunity in a mammal, comprising administering to the mammal an effective amount of a cell described herein.

[0114] In another aspect, the invention relates to a kit comprising a cell or nucleic acid described herein.

[0115] In another aspect, the present invention pertains to an isolated nucleic acid sequence encoding a KIR-CAR (Killer Cell Immunoglobulin Receptor-Like - Chimeric Antigen Receptor), wherein the isolated nucleic acid sequence comprises an antigen-binding domain and the nucleic acid sequence of a KIR, or a fragment thereof. In some embodiments, the antigen-binding domain is selected from the group consisting of a murine antibody, a humanized antibody, a human antibody, a chimeric antibody, and fragments thereof. In some embodiments, the fragment is a Fab or scFv. In some embodiments, the antigen-binding domain is an antigen-binding domain described herein, e.g., in Table 4. In some embodiments, the KIR is selected from the group consisting of an activating KIR, an inhibitory KIR, and any combination thereof. In some embodiments, at least one hinge region is removed from the activating KIR.

[0116] In another aspect, the present invention relates to an isolated KIR-CAR (Killer Cell Immunoglobulin-Like Receptor-Chimeric Antigen Receptor) comprising an antigen-binding domain and a KIR or a fragment thereof. In some embodiments, the antigen-binding domain is selected from the group consisting of a murine antibody, a humanized antibody, a human antibody, a chimeric antibody, and fragments thereof. In some embodiments, the fragment is a Fab or scFv. In some embodiments, the antigen-binding domain is an antigen-binding domain described herein, e.g., in Table 4. In some embodiments, the KIR is selected from the group consisting of an activating KIR, an inhibitory KIR, and any combination thereof. In some embodiments, at least one hinge region is removed from the activating KIR.

[0117] In another aspect, the invention pertains to a composition comprising at least two KIR-CARs, wherein a first KIR-CAR comprises an antigen-binding domain and an activating KIR or fragment thereof, and a second KIR-CAR comprises an antigen-binding domain and an inhibitory KIR or fragment thereof. In one embodiment, the antigen-binding domain in the first KIR-CAR is specific for an antigen presented on a tumor, e.g., an antigen-binding domain described herein, e.g., in Table 4, and the antigen-binding domain in the second KIR-CAR is specific for an antigen presented on a normal cell.

[0118] In another aspect, the invention pertains to a genetically modified T cell comprising at least two KIR-CARs, wherein the first KIR-CAR comprises an antigen-binding domain and an activating KIR or fragment thereof, and the second KIR-CAR comprises an antigen-binding domain and an inhibitory KIR or fragment thereof. In one embodiment, the antigen-binding domain in the first KIR-CAR is specific for an antigen presented on a tumor, e.g., an antigen-binding domain described herein, e.g., in Table 4, and the antigen-binding domain in the second KIR-CAR is specific for an antigen presented on a normal cell. In one embodiment, the cell is a T cell.

[0119] In another aspect, the invention relates to a method of providing anti-tumor immunity in a mammal, comprising administering to the mammal an effective amount of cells comprising at least two KIR-CARs, wherein a first KIR-CAR comprises an antigen-binding domain and an activating KIR or fragment thereof, and a second KIR-CAR comprises an antigen-binding domain and an inhibitory KIR or fragment thereof. In some embodiments, the antigen-binding domain in the first KIR-CAR is specific for an antigen presented on a tumor, e.g., an antigen-binding domain described herein, e.g., in Table 4, and the antigen-binding domain in the second KIR-CAR is specific for an antigen presented on a normal cell, thereby controlling off-target activity of the cell. In some embodiments, the cell is a T cell.

[0120] In another aspect, the invention features a method of treating a subject having a disease associated with expression of a tumor antigen (e.g., a proliferative disease, precancerous condition, and non-cancer indication associated with expression of a tumor antigen) comprising administering to a mammal an effective amount of cells comprising at least two KIR-CARs, wherein the first KIR-CAR comprises an antigen-binding domain and an activating KIR or fragment thereof, and the second KIR-CAR comprises an antigen-binding domain and an inhibitory KIR or fragment thereof. In one embodiment, the first KIR-CAR comprises an antigen-binding domain described herein, e.g., in Table 4.

[0121] In some embodiments, the disease associated with tumor antigen expression is cancer, e.g., a cancer described herein. In some embodiments, the cancer is a solid tumor, e.g., a solid tumor described herein, e.g., mesothelioma (e.g., malignant pleural mesothelioma), lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, squamous cell lung cancer, or large cell lung cancer), pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), ovarian cancer, colorectal cancer, and bladder cancer, or any combination thereof. In some embodiments, the disease is pancreatic cancer, e.g., metastatic pancreatic ductal adenocarcinoma (PDA), e.g., in a subject that has progressed on at least one prior standard of care. In some embodiments, the disease is mesothelioma (e.g., malignant pleural mesothelioma), e.g., in a subject that has progressed on at least one prior standard of care regimen. In some embodiments, the disease is ovarian cancer, e.g., serous epithelial ovarian cancer, in a subject that has progressed after at least one prior standard of care regimen.

[0122] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Additionally, the materials, methods, and examples are intended to be illustrative only and not limiting. Titles, subtitles, or numbered or lettered designations, e.g., (a), (b), (i), etc., are for ease of reading only. The use of titles or numbered or lettered designations herein does not require that the steps or elements be performed in alphabetical order or that the steps or elements are necessarily separated from one another. Other features, objects, and advantages of the present invention will be apparent from the specification and drawings, and from the claims.

[0123] Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0124] BRIEF DESCRIPTION OF THE DRAWINGS The following detailed description of preferred embodiments of the present invention will be better understood when viewed in conjunction with the accompanying drawings. For purposes of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and equipment shown in the drawings. [Brief explanation of the drawings]

[0125] [Figure 1] FIG. 1, comprising FIGS. 1A and 1B, is a series of schematic diagrams showing the structures of naturally occurring inhibitory and activating KIRs (FIG. 1A) and scFv-based activating KIR-CARs (FIG. 1B).

[0126] [Figure 2] Figure 2 is a general schematic diagram of the lentiviral vector used to deliver an activated KIR-based CAR in combination with the DAP12 signaling molecule.

[0127] [Figure 3] Figure 3 shows that mesothelin-specific actKIR-CARs can be efficiently expressed on the surface of primary human T cells. Human T cells were stimulated with anti-CD3 / anti-CD28 microbeads, transduced with the indicated CARs, or mock transduced, and expanded ex vivo. Expression was detected using biotinylated goat anti-mouse F(ab)2-specific polyclonal IgG (Jackson Immunologics) followed by staining with streptavidin-PE.

[0128] [Figure 4]Figure 4 shows that T cells expressing the SS1 actKIR-CAR exhibit cytotoxic activity against target K562 cells (KT-meso) engineered to express a mesothelin ligand. Human T cells were stimulated with anti-CD3 / anti-CD28 microbeads, transduced with the indicated CARs, or mock transduced, and expanded ex vivo. 10 CFSE-labeled K562 cells expressing mesothelin (KT-meso) or wild-type control K562 were incubated with various ratios of CAR-expressing T cells for 16 hours at 37°C and 5% CO2. K562 target cells were then counted by flow cytometry using countbright beads and a viability stain (7AAD). The percentage of K562 cells lysed (percent lysis) was calculated by subtracting the number of viable target cells remaining after incubation with effector T cells from the number of viable K562 cells remaining after overnight culture without effector T cells, then dividing by the number of viable K562 cells remaining after overnight culture without effector T cells.

[0129] [Figure 5] Figure 5, comprising Figures 5A and 5B, is a series of schematic diagrams showing an activating KIR CAR (KIR2S CAR) with the KIR2DS2 hinge removed. Based on the kinetic sequestration model of TCR activation shown in Figure 5A, the mesothelin-specific SS1 KIR CAR would have a hinge that is too long to allow proper sequestration. Therefore, shortening the mesothelin-specific KIR CAR hinge would improve function. Figure 5B is a schematic diagram showing the SS1 scFv fused to the KIR transmembrane domain without the 2Ig-like domain from KIR2DS2 as a hinge.

[0130] [Figure 6]Figure 6, including Figures 6A and 6B, is a series of images showing the enhanced cytolytic activity of the SS1 scFv-based KIRS2 CAR against mesothelin-expressing target cells compared to a CAR formed by fusing the SS1 scFv to full-length wild-type KIR2DS2. Primary human T cells were stimulated with CD3 / 28 microbeads and subsequently lentivirally transduced with either the SS1-KIR2DS2-activated KIR-CAR, the SS1-KIRS2-activated KIR CAR, or the SS1 zeta CAR. Mock non-transduced T cells (NTD) were used as a control. T cells were expanded to the end of the logarithmic growth phase. Surface expression of the SS1-specific CAR was determined by flow cytometry using a biotinylated goat anti-mouse F(ab)2-specific polyclonal antibody, followed by streptavidin-PE detection, as shown in Figure 6A. As shown in Figure 6B, K562 target cells stained with CFSE in the presence or absence of mesothelin were mixed with effector T cells characterized in Figure 6A as indicated, using various effector T cell-to-target ratios ranging from 10:1 to 1:1. Target K562 cell lysis was assessed using flow cytometry to determine the percent of viable CFSE+ cells, as described for Figure 4. Data shown are calculated percent target cell lysis compared to target cells in the absence of effector cells.

[0131] [Figure 7] Figure 7, including Figures 7A and 7B, is a series of images showing coexpression of CD19 actKIR-CAR and SS1 inhKIR-CAR. Jurkat NFAT-GFP reporter cells, transduced with the indicated KIR CAR or non-transduced (NDT), were mixed 1:1 with target cells in the presence or absence of CD19 and mesothelin antigens, as indicated. Results show GFP expression 24 hours after mixing of Jurkat and target cells (Figure 7A). Figure 7B shows surface expression of mesothelin and CD19 idiotype, as determined by staining with a monoclonal antibody specific for mesothelin-Fc fusion protein and the FMC63 anti-CD19 scFv idiotype.

[0132] [Figure 8] Figure 8, including Figures 8A, 8B, and 8C, is a series of images showing the coexpression of wild-type PD-1 with both activating KIR-based CARs or TCR zeta-based CARs targeting mesothelin. Primary human T cells were stimulated with CD3 / 28 microbeads and subsequently lentivirally transduced with SS1-KIRS2-activating KIR CARs or SS1 zeta CARs. Mock-nontransduced cells (NTD) were used as a negative control. T cells were expanded for 9 days, and surface CAR expression was determined by staining with mesothelin-Fc followed by a goat anti-human Fc-specific antibody conjugated to PE (Figure 8A). K562 cell lines (wild-type [wt], mesothelin-expressing [meso], or mesothelin and PD-L1 coexpressing [meso-PDL1]) were stained with the CAK1 anti-mesothelin-specific monoclonal antibody to confirm mesothelin expression in the targets (Figure 8B). Primary human T cells transduced as shown in Figure 8A were electroporated with 10 μg of in vitro transcribed RNA encoding wild-type PD1 using a BTX ECM830 electroporator (PD1+) or mock transfected (PD1-). Surface expression of PD-1 was expressed using an anti-PD1 monoclonal antibody conjugated to APC (Figure 8C).

[0133] [Figure 9]Figure 9 shows that coexpression of wild-type PD-1 with both an activating KIR-based CAR and a TCR zeta-based CAR targeting mesothelin leads to PD-1 ligand 1 (PDL-1)-dependent inhibition of mesothelin-specific activating KIR-CAR cytotoxicity. Primary human T cells were stimulated with CD3 / 28 microbeads and subsequently lentivirally transduced with an SS1-KIRS2-activating KIR CAR, an SS1 zeta CAR, or a mock transduction (NTD). T cells were expanded for 9 days, followed by electroporation of 5 x 10 T cells with 10 μg of in vitro transcribed RNA encoding wild-type PD1 using a BTX ECM830 electroporator (PD1+) or mock transduction (PD1-). Surface expression of the SS1-specific CAR and PD-1 was determined as shown in Figure 8. K562 target cells expressing mesothelin without or with or without PDL-1 were mixed in different T cell conditions, as indicated, using various effector T cell-to-target ratios ranging from 30:1 to 1:1. Target K562 cell lysis was assessed using the calcein AM dye method to quantify viable cells remaining after 4 hours of incubation. Data shown are calculated % target cell lysis compared to target cells in the absence of effector cells.

[0134] [Figure 10]Figure 10 shows images of interferon gamma (IFN-γ) and interleukin-2 production by T cells from donors expressing mesothelin-specific activating KIR-based CARs (SS1-KIRS2) or TCR zeta-based CARs in the presence or absence of costimulatory domains (SS1-z, SS1-28z, or SS1-BBz). Primary human T cells were stimulated and subsequently lentivirally transduced with the indicated activating KIR CARs or TCR zeta-based CARs. After expression, transduced T cells were mixed with K562 (Kwt) or K562-mesothelin cells (Kmeso) at a 2:1 ratio. Cytokine concentrations were determined in supernatants 24 hours after stimulation by ELISA for the indicated cytokines in multiple independent donors. Repeated-measures ANOVA demonstrated significant CAR effects for IFN-γ (p=0.002) and IL-2 (p=0.0156). SS1-KIRS2 / DAP12 (SS1KIR) vs. sham for IFN-γ (post-hoc paired t-test, p=0.0162). SS1-KIR vs. SS1-28z for IL-2 (post-hoc paired t-test, p=0.0408).

[0135] [Figure 11] Figure 11 shows a heat map of cytokine concentrations in supernatants assessed by a multiplex luminex-based immunoassay (Cytokine Human 10-Plex Panel, Life Technologies). Heat maps of relative concentrations after normalization across donors and conditions to the lowest concentration for each cytokine were generated using the heatmap package included in the R totalization software.

[0136] [Figure 12]Figure 12, including Figures 12A-12B, describes the construction of mesothelin-specific KIR-based chimeric antigen receptor (KIR-CAR)-engineered T cells with potent cytotoxic activity. Primary human T cells were stimulated with CD3 / 28 microbeads and subsequently transduced with lentiviral vectors expressing GFP and dsRed (control) or DAP12 and dsRed (DAP12). Cells were expanded ex vivo until the end of logarithmic growth phase. 5 x 10 T cells from each transduced population were electroporated with 10 µg of in vitro transcribed RNA encoding SS1-KIRS2 using a BTX ECM830 electroporator. Expression of both dsRed and SS1-KIRS2 was assessed by flow cytometry, with SS1-KIRS2 detected using a biotinylated goat anti-mouse F(ab)2-specific polyclonal antibody followed by streptavidin-PE. The top panel of Figure 12A shows the gating strategy for identifying T cells expressing dsRed, which were then analyzed for SS1-KIRS2 expression as indicated by the strains in the panel. Figure 12B shows the ability of the cells characterized in Figure 12A to mediate cytotoxicity against wild-type K562 cells (K562-wt) or K562 cells expressing mesothelin (K562-mesothelin), as assessed using a 4-hour 51Cr release assay.

[0137] [Figure 13] Figure 13 shows that endogenous TCR expression is not affected by SS1-KIRS2 and DAP12 expression. 5 x 10 primary human T cells were electroporated with 10 μg of in vitro transcribed RNA encoding SS1-KIRS2 or mock transfection using a BTX ECM830 electroporator. After overnight incubation, transfected T cells were stained for SS1-KIRS2 expression using a biotinylated goat anti-mouse F(ab)2-specific polyclonal antibody followed by streptavidin-PE. Vβ13.1 expression was assessed using a PE-conjugated monoclonal antibody specific for this Vβ chain of the TCR.

[0138] [Figure 14]Figure 14 shows that the ability of mesothelin-specific KIR-based CAR (SS1-KIRS2) to stimulate T cell proliferation is antigen-dependent but independent of additional CD28 costimulation. Primary human T cells were stimulated with CD3 / 28 microbeads and subsequently lentivirally transduced with SS1-KIRS2 and DAP12 or mesothelin-specific TCR zeta CAR (SS1 zeta). Mock-untransduced cells (NTD) were used as a negative control. Mesothelin-free (K562 wt) or mesothelin-expressing (K562-mesothelin) K562 target cells were mixed at a 2:1 ratio of effector T cells to target cells under various T cell conditions as indicated. K562-mesothelin-stimulated T cells were further divided into conditions without or with 1 μg / mL of monoclonal anti-CD28 agonist antibody (clone 9.3). Viable T cell numbers were enumerated by flow cytometry using bead-based counting at the indicated time points, and the number of population doublings after antigen stimulation was calculated.

[0139] [Figure 15]Figure 15, including Figures 15A, 15B, 15C, 15D, and 15E, shows that mesothelin-specific KIR-CAR-modified T cells exhibit enhanced antitumor activity in vivo compared to second-generation TCR-ζ-based CARs carrying CD28 or CD137 (4-1BB) costimulatory domains. Figure 15A shows an experiment in which NOD-SCID-γc − / − (NSG) mice were subcutaneously implanted with 2×10 mesothelin-expressing mesothelioma-derived cells (EM-meso cells). Twenty days after tumor implantation, each animal was stimulated with anti-CD3 / anti-CD28 stimulator beads and subsequently injected intravenously with 5 x 10 T cells lentivirally transduced with a series of CD3ζ-based CARs with or without costimulatory domains (SS1-ζ, SS1-BBζ, and SS1-28ζ) or the mesothelin-specific KIR-based CAR, SS1-KIRS2, carrying DAP12. Mock-transduced T cells (NTD) were used as controls. Tumor volumes were measured with calipers at the indicated times (n = 7 mice per group). Figure 15B shows that the in vivo activity of KIR-CARs is independent of T cell engraftment in the blood, spleen, or tumor. Human CD45+ T cell frequencies were assessed by flow cytometry at the end of the experiment, and data are expressed as the percentage of total viable cells in the blood, spleen, and tumor digesta. Figure 15C shows that comparable frequencies of CD3+ TILs were observed in the SS1-28ζ and SS1-KIRS2 / DAP12 CAR T cell-treated groups. The same model as shown in Figure 15A was used. CD3+ human lymphocyte frequencies in tumors at day 30 (10 days after CAR T injection) were assessed by flow cytometry. Figure 15D shows that DAP12-modified T cells require a mesothelin-specific KIR-based CAR for tumor eradication. The same model as shown in Figure 15A was used. Four million T cells expressing DAP12 and dsRed (DAP12), SS1-28z, or SS1-KIRS2 and DAP12 (SS1-KIRS2) were intravenously injected on day 20, and tumor volume was assessed over time by caliper measurement. The arrow indicates the time of TIL isolation used for functional and phenotypic analysis. FIG. 15E shows the antigen-specific cytotoxic activity of TILs isolated from the mice described in FIG. 15D.Antigen-specific cytotoxicity was assessed by co-culturing with firefly luciferase-expressing EM-meso cells or EMp cells (parental EM cells lacking mesothelin expression) at the indicated E:T ratios for 18 hours.

[0140] [Figure 16] Figure 16, including Figures 16A and 16B, shows that KIR-based CARs with CD19 specificity can induce antigen-specific target cytotoxicity. After anti-CD3 / anti-CD28 bead activation, T cells were transduced with a bicistronic lentiviral vector expressing DAP12, along with a CD19-specific KIR-based CAR (CD19-KIR2DS2) in which FMC63-derived scFv was fused to the full-length KIR2DS2, or a KIR-based CAR (CD19-KIRS2) produced by fusing FMC63 scFv to the transmembrane and cytoplasmic domains of KIR2DS2 via a short linker, [Gly]4-Ser. The transduced T cells were cultured until the end of the logarithmic growth phase, and expression of the CD19-specific KIR-based CAR was assessed by flow cytometry using a biotinylated goat anti-mouse F(ab)2 polyclonal antibody followed by SA-PE. 51Cr-labeled K562 target cells with or without CD19 expression (K562-CD19) were mixed at various T cell-to-target cell (E:T) ratios. Cytotoxicity was determined by measuring the fraction of 51Cr released from the supernatant at 4 hours. Mock-transfected (NTD) or CD19-specific CD3ζ-based CAR-transduced (CD19-z) control T cells were also included as negative and positive controls, respectively.

[0141] [Figure 17]Figure 17, including Figures 17A and 17B, shows the in vivo activity of CD19-KIRS2. NOD-SCID-γc − / − (NSG) mice were intravenously implanted with 1 million Nalm-6 CBG tumor cells, a CD19-expressing leukemia cell line, via tail vein injection on day 0. T cells were stimulated with anti-CD3 / anti-CD28 stimulator beads and then lentivirally transduced on day 1 with a series of CD19-specific CD3ζ-based CARs or a CD19-specific KIR-based CAR, CD19-KIRS2 (19KIRS2), bearing DAP12, in the presence or absence of costimulatory domains (CD19z, 19BBz). Mock non-transduced T cells (NTD) were used as a control. T cells were expanded ex vivo to the end of logarithmic growth phase and then intravenously injected at 2 million CAR T cells per mouse 5 days after leukemia cell line injection. Tumor burden was assessed by bioluminescence imaging. Five animals were analyzed for each T cell condition. Figure 17A shows the individual bioluminescence photon flux for each animal at day 5 (baseline before T cell injection) and 15 days after leukemia cell transplantation. Figure 17B shows the median total flux over time for each treatment group.

[0142] [Figure 18] Figure 18 shows that NKp46-based NCR CARs bearing mesothelin specifically induce antigen-specific cytotoxicity. After anti-CD3 / anti-CD28 bead activation, T cells were transduced with bicistronic lentiviral vectors expressing DAP12 and SS1-KIRS2 (control), or FcεRγ and mesothelin-specific NKp46-based CARs (SS1-NKp46), or FcεRγ and mesothelin-specific NKp46 CARs in which the native NKp46 extracellular domain was truncated (SS1-TNKp46). Expression of the mesothelin-specific CAR was assessed by flow cytometry using a biotinylated goat anti-mouse F(ab)2 polyclonal antibody followed by SA-PE, as shown in Figure 18A. T cells were mixed with 51Cr-labeled K562 target cells expressing mesothelin at various effector T cell to target K562 cell ratios (E:T ratios). Cytotoxicity was determined by measuring the fraction of 51Cr released from the supernatant at 4 hours compared to spontaneous release, as shown in Figure 18B.

[0143] [Figure 19] FIG. 19 shows a schematic diagram of the receptors used in the experiments shown in FIGS.

[0144] [Figure 20] Figure 20 shows the generation and characterization of the K562-meso cell line, which expresses the KIR2DL3 ligand HLA-Cw. K562 cells (K562) or K562 cells expressing mesothelin (K562-meso) were transduced with the HLA-Cw3 ​​allele, followed by fluorescence-activated cell sorting to obtain K562 cells expressing HLA-Cw with or without mesothelin expression (K562-meso-HLACw). HLA-Cw3 ​​expression was assessed by flow cytometry using an APC-conjugated monoclonal antibody that recognizes HLA-A, B, and C alleles (clone W6 / 32).

[0145] [Figure 21] Figure 21 shows coexpression of SS1-KIRS2 and KIR2DL3 in primary human T cells. Primary human T cells were stimulated with CD3 / 28 microbeads and then lentivirally transduced with SS1-KIRS2 and DAP12 (SS1-KIRS2) or a pseudotransduction (NTD) along with wild-type KIR2DL3. T cells were expanded to the end of the logarithmic growth phase. Expression of surface mesothelin-specific CAR and KIR2DL3 was determined by staining with mesothelin-Fc, followed by PE-conjugated goat anti-human Fc and a monoclonal antibody against the KIR2DL3 ectodomain.

[0146] [Figure 22]Figure 22 shows that KIR2DL3 co-expressed with KIR CAR can suppress antigen-specific cytotoxicity in the presence of HLA-Cw on target cells. T cells generated and characterized as described in Figure 21 were mixed with 51Cr-targeted K562 cells generated and treated as described in Figure 22. Cytotoxicity was determined by measuring the fraction of 51Cr released from the supernatant at 4 hours compared to target cells in the absence of effector cells.

[0147] [Figure 23] FIG. 23 shows a schematic diagram of the receptors used in the experiments shown in FIG.

[0148] [Figure 24] Figure 24 shows that it is not possible to co-express two scFv-based chimeric receptors on the T cell surface while maintaining the binding specificity of each receptor. Jurkat T cells were transduced with a lentiviral vector encoding SS1-KIR2DL3. These cells were subsequently transduced with a second lentiviral vector encoding CD19-KIR2DS2 at various vector dilutions. Expression of the SS1-specific scFv was assessed using mesothelin-Fc followed by PE-conjugated goat anti-human Fc. CD19-specific scFv expression was assessed using a PE-conjugated monoclonal antibody specific for the FMC63 idiotype.

[0149] [Figure 25]Figure 25 shows that expression of a CD19-specific CAR also reduces the expression of mesothelin-binding sites on the surface of cells co-expressing an SS1 zeta-mCherry fusion CAR. Primary human T cells were stimulated with CD3 / 28 microbeads and then lentivirally transduced with SS1 scFv zeta CAR carrying a C-terminal mCherry fusion (SS1z-mCh) or the FMC63-derived CD19-specific 41BB zeta CAR (19bbz), alone or in combination. Mock-transfected cells were used as controls. T cells were expanded to the end of the logarithmic growth phase, and dsRed and surface CAR expression were determined by flow cytometry after staining with mesothelin-Fc followed by a goat anti-human Fc-specific polyclonal antibody conjugated to FITC.

[0150] [Figure 26] Figure 26 shows that the mutually exclusive expression of the binding site of SS1 scFv is not unique to FMC63 scFv. Primary human T cells were stimulated with CD3 / 28 microbeads and then lentivirally transduced with SS1 scFv zeta CAR or various CD19-specific 41BB zeta CARs (19BBz [FMC63 scFv, 214d scFv, or BL22 scFv CAR] with alternating VH and VL orientations [H2L and L2H]). NTD refers to mock-transduced cells used as a staining control. Additionally, another set of T cells was co-transduced with SS1 scFv zeta CAR and different CD19-specific CARs as described above. T cells were expanded to the end of logarithmic growth phase, and surface CAR expression was determined with biotinylated protein L (kappa light chain recognition), followed by streptavidin APC staining, followed by mesothelin-Fc, followed by a goat anti-human Fc-specific polyclonal antibody conjugated to PE. Co-transduced cells demonstrate the mutually exclusive expression seen with FMC63-based CARs, as well as with other scFv-CARs.

[0151] [Figure 27]Figure 27, comprising Figures 27A and 27B, shows the putative mechanism of loss of scFv binding when two scFv molecules are expressed on the cell surface (Figure 27A) and the putative avoidance of this interaction when a camelid single VHH domain-based CAR is expressed on the T cell surface in combination with an scFv-based CAR.

[0152] [Figure 28] Figure 28 shows that camelid single VHH domain-based CARs can be expressed on the T cell surface in combination with scFv-based CARs without any apparent receptor interaction. Jurkat T cells expressing GFP under an NFAT-dependent promoter (NF-GFP) were transduced with a mesothelin-specific activating CAR (SS1-CAR), a CD19-specific activating CAR (19-CAR), or a CAR produced using a camelid VHH domain specific for EGFR (VHH-CAR). After transduction with the activating CAR, the cells were transduced with an additional inhibitory CAR that recognizes CD19 (19-PD1) to produce cells co-expressing both the activating and inhibitory CARs (SS1+19PD1, 19+19PD1, or VHH+19PD1). Transduced Jurkat T cells were cocultured for 24 hours with various cell lines that either lacked all target antigens (K562), expressed only mesothelin (K-meso), CD19 (K-19), or EGFR (A431), expressed a combination of EGFR and mesothelin (A431-mesothelin) or CD19 (A431-CD19), or expressed a combination of CD19 and mesothelin (K-19 / meso). Additional conditions, including no stimulator cells (no stim) or K562 with 1 μg / mL OKT3 (OKT3), were included as negative and positive controls for NFAT activation, respectively. GFP expression, as a marker of NFAT activation, was assessed by flow cytometry.

[0153] [Figure 29-1] Figure 29 shows the KIR2DS2 sequence annotation. The nucleotide sequence provided in Figure 29 is designated SEQ ID NO: 342. The amino acid sequence provided in Figure 29 is designated SEQ ID NO: 343. [Figure 29-2] Figure 29 shows the KIR2DS2 sequence annotation. The nucleotide sequence provided in Figure 29 is designated SEQ ID NO: 342. The amino acid sequence provided in Figure 29 is designated SEQ ID NO: 343.

[0154] [Figure 30-1] Figure 30 shows the KIR2DL3 sequence annotation. The nucleotide sequence provided in Figure 30 is designated SEQ ID NO: 344. The amino acid sequence provided in Figure 30 is designated SEQ ID NO: 345. [Figure 30-2] Figure 30 shows the KIR2DL3 sequence annotation. The nucleotide sequence provided in Figure 30 is designated SEQ ID NO: 344. The amino acid sequence provided in Figure 30 is designated SEQ ID NO: 345. [Figure 30-3] Figure 30 shows the KIR2DL3 sequence annotation. The nucleotide sequence provided in Figure 30 is designated SEQ ID NO: 344. The amino acid sequence provided in Figure 30 is designated SEQ ID NO: 345.

[0155] [Figure 31-1] Figure 31 shows the NKp46 sequence annotation. The nucleotide sequence provided in Figure 31 is designated SEQ ID NO: 346. The amino acid sequence provided in Figure 31 is designated SEQ ID NO: 347. [Figure 31-2] Figure 31 shows the NKp46 sequence annotation. The nucleotide sequence provided in Figure 31 is designated SEQ ID NO: 346. The amino acid sequence provided in Figure 31 is designated SEQ ID NO: 347.

[0156] [Figure 32-1] Figure 32 shows the SS1-KIRS2 sequence annotation. The nucleotide sequence provided in Figure 32 is designated SEQ ID NO: 348. The amino acid sequence provided in Figure 32 is designated SEQ ID NO: 349. [Figure 32-2] Figure 32 shows the SS1-KIRS2 sequence annotation. The nucleotide sequence provided in Figure 32 is designated SEQ ID NO: 348. The amino acid sequence provided in Figure 32 is designated SEQ ID NO: 349.

[0157] [Figure 33-1] Figure 33 shows the SS1-KIR2DS2 sequence annotation. The nucleotide sequence provided in Figure 33 is designated SEQ ID NO: 350. The amino acid sequence provided in Figure 33 is designated SEQ ID NO: 351. [Figure 33-2] Figure 33 shows the SS1-KIR2DS2 sequence annotation. The nucleotide sequence provided in Figure 33 is designated SEQ ID NO: 350. The amino acid sequence provided in Figure 33 is designated SEQ ID NO: 351. [Figure 33-3] Figure 33 shows the SS1-KIR2DS2 sequence annotation. The nucleotide sequence provided in Figure 33 is designated SEQ ID NO: 350. The amino acid sequence provided in Figure 33 is designated SEQ ID NO: 351.

[0158] [Figure 34-1] Figure 34 shows the SS1-tNKp46 sequence annotation. The amino acid sequence provided in Figure 34 is designated number 352. The amino acid sequence provided in Figure 34 is designated number 353. [Figure 34-2] Figure 34 shows the SS1-tNKp46 sequence annotation. The amino acid sequence provided in Figure 34 is designated number 352. The amino acid sequence provided in Figure 34 is designated number 353.

[0159] [Figure 35-1] Figure 35 shows the SS1-KIRL3 sequence annotation. The nucleotide sequence provided in Figure 35 is designated SEQ ID NO: 354. The amino acid sequence provided in Figure 35 is designated SEQ ID NO: 355. [Figure 35-2] Figure 35 shows the SS1-KIRL3 sequence annotation. The nucleotide sequence provided in Figure 35 is designated SEQ ID NO: 354. The amino acid sequence provided in Figure 35 is designated SEQ ID NO: 355.

[0160] [Figure 36]Figure 36 shows that mesothelin-specific CD3ζ- and KIR-based CARs have similar antigen-specific in vitro cytotoxicity against mesothelin-expressing mesothelioma-derived cells (EM-meso cells). Primary human T cells were stimulated with anti-CD3 / CD28 stimulator beads and transduced with a lentiviral vector expressing the SS1-KIRS2 mesothelin-specific CAR. After expression, T cells were mixed with 51Cr-labeled K562 cells expressing EM-meso at the indicated effector-to-target (E:T) ratio. % lysis was determined.

[0161] [Figure 37] Figure 37 shows that TILs from 28ζ CART-treated mice lose IFNγ secretion upon stimulation with mesothelioma-derived cells expressing mesothelin (EM-meso cells). NOD-SCID-γc − / − (NSG) mice were subcutaneously injected with 2×10 EM-meso cells. Five-hundred-five (5×10 6 ) primary human T cells transduced with the indicated CARs were injected IV on day 16. Eighteen days after CAR T cell infusion, TILs were isolated with CD45 magnetic beads and mixed with EM-meso at the indicated effector-to-target (E:T) ratio. Cytokine concentrations were determined in the supernatants by ELISA.

[0162] [Figure 38] Figure 38 shows that SS1-KIRS2 / DAP12 T cells mediate robust antitumor activity in vivo. NOD-SCID-γc − / − (NSG) mice were subcutaneously injected with 2×10 mesothelioma-derived cells expressing mesothelin (EM-meso cells). 5×10 primary human T cells transduced with the indicated CAR were injected IV on day 20. Tumor volumes were measured by caliper at the indicated times.

[0163] [Figure 39]Figure 39, comprising Figures 39A, 39B, and 39C, is a schematic diagram showing the structure of a mesothelin-specific CAR. Figure 39A is a mesothelin-specific multi-chain KIR-CAR. Figure 39B is a mesothelin-specific single-chain KIR-CAR containing DAP12. Figure 39C is a mesothelin-specific CAR containing CD28 and CD3 zeta signaling domains.

[0164] [Figure 40] Figure 40 includes the in vivo antitumor activity of mesothelin-based CAR constructs, including multi-chain KIRS / DAP12 and single-chain DAP12 constructs.

[0165] [Figure 41-1] Figure 41 shows surface expression of mesothelin-based KIR-CAR on human primary T cells as detected by flow cytometry analysis. [Figure 41-2] Figure 41 shows surface expression of mesothelin-based KIR-CAR on human primary T cells as detected by flow cytometry analysis. [Figure 41-3] Figure 41 shows surface expression of mesothelin-based KIR-CAR on human primary T cells as detected by flow cytometry analysis. [Figure 41-4] Figure 41 shows surface expression of mesothelin-based KIR-CAR on human primary T cells as detected by flow cytometry analysis. [Figure 41-5] Figure 41 shows surface expression of mesothelin-based KIR-CAR on human primary T cells as detected by flow cytometry analysis. [Figure 41-6] Figure 41 shows surface expression of mesothelin-based KIR-CAR on human primary T cells as detected by flow cytometry analysis.

[0166] [Figure 42] Figure 42, comprising Figures 42A and 42B, shows the antigen-specific cytotoxic activity of mesothelin-based KIR-CAR against target cells that do not express mesothelin (K562) (Figure 42A) or target cells engineered to express mesothelin (K562-meso) (Figure 42B).

[0167] [Figure 43A] Figure 43, comprising Figures 43A and 43B, shows cytokine production of mesothelin-based KIR-CARs when cultured in the presence of mesothelin-expressing target cells (K562-meso, black bars) or control target cells that do not express mesothelin (K562, open bars). Figure 43A shows IFN-gamma production; Figure 43B shows IL-2 cytokine production. [Figure 43B] Figure 43, comprising Figures 43A and 43B, shows cytokine production of mesothelin-based KIR-CARs when cultured in the presence of mesothelin-expressing target cells (K562-meso, black bars) or control target cells that do not express mesothelin (K562, open bars). Figure 43A shows IFN-gamma production; Figure 43B shows IL-2 cytokine production.

[0168] [Figure 44] Figure 44, including Figures 44A, 44B, 44C, 44D, and 44E, shows various configurations in a single vector, for example, when a U6-controlled shRNA is located upstream or downstream of an EF1 alpha-controlled CAR coding element. In the example constructs shown in Figures 44A and 44B, transcription occurs via the U6 and EF1 alpha promoters in the same direction. In the example constructs in Figures 44C and 44D, transcription occurs via the U6 and EF1 alpha promoters in different directions. In Figure 44E, the shRNA (and the corresponding U6 promoter) is in a first vector, and the CAR (and the corresponding EF1 alpha promoter) is in a second vector.

[0169] [Figure 45]Figure 45 depicts the structure of an exemplary two-RCAR configuration. The antigen-binding member comprises an antigen-binding domain, a transmembrane domain, and a switch domain. The intracellular binding member comprises a switch domain, a costimulatory signaling domain, and a primary signaling domain. The two-configuration illustrates that the first and second switch domains described herein can be in different orientations relative to the antigen-binding member and the intracellular binding member. Other RCAR configurations are further described herein.

[0170] [Figure 46] Figure 46 shows that proliferation of CAR-expressing, transduced T cells is stimulated by the presence of low doses of RAD001 in a cell culture system. CARs were co-cultured with Nalm-6 cells in the presence of various concentrations of RAD001. The number of CAR-positive CD3-positive T cells (black) and total T cells (gray) was assessed after 4 days of co-culture.

[0171] [Figure 47] Figure 47 shows tumor growth measurements of NALM6-luc cells with daily dosing of RAD001 at 0.3 mg / kg, 1 mg / kg, 3 mg / kg, and 10 mg / kg (mpk) or vehicle dosing. Circles represent vehicle; squares represent a 10 mg / kg dose of RAD001; triangles represent a 3 mg / kg dose of RAD001; inverted triangles represent a 1 mg / kg dose of RAD001; and diamonds represent a 0.3 mg / kg dose of RAD001.

[0172] [Figure 48] Figure 48, comprising Figures 48A and 48B, shows pharmacokinetic curves showing the amount of RAD001 in the blood of NSG mice bearing NALM6 tumors. Figure 48A shows day 0 PK after the first dose of RAD001. Figure 48B shows day 14 PK after the final RAD001 administration. Diamonds represent a 10 mg / kg dose of RAD001; squares represent a 1 mg / kg dose of RAD001; triangles represent a 3 mg / kg dose of RAD001; and x represents a 10 mg / kg dose of RAD001.

[0173] [Figure 49]Figure 49, showing Figures 49A and 49B, shows the in vivo expansion of humanized CD19 CART cells in the presence and absence of RAD001 dosing. Daily low doses of RAD001 (0.003 mg / kg) are shown to result in enhanced CAR T cell proliferation beyond normal levels of huCAR19 proliferation. Figure 49A shows CD4+ CAR T cells; Figure 49B shows CD8+ CAR T cells. Circles represent PBS; squares represent huCTL019; triangles represent huCTL019 and 3 mg / kg RAD001; inverted triangles represent huCTL019 and 0.3 mg / kg RAD001; diamonds represent huCTL019 and 0.03 mg / kg RAD001; circles represent huCTL019 and 0.003 mg / kg RAD001. DETAILED DESCRIPTION OF THE INVENTION

[0174] Detailed Description In one aspect, the present invention provides compositions and methods for controlling the specificity and activity of T cells or other cytotoxic cells, e.g., NK cells. In one embodiment, chimeric antigen receptors (CARs) based on NK cell receptors (NKRs), e.g., KIR-CARs, NCR-CARs, SLAMF-CARs, FcR-CARs, or Ly49-CARs, e.g., NK cell receptor-CARs (NKR-CARs) are provided. In one embodiment, the present invention provides a type of chimeric antigen receptor (CAR), wherein the CAR is an NKR, e.g., referred to as a "KIR-CAR," which is a CAR design that includes components of a receptor found on natural killer (NK) cells. In one embodiment, NK receptors include, but are not limited to, killer cell immunoglobulin-like receptors (KIRs). KIRs can function as activating or inhibitory KIRs.

[0175] One advantage of the NKR-CAR, e.g., KIR-CAR, of the present invention is that the NKR-CAR, e.g., KIR-CAR, provides a method for controlling cytotoxic cell, e.g., T cell, specificity to control off-target activity of engineered T cells. In some instances, the KIR-CAR of the present invention does not require costimulation to proliferate.

[0176] NKR-CARs deliver signals through adaptor proteins, e.g., ITAM-containing adaptor proteins. In some embodiments, the KIR-CARs of the present invention comprise an activating KIR, which delivers its signal through interaction with the immunotyrosine-based activation motif (ITAM)-containing membrane protein, DAP12, which is mediated by residues in the transmembrane domain of these proteins.

[0177] In some embodiments, NKR-CARs can deliver inhibitory signals through inhibitory motifs. In some embodiments, KIR-CARs of the present invention comprise inhibitory KIRs that deliver their signals through interaction with immunotyrosine-based inhibitory motifs (ITIMs). KIRs bearing cytoplasmic domains containing ITIMs neutralize activation signals that lead to the inhibition of NK cell lytic and cytokine production activity. However, the present invention should not be limited to inhibitory KIRs. Rather, any inhibitory protein having a cytoplasmic domain associated with an inhibitory signal can be used to construct the CARs of the present invention.

[0178] Thus, the present invention provides compositions comprising an NKR-CAR, e.g., a KIR-CAR, a vector comprising the same, a composition comprising an NKR-CAR packaged in a viral particle, e.g., a KIR-CAR vector, and recombinant T cells or other cytotoxic cells comprising an NKR-CAR, e.g., a KIR-CAR. The present invention also includes methods for producing genetically modified T cells or other cytotoxic cells, e.g., NK cells, or cultured NK cells, e.g., NK92 cells, that express an NKR-CAR, e.g., a KIR-CAR (KIR-CART), wherein the expressed NKR-CAR, e.g., KIR-CAR, comprises an antigen-recognition domain of a specific antibody bearing an intracellular signaling molecule from an NKR, e.g., a KIR. For example, in some embodiments, the intracellular signaling molecule includes, but is not limited to, KIR ITAM, KIR ITIM, etc.

[0179] Thus, the present invention provides compositions and methods for controlling the specificity and activity of T cells or other cytotoxic cells modified to express an NKR-CAR, e.g., a KIR-CAR. The present invention also provides cells comprising multiple types of NKR-CARs, e.g., KIR-CARs (e.g., an activating NKR-CAR, e.g., a KIR-CAR, and an inhibitory NKR-CAR, e.g., a KIR-CAR), where the multiple types of NKR-CARs, e.g., KIR-CARs, participate in signal transduction to control T cell activation. In this regard, it is advantageous to efficiently regulate and control NKR-CAR cytotoxic cells, e.g., KIR-CAR T cells, so as to kill tumor cells but not affect normal bystander cells. Thus, in certain embodiments, the present invention also provides methods for killing cancerous cells while minimizing depletion of normal, non-cancerous cells, thereby improving the specificity of NKR-CAR, e.g., KIR-CAR, therapy.

[0180] In some embodiments, the NKR-CAR, e.g., KIR-CAR, approach involves the physical separation of multiple types of CARs expressed in cells, where binding of the multiple types of NKR-CAR, e.g., KIR-CAR, to its target antigen is required for NKR-CAR cytotoxic cell, e.g., KIR-CAR T cell, activation. For example, in the KIR-CAR approach, each KIR-CAR from the multiple types of KIR-CARs has a different intracellular signaling domain. For example, when multiple types of KIR-CARs are used to induce KIR-CAR T cell activation, the first type of KIR-CAR may contain only the intracellular domain from an activating KIR, and the second type of CAR may contain only the intracellular domain from an inhibitory KIR. In this way, conditional activation of T cells is produced by binding of the activating KIR-CAR (actKIR-CAR) to an antigen on the malignant cell of interest. Inhibitory KIR-CARs (inhKIR-CARs), which carry antigen-binding domains directed against antigens present on normal cells but not on malignant cells, provide attenuation of the activating effect from actKIR-CARs when T cells encounter normal cells.

[0181] In some embodiments, the present invention provides a T cell or other cytotoxic cell engineered to express at least two NKR-CARs, e.g., at least two KIR-CARs, wherein the first NKR-CAR, e.g., KIR-CAR, is an actNKR-CAR, e.g., actKIR-CAR, and the second NKR-CAR, e.g., KIR-CAR, is an inhNKR-CAR, e.g., inhKIR-CAR. In some embodiments, the present invention provides an inhNKR-CAR, e.g., inhKIR-CAR, wherein binding of the inhNKR-CAR, e.g., inhKIR-CAR, to a normal cell results in inhibition of the cytotoxic cell, e.g., inhibition of KIR-CAR T cell activity. In some embodiments, binding of the inhNKR-CAR, e.g., inhKIR-CAR, to an antigen associated with a non-cancerous cell results in the death of the NKR-CAR cytotoxic cell, e.g., KIR-CAR T cell.

[0182] In some embodiments, the inhNKR-CARs of the present invention, such as actKIR-CARs, can be used in combination with existing CARs to regulate the activity of the CAR. Exemplary CARs are described in PCT / US11 / 64191, the entire contents of which are incorporated herein by reference.

[0183] It has also been found that in cells having multiple chimeric membrane-embedded receptors containing antigen-binding domains (CMERs), interactions between the antigen-binding domains of the CMERs may be undesirable, for example, because the interactions may inhibit the ability of one or more of the antigen-binding domains to bind to its cognate antigen or create novel binding sites with an unknown cognate antigen. Accordingly, disclosed herein are cells having first and second non-naturally occurring CMERs, wherein the antigen-binding domains exhibit minimal such interactions. Also disclosed herein are nucleic acids encoding such first and second non-naturally occurring CMERs, as well as methods of making and using such cells and nucleic acids. In some embodiments, the antigen-binding domain of one of the first and second non-naturally occurring CMERs comprises an scFv, and the other comprises a single VH domain, e.g., a camelid, shark, or lamprey single VH domain, or a single VH domain derived from a human or mouse sequence, or a non-antibody scaffold.

[0184] definition Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those disclosed herein can be used in the practice and / or testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terms will be used according to how they are defined, if any.

[0185] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting.

[0186] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object. By way of example, "an element" means one element or more than one element.

[0187] As used herein, "about," when referring to a measurable value such as an amount, duration, etc., is meant to encompass variations of ±20%, or ±10%, in some cases ±5%, in some cases ±1%, and in some cases ±0.1% from the specified value, where such variations are reasonable in the practice of the disclosed methods.

[0188] As used herein, the term "adapter molecule" refers to a polypeptide having a sequence that allows for the interaction of two or more molecules, which in some embodiments promotes the activation or inactivation of cytotoxic cells. For example, in the case of DAP12, this includes interaction with activating KIRs through a charged interaction within the transmembrane domain and with signaling molecules such as ZAP70 or Syk through a phosphorylated ITAM sequence within the cytoplasmic domain.

[0189] The term "antigen" or "Ag" refers to a molecule that elicits an immune response. This immune response may involve antibody production, activation of specific immunocompetent cells, or both. Those skilled in the art will understand that any macromolecule, including substantially an entire protein or peptide, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. Those skilled in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an "antigen," as that term is used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It will be readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, with these nucleotide sequences arranged in various combinations to encode a polypeptide that elicits the desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It will be readily apparent that antigens can be synthetically produced or derived from biological samples. Such biological samples may include, but are not limited to, tissue samples, tumor samples, and fluids containing cells or other biological components.

[0190] As used herein, the term "anti-tumor effect" refers to a biological effect that can be manifested by various means, including, but not limited to, a reduction in tumor volume, a reduction in tumor cell number, a reduction in the number of metastases, an increase in life expectancy, a reduction in tumor cell proliferation, a reduction in tumor cell survival, or an improvement in various physiological symptoms associated with a cancerous condition. An "anti-tumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells, and antibodies of the invention in preventing the development of tumors in the first place.

[0191] As used herein, the term "apheresis" refers to the art-recognized extracorporeal process in which a donor's or patient's blood is withdrawn from the donor or patient, passed through a device that separates selected components, and the remainder is returned to the donor's or patient's circulation, e.g., by transfusion. Thus, the term "apheresis sample," in the context of the present invention, refers to a sample obtained using apheresis.

[0192] The term "autoantigen" according to the present invention refers to any self-antigen that is recognized by the immune system as if it were foreign. Autoantigens include, but are not limited to, cellular proteins, phosphoproteins, cell surface proteins, cellular lipids, nucleic acids, and glycoproteins, including cell surface receptors.

[0193] As used herein, the term "autoimmune disease" is defined as a disorder caused by an autoimmune response. Autoimmune diseases are the result of an inappropriate and excessive response to self-antigens. Examples of autoimmune diseases include, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes mellitus (type 1), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, leukoplakia, myxedema, pernicious anemia, and ulcerative colitis, among others.

[0194] As used herein, the term "autologous" refers to any material derived from the same individual into which it is subsequently reintroduced.

[0195] As used herein, the term "allogeneic" refers to any material derived from a different animal of the same species as the individual into whom the material is being introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some aspects, allogeneic material derived from individuals of the same species may not be sufficiently genetically similar to interact antigenically. In some embodiments, allogeneic refers to a graft derived from a different animal of the same species.

[0196] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a chimeric polypeptide that shares structural and functional properties with a cellular immune function receptor or adapter molecule, e.g., from a T cell or NK cell. CARs include TCARs and NKR-CARs. In some embodiments, a CAR comprises an antigen-binding domain that binds to a cognate antigen, e.g., a tumor antigen described herein. By binding to the cognate antigen, the CAR can activate or inactivate cytotoxic cells to which it is placed, regulate the anti-tumor activity of the cell, or otherwise regulate the immune response of the cell.

[0197] In some embodiments, the domains in a CAR polypeptide construct are on the same polypeptide chain, e.g., comprising a chimeric fusion protein. In some embodiments, the domains in a CAR polypeptide construct are not adjacent to each other, e.g., are on different polypeptide chains, e.g., as provided in an RCAR as described herein.

[0198] As used herein, the term "natural killer cell immune function receptor-chimeric antigen receptor" or "NKR-CAR" refers to a CAR that shares functional and structural properties with a natural killer cell immune function receptor (NKR) or adapter molecule from an NK cell. In some embodiments, the NKR-CAR comprises two or all of an antigen binding domain, a transmembrane domain, e.g., an NKR transmembrane domain, and / or a cytoplasmic domain, e.g., an NKR cytoplasmic domain.

[0199] As used herein, the term "Fc receptor-chimeric antigen receptor" or "FcR-CAR" refers to a CAR that shares functional and structural properties with an Fc receptor (FcR). As used herein, the term "killer cell immunoglobulin-like receptor-chimeric antigen receptor" or "KIR-CAR" refers to a CAR that shares functional and structural properties with a killer cell immunoglobulin-like receptor (KIR).

[0200] As used herein, the term "Ly49 receptor-chimeric antigen receptor" or "Ly49-CAR" refers to a CAR that shares functional and structural properties with the Ly49 receptor (Ly49).

[0201] As used herein, the term "natural cytotoxic receptor-chimeric antigen receptor" or "NCR-CAR" refers to a CAR that shares functional and structural properties with a natural cytotoxic receptor (NCR).

[0202] As used herein, the term "signaling lymphocyte activation molecule family-chimeric antigen receptor" or "SLAM-CAR" or "SLAMF-CAR" refers to a CAR that shares functional and structural properties with SLAM or SLAMF.

[0203] As used herein, the term "T cell-based chimeric antigen receptor" or "TCAR" refers to a CAR that shares functional and structural properties with a cellular immune function receptor or adapter molecule from a T cell. In some embodiments, a TCAR comprises an antigen domain, a primary intracellular signaling domain, and optionally one or more costimulatory signaling domains.

[0204] As used herein, the term "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to a target antigen. An antibody can be a complete immunoglobulin from natural or recombinant sources, or an immunoreactive portion of a complete immunoglobulin. Antibodies can be polyclonal or monoclonal, multi-chain or single-chain, or a complete immunoglobulin, and can be derived from natural or recombinant sources. Antibodies are generally tetramers of immunoglobulin molecules. The antibody molecules described herein can exist in a variety of forms in which the antigen-binding portion of the antibody is expressed as part of a continuous polypeptide chain, including, for example, single-domain antibody fragments (sdAbs), single-chain antibodies (scFvs), and humanized or human antibodies, as described herein.

[0205] The term "antibody fragment" refers to at least a portion of an intact antibody or a recombinant variant thereof, including the antigen-determining variable regions of the intact antibody sufficient to confer recognition and specific binding of the antibody fragment to a target, such as an antigen. Examples of antibody fragments include single-chain domain antibodies (sdAbs), Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, scFv antibody fragments, linear antibodies, single domain antibodies such as sdAbs (either VL or VH), multispecific antibodies formed from antibody fragments such as camelid VHH domains and bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region, and isolated CDRs or other epitope-binding fragments of antibodies. Antigen-binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen-binding fragments can also be grafted onto scaffolds based on polypeptides such as fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199, which describes fibronectin polypeptide minibodies).

[0206] The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked by a short flexible polypeptide linker and can be expressed as a single polypeptide chain, and the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, an scFv as used herein may comprise the VL and VH variable regions in either order, e.g., with respect to the N- and C-termini of the polypeptide, an scFv may comprise VL-linker-VH or VH-linker-VL.

[0207] As used herein, the term "complementarity-determining region" or "CDR" refers to the sequence of amino acids within an antibody variable region that confers antigen specificity and binding affinity. For example, there are typically three CDRs (e.g., HCDR1, HCDR2, and HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, and LCDR3) in each light chain variable region. The exact amino acid sequence boundaries of a CDR can be determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme), or a combination thereof. Under the Kabat numbering scheme, in some embodiments, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under the Chothia numbering scheme, in some embodiments, the CDR amino acids in the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). In a combined Kabat and Chothia numbering scheme, in certain embodiments, the CDRs correspond to amino acid residues that are part of a Kabat CDR, a Chothia CDR, or both.For example, in one embodiment, the CDRs correspond to amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in a VH, e.g., a mammalian VH, e.g., a human VH; and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in a VL, e.g., a mammalian VL, e.g., a human VL.

[0208] The portion of the CAR composition of the invention comprising an antibody or antibody fragment thereof can exist in a variety of forms in which the antigen-binding domain is expressed as part of a contiguous polypeptide chain, including, for example, single-domain antibody fragments (sdAbs), single-chain antibodies (scFvs), and humanized or human antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In one aspect, the antigen-binding domain of the CAR composition of the invention comprises an antibody fragment. In a further aspect, the CAR comprises an antibody fragment comprising an scFv.

[0209] As used herein, the term "binding domain" or "antibody molecule" (also referred to herein as "anti-target (e.g., mesothelin) binding domain") refers to a protein, e.g., an immunoglobulin chain or fragment thereof, that comprises at least one immunoglobulin variable domain sequence. The term "binding domain" or "antibody molecule" encompasses antibodies and antibody fragments. In certain embodiments, an antibody molecule is a multispecific antibody molecule, e.g., comprises a plurality of immunoglobulin variable domain sequences, where a first immunoglobulin variable domain sequence in the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence in the plurality has binding specificity for a second epitope. In certain embodiments, a multispecific antibody molecule is a bispecific antibody molecule. Bispecific antibodies have specificity for no more than two antigens. Bispecific antibody molecules are characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope.

[0210] As used herein, "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.

[0211] As used herein, "antibody light chain" refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations. Kappa and lambda light chains refer to the two major antibody light chain isotypes.

[0212] As used herein, the term "synthetic antibody" or "recombinant antibody" refers to an antibody molecule produced using recombinant DNA technology, such as, for example, an antibody molecule expressed by a bacteriophage as described herein. The term should also be construed to refer to an antibody produced by synthesis of a DNA molecule encoding the antibody molecule, which DNA molecule expresses an antibody protein or amino acid sequence specifying the antibody, where the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology that is available and well known in the art.

[0213] As used herein, the term "cancer" refers to a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or via the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein and include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, glioma, and the like. The terms "tumor" and "carcinoma" are used interchangeably herein; e.g., both terms include solid and liquid, e.g., diffuse or circulating tumors. As used herein, the term "cancer" or "tumor" includes pre-malignant and malignant cancers and tumors.

[0214] The term "combination" refers to a fixed combination in a single dosage unit form or a combined administration in which a compound of the present invention and a combination partner (e.g., other agents, as described below, also referred to as "therapeutic agents" or "combination agents") can be administered independently, simultaneously, or at intervals that allow the combination partners to exhibit a coordinated, e.g., synergistic, effect. The single components can be packaged in a kit or separately. One or both of the components (e.g., powder or liquid) can be reconstituted or diluted to the desired dosage before administration. As used herein, the terms "co-administration" or "administration in combination," and the like, are intended to encompass the administration of selected combination partners to a subject (e.g., patient) in need thereof, and are intended to encompass treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time. As used herein, the term "pharmaceutical combination" refers to a product resulting from the mixing or combining of more than one active ingredient, and includes both fixed and non-fixed combinations of the active ingredients. The term "fixed combination" means that the active ingredients, e.g., a compound of the present invention and a combination partner, are both administered to a patient simultaneously as a single entity or dosage. The term "unfixed combination" means that the active ingredients, e.g., a compound of the present invention and a combination partner, are administered to a patient as separate entities simultaneously, together or separately, with no specific time limits, wherein such administration provides therapeutically effective levels of the two compounds in the patient's body. The latter also applies to cocktail therapy, e.g., the administration of three or more active ingredients.

[0215] The term "conservative sequence modifications" refers to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody or antibody fragment containing that amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies or antibody fragments of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in a CAR of the invention can be replaced with other amino acid residues from the same side chain family, and the ability of the modified CAR to bind to FRβ can be tested using the functional assays described herein.

[0216] A "constitutive" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes production of the gene product in a cell under most or all physiological conditions of the cell.

[0217] Cytoplasmic and intracellular, as applied to adapter molecules and signaling domains, are used interchangeably herein.

[0218] As used herein, the term "derived from" refers to the relationship between a first molecule and a second molecule. It generally refers to the structural similarity between the first molecule and the second molecule and does not imply or include limitations on the process or source from which the first molecule is derived from the second molecule. For example, in the case of an intracellular signaling domain derived from a CD3 zeta molecule, the intracellular signaling domain retains sufficient CD3 zeta structure so that it has the required function, i.e., the ability to produce a signal under appropriate conditions. It does not imply or include limitations on the particular process by which the intracellular signaling domain is produced, e.g., it does not mean that one must start from the CD3 zeta sequence and delete or mutate unwanted sequences to arrive at the intracellular signaling domain in order to provide the intracellular signaling domain.

[0219] The term "stimulation" refers to a primary response elicited by the binding of a stimulatory molecule (e.g., the TCR / CD3 complex) to its cognate ligand, thereby mediating a signal transduction event, such as, but not limited to, signal transduction by the TCR / CD3 complex. Stimulation may also mediate altered expression of certain molecules, such as downregulation of TGF-β and / or cytoskeletal reorganization.

[0220] The term "stimulatory molecule" refers to a molecule expressed by a T cell that provides the primary cytoplasmic signaling sequences that regulate the primary activation of the TCR complex in a stimulatory direction for at least some aspect of the T cell signaling pathway. In one aspect, the primary signal is initiated, for example, by binding of the TCR / CD3 complex to a peptide-loaded MHC molecule, which culminates in mediating a T cell response, including, but not limited to, proliferation, activation, differentiation, etc. Primary cytoplasmic signaling sequences (also referred to as "primary signaling domains") that act in a stimulatory direction can contain signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs. Examples of ITAM-containing primary cytoplasmic signaling sequences that are particularly useful in the present invention include, but are not limited to, those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FcεRI and CD66d, DAP10, and DAP12. In certain CARs of the invention, the intracellular signaling domain in any one or more CARs of the invention comprises an intracellular signaling sequence, e.g., the primary signaling sequence of CD3 zeta. In certain CARs of the invention, the primary signaling sequence of CD3 zeta is the sequence provided as SEQ ID NO: 9 or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc. In certain CARs of the invention, the primary signaling sequence of CD3 zeta is the sequence provided as SEQ ID NO: 10 or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc.

[0221] The term "antigen-presenting cell" or "APC" refers to a cell of the immune system, such as an accessory cell (e.g., a B cell, a dendritic cell, etc.), that displays foreign antigens complexed with major histocompatibility complexes (MHC) on its surface. T cells can recognize these complexes using their T cell receptors (TCRs). APCs process antigens and present them to T cells.

[0222] As used herein, the term "intracellular signaling domain" refers to the intracellular portion of a molecule. The intracellular signaling domain can generate a signal that promotes immune effector function of a CAR-containing cell, e.g., a CART cell or a CAR-expressing NK cell. For example, examples of immune effector function in a CART cell or a CAR-expressing NK cell include cytolytic activity and helper activity, including cytokine secretion. In some embodiments, the intracellular signaling domain transmits an effector function signal, directing the cell to perform a specialized function. While the entire intracellular signaling domain can be used, it is often not necessary to use the entire chain. To the extent that a truncated portion of an intracellular signaling domain is used, such a truncated portion may be used in place of the complete chain, so long as it transmits the effector function signal. The term intracellular signaling domain, therefore, is intended to include any truncated portion of the intracellular signaling domain sufficient to transmit the effector function signal.

[0223] In some embodiments, the intracellular signaling domain can comprise a primary intracellular signaling domain. Examples of primary intracellular signaling domains include those derived from molecules responsible for primary stimulation or antigen-dependent stimulation. In some embodiments, the intracellular signaling domain can comprise a costimulatory intracellular domain. Examples of costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals or antigen-independent stimulation. For example, in the case of CAR-expressing immune effector cells, such as CART cells or CAR-expressing NK cells, the primary intracellular signaling domain can comprise the cytoplasmic sequence of a T cell receptor, and the costimulatory intracellular signaling domain can comprise the cytoplasmic sequence of a co-receptor or costimulatory molecule.

[0224] The primary intracellular signaling domain can contain a signaling motif known as an immunoreceptor tyrosine-based activation motif, or ITAM. Examples of ITAM-containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 ("ICOS"), FcεRI, CD66d, DAP10, and DAP12.

[0225]

[0033] The term "zeta" or alternatively "zeta chain," "CD3 zeta," or "TCR zeta" is defined as the protein provided as GenBank Accession No. BAG36664.1, or the equivalent bases from a non-human species, e.g., mouse, rodent, monkey, ape, etc., and a "zeta stimulatory domain" or alternatively "CD3 zeta stimulatory domain" or "TCR zeta stimulatory domain" is defined as the amino acid residues from the cytoplasmic domain of the zeta chain that are sufficient to functionally transmit an early signal necessary for T cell activation. In one aspect, the cytoplasmic domain of zeta comprises residues 52-164 of GenBank Accession No. BAG36664.1, or the equivalent residues from a non-human species that is a functional ortholog thereof, e.g., mouse, rodent, monkey, ape, etc. In one aspect, a "zeta stimulatory domain" or "CD3 zeta stimulatory domain" is defined as SEQ ID NO:9. In one aspect, the "zeta stimulatory domain" or "CD3 zeta stimulatory domain" is the sequence provided as SEQ ID NO: 10.

[0226] The term "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are necessary for an efficient immune response. Costimulatory molecules include MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), B7-H, and other T cell receptors. 3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, C D49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA- 1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, C Ligands that specifically bind to RTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and CD83 are included, but are not limited to these.

[0227] A costimulatory intracellular signaling domain refers to the intracellular portion of a costimulatory molecule. The intracellular signaling domain can include the entire intracellular portion of the molecule from which it is derived, or the entire native intracellular signaling domain, or a functional fragment thereof.

[0228] The term "4-1BB" refers to a member of the TNFR superfamily having the amino acid sequence provided as GenBank Accession No. AAA62478.2, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc.; and a "4-1BB costimulatory domain" is defined as amino acid residues 214-255 of GenBank Accession No. AAA62478.2, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc. In one aspect, the "4-1BB costimulatory domain" is the sequence provided as SEQ ID NO:7, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc.

[0229] As used herein, the term "immune effector cell" refers to a cell that is involved in an immune response, e.g., enhancing an immune effector response. Examples of immune effector cells include T cells, e.g., alpha / beta T cells and gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived phagocytes.

[0230] As used herein, the term "immune effector function or immune effector response" refers to a function or response, e.g., of an immune effector cell, that enhances or promotes immune attack of a target cell. For example, an immune effector function or response refers to a property of a T cell or NK cell that promotes killing or inhibits growth or proliferation of a target cell. In the case of T cells, primary stimulation and costimulation are examples of immune effector functions or responses.

[0231] The term "effector function" refers to a specialized function of a cell. The effector function of a T cell can be, for example, cytolytic activity or helper activity, including secretion of cytokines.

[0232] The term "encoding" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand and the non-coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, used as a template for transcription of the gene or cDNA, can be referred to as encoding a protein or other product of the gene or cDNA.

[0233] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNAs can contain introns.

[0234] The terms "effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, substance, or composition as described herein effective to achieve a particular biological result, including, but not limited to, inhibition of viral infection, as determined by any means suitable in the art.

[0235] As used herein, the term "endogenous" refers to any substance that is native to or produced within an organism, cell, tissue, or system.

[0236] As used herein, the term "exogenous" refers to any substance introduced from or produced outside an organism, cell, tissue, or system.

[0237] As used herein, the term "expression" is defined as the transcription and / or translation of a particular nucleotide sequence driven by a promoter.

[0238] The term "expression" refers to the transcription and / or translation of a particular nucleotide sequence driven by a promoter.

[0239] The term "transfer vector" refers to a composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides bound to ionic or amphipathic compounds, plasmids, and viruses. Thus, the term "transfer vector" includes autonomously replicating plasmids or viruses. The term should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid into a cell, such as, for example, polylysine compounds, liposomes, etc. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, etc.

[0240] An "expression vector" refers to a vector containing a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by a host cell or in an in vitro expression system. Expression vectors include all such vectors known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0241] As used herein, the term "vector" refers to any vehicle that can be used to deliver and / or express a nucleic acid molecule. It may be a transfer vector or an expression vector, as described herein.

[0242] The term "homologous" or "identity" refers to the subunit sequence identity between two polymer molecules, e.g., between two nucleic acid molecules, e.g., between two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit in both of the two molecules is occupied by the same monomer subunit; for example, if a position in each of the two DNA molecules is occupied by adenine, they are homologous or identical at that position. The homology between two sequences is a direct function of the number of matched or homologous positions; for example, if half of the positions in two sequences (e.g., 5 positions in a 10-subunit long polymer) are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 out of 10) are matched or homologous, the two sequences are 90% homologous.

[0243] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further tailor and optimize antibody performance. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions correspond to those of a human immunoglobulin sequence. The humanized antibody may also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332: 323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

[0244] "Fully human" refers to an immunoglobulin, such as an antibody or antibody fragment, where the entire molecule is of human origin or consists of an amino acid sequence identical to the human form of the antibody or immunoglobulin.

[0245] As used herein, "instructions" refers to publications, records, diagrams, or any other medium of expression that can be used to describe the utility of the compositions and methods of the invention. Instructions for kits of the invention may, for example, be affixed to a container containing the nucleic acids, peptides, and / or compositions of the invention or shipped with a container containing the nucleic acids, peptides, and / or compositions. Alternatively, the instructions may be shipped separately from the container, with instructions for the instructions and the compound to be used by the recipient in a complementary manner.

[0246] The term "isolated" means altered or removed from its natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in substantially purified form, or it can exist in a non-native environment, such as, for example, a host cell.

[0247] In the context of the present invention, the following abbreviations are used for commonly occurring nucleobases: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0248] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The term nucleotide sequence encoding a protein or RNA can also include introns if the nucleotide sequence encoding the protein, in some version, contains introns.

[0249] As used herein, "lentivirus" refers to a genus in the Retroviridae family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells; they can deliver significant amounts of genetic information into the DNA of host cells, making them one of the most efficient gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses. Lentivirus-derived vectors provide a means to achieve significant levels of gene transfer in vivo.

[0250] The term "lentiviral vector" refers to a vector derived at least in part from a lentiviral genome, and specifically includes self-inactivating lentiviral vectors such as those provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentiviral vectors that may be used clinically include LENTIVECTOR® Gene Delivery Technology from Oxford BioMedica and LENTIMAX® from Lentigen. TM Non-clinical types of lentiviral vectors are also available and known to those skilled in the art, including, but not limited to:

[0251] As used herein, the term "flexible polypeptide linker" or "linker" in the context of scFvs refers to a peptide linker composed of amino acids such as glycine and / or serine residues used alone or in combination to link the variable heavy and variable light chain regions together. In one embodiment, the flexible polypeptide linker is a Gly / Ser linker, having the amino acid sequence (Gly-Gly-Gly-Ser). n(SEQ ID NO: 38) (wherein n is a positive integer greater than or equal to 1). For example, n=1, n=2, n=3, n=4, n=5, and n=6, n=7, n=8, n=9, and n=10. In some embodiments, the flexible polypeptide linker includes, but is not limited to, (Gly4Ser)4 (SEQ ID NO: 27) or (Gly4Ser)3 (SEQ ID NO: 28). In other embodiments, the linker includes multiple repeats of (Gly2Ser), (GlySer), or (Gly3Ser) (SEQ ID NO: 29). Linkers described in WO2012 / 138475 (incorporated herein by reference) are also within the scope of the present invention.

[0252] As used herein, "NK cell immune function receptor" or "NKR" refers to an endogenous, naturally occurring transmembrane protein expressed in NK cells that binds to a ligand on an antigen-presenting cell and can modulate NK cell immune function responses, e.g., regulating NK cell cytolytic activity or cytokine secretion. NKRs contribute to activation (activating NKR, or actNKR) or inhibition (inhibitory NKR, or inhNKR). Generally, NKRs contain an extracellular ligand-binding domain (ECD), a transmembrane domain (TM), and an intracellular cytoplasmic domain (ICD). NKRs include the killer immunoglobulin-like receptor (KIR) family of receptors, such as KIR2DS2; the NK cell receptor (NCR) family of receptors, such as NKp46 (NCR1); the signaling lymphocyte-activating receptor (SLAM) family of receptors, such as 2B4; and Fc-binding receptors, such as CD16 (FcγRIII). Examples of NK cell immune function responses regulated by NKR are target cell killing (often also referred to as cytotoxicity or cytolysis), cytokine secretion and / or proliferation.Generally, the NKR suitable for use in the methods and compositions described herein is human NKR (or hNKR).In some embodiments, the Ly49 receptor family in mice is also included, which appears by convergent evolution to provide the same function as KIR in mouse NK and T cells.

[0253] The term "operably linked" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence that results in expression of the latter. For example, a first nucleic acid sequence is operably linked when the first nucleic acid sequence is placed into a functional relationship with a second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

[0254] The term "parenteral" administration of an immunogenic composition includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im) or intrasternal injection or infusion techniques.

[0255] As used herein, the terms "nucleic acid," "nucleic acid molecule," or "polynucleotide" are used interchangeably and define a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Therefore, as used herein, nucleic acid and polynucleotide are interchangeable. Those skilled in the art have the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into monomeric "nucleotides." Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotides can be synthesized using conventional cloning technologies and PCR. TMThe term "nucleic acid" refers to any nucleic acid sequence obtained by any means available in the art, including, but not limited to, recombinant means, i.e., cloning a nucleic acid sequence from a recombinant library or a cellular genome, and synthetic means, using techniques such as those described herein. In certain embodiments, nucleic acid herein includes deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or combinations of DNA or RNA, and combinations of such polymers in either single- or double-stranded form. The term "nucleic acid" includes genes, cDNAs, or mRNAs. In certain embodiments, nucleic acid molecules are synthetic (e.g., chemically synthesized) or recombinant. Unless otherwise specified, the term encompasses nucleic acids containing analogs or derivatives of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, a particular nucleic acid sequence also encompasses implicitly conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0256] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no set upper limit to the number of amino acids that may comprise a protein or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains, commonly referred to as peptides, oligopeptides, and oligomers, and longer chains, commonly referred to in the art as proteins, of which there are many types. "Polypeptide" includes, inter alia, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins. A polypeptide includes natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0257] The term "promoter" as used herein is defined as a DNA sequence recognized by the synthetic machinery of a cell or introduced synthetic machinery necessary for initiating the specific transcription of a polynucleotide sequence.

[0258] As used herein, the term "promoter / regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product operably linked to the promoter / regulatory sequence. In some instances, this sequence may be the core promoter sequence, and in other instances, this sequence may also include enhancer sequences and other regulatory elements required for expression of the gene product. The promoter / regulatory sequence may, for example, be one that expresses the gene product in a tissue-specific manner.

[0259] The term "inducible" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell substantially only when an inducer corresponding to the promoter is present in that cell.

[0260] The term "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoded or specified by a gene, causes a gene product to be produced in a cell substantially only if that cell is a cell of that tissue type.

[0261] The term "signal transduction pathway" refers to the biochemical relationships between various signaling molecules that are responsible for transmitting a signal from one part of a cell to another part of the cell. The term "cell surface receptor" includes molecules and molecular complexes that can receive a signal and transmit the signal across the cell membrane.

[0262] The term "subject" is intended to include living organisms in which an immune response can be elicited (eg, mammals, humans).

[0263] As used herein, "substantially purified" cells refer to cells that are essentially free of other cell types. Substantially purified cells also refer to cells that have been separated from other cell types with which they are normally associated in their naturally occurring state. In some instances, a population of substantially purified cells refers to a homogenous population of cells. In other instances, the term simply refers to cells that have been separated from the cells with which they are naturally associated in their natural state. In some aspects, the cells are cultured in vitro. In other aspects, the cells are not cultured in vitro.

[0264] The 5' cap used here (also known as RNA cap, RNA 7-methylguanosine cap or RNA m 7The 5' cap (G-cap) is a modified guanine nucleotide added to the "front," or 5' end, of eukaryotic messenger RNA immediately after transcription initiation. The 5' cap consists of a terminal group attached to the first transcribed nucleotide. Its presence is important for ribosomal recognition and protection from RNases. Capping is coupled to transcription and occurs co-transcriptionally, interacting with transcription. Shortly after transcription initiation, the 5' end of the synthesizing mRNA is attached by a cap-synthesizing complex associated with RNA polymerase. This enzyme complex catalyzes the chemical reactions required for mRNA capping. Synthesis proceeds as a multi-step biochemical reaction. The capping moiety can be modified to modulate mRNA functionality, such as stability or translation efficiency.

[0265] As used herein, "in vitro transcribed RNA" refers to RNA, preferably mRNA, that has been synthesized in vitro. Generally, in vitro transcribed RNA is produced from an in vitro transcription vector. The in vitro transcription vector contains a template used to produce the in vitro transcribed RNA.

[0266] As used herein, "poly(A)" refers to a series of adenosines attached to mRNA by polyadenylation. In preferred embodiments of constructs for transient expression, the poly(A) is 50-5000 (SEQ ID NO: 30), preferably greater than 64, more preferably greater than 100, and most preferably greater than 300 or 400. The poly(A) sequence can be chemically or enzymatically modified to modulate mRNA functionality such as localization, stability, or translation efficiency.

[0267] As used herein, "polyadenylation" refers to the covalent attachment of a polyadenylyl moiety or its modified variants to a messenger RNA molecule. In eukaryotes, most messenger RNA (mRNA) molecules are polyadenylated at their 3' ends. The 3' poly(A) tail is a long sequence (often several hundred) of adenine nucleotides added to pre-mRNA by the action of the enzyme polyadenylate polymerase. In higher eukaryotes, the poly(A) tail is added to transcripts that contain a specific sequence, the polyadenylation signal. The poly(A) tail and the proteins associated with it help protect the mRNA from exonucleolytic degradation. Polyadenylation is also important for transcription termination, nuclear export of mRNA, and translation. Polyadenylation can occur not only immediately after transcription of DNA into RNA in the nucleus, but also later in the cytoplasm. After transcription is terminated, the mRNA strand is cleaved by the action of an endonuclease complex associated with RNA polymerase. The cleavage site is usually characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA is cleaved, an adenosine residue is added to the free 3' end of the cleavage site.

[0268] As used herein, "transient" refers to expression of a non-integrated transgene for a period of hours, days, or weeks, where the period of expression is shorter than the period of expression of the gene when integrated into the genome or contained within a stable plasmid replicon in a host cell.

[0269] As used herein, the term "therapy" refers to treatment and / or prophylaxis. A therapeutic effect is achieved by suppressing, ameliorating, or eradicating the disease state.

[0270] The terms "transfection" or "transformation" or "transduction" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transgenic" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0271] As used herein, the terms "treatment" and "treating" refer to a reduction or amelioration of the progression, severity, and / or duration of a proliferative disorder or an amelioration of one or more symptoms (preferably one or more discernible symptoms) of a proliferative disorder resulting from the administration of one or more therapeutic agents (e.g., one or more therapeutic agents such as a CAR of the invention). In certain embodiments, the terms "treatment" and "treating" refer to an improvement in at least one measurable physical parameter of a proliferative disorder, such as tumor growth, which is not necessarily discernible by the patient. In other embodiments, the terms "treatment" and "treating" refer to an inhibition of progression of a proliferative disorder, either physical, e.g., stabilization of discernible symptoms, physiological, e.g., stabilization of physical parameters, or both. In other embodiments, the terms "treatment" and "treating" refer to a reduction or stabilization of tumor size or cancerous cell number.

[0272] As used herein, the term "prevention" refers to preventative or prophylactic treatment against a disease or disease state.

[0273] As used herein, the term "tumor antigen" refers to a molecule (generally a protein, carbohydrate, or lipid) that is expressed on the surface of cancer or tumor cells, either entirely or as fragments (e.g., MHC / peptides), and that is useful for preferential targeting of agents to cancer or tumor cells. In some embodiments, a tumor antigen is a marker expressed by both normal and cancer cells, e.g., a lineage marker, e.g., CD19 or CD123 on B cells. In some embodiments, a tumor antigen is a cell surface molecule that is overexpressed on cancer cells compared to normal cells, e.g., 1-fold overexpression, 2-fold overexpression, 3-fold overexpression, or more overexpression compared to normal cells. In some embodiments, a tumor antigen is a cell surface molecule that is inappropriately synthesized on cancer cells compared to molecules expressed on normal cells, e.g., a molecule containing deletions, additions, or mutations. In some embodiments, a tumor antigen is expressed exclusively on the surface of cancer cells, whether entirely or as fragments (e.g., MHC / peptides), and is not synthesized or expressed on the surface of normal cells.

[0274] In some embodiments, the CARs of the present invention include CARs that contain an antigen-binding domain (e.g., an antibody or antibody fragment) that binds to an MHC-presented peptide. Typically, peptides derived from endogenous proteins fill the pocket of a major histocompatibility complex (MHC) class I molecule and bind to CD8 + They are recognized by the T cell receptor (TCR) of T lymphocytes. MHC class I complexes are constitutively expressed by all nucleated cells. In cancer, virus-specific and / or tumor-specific peptide / MHC complexes represent a unique class of cell surface targets for immunotherapy. TCR-like antibodies targeting peptides derived from viral or tumor antigens in the context of human leukocyte antigen (HLA)-A1 or HLA-A2 have been described (see, e.g., Sastry et al., J Virol. 2011 85(5):1935-1942; Sergeeva et al., Blood, 2011 117(16):4262-4272; Verma et al., J Immunol 2010 184(4):2156-2165; Willemsen et al., Gene Ther 2001 8(21):1601-1608; Dao et al., Sci Transl Med 2013 5(176):176ra33; Tassev et al., Cancer Gene Ther 2012 19(2):84-100). For example, TCR-like antibodies can be identified by screening libraries such as human scFv phage display libraries.

[0275] As used herein, the term "under transcriptional control" or "operably linked" means that the promoter is in the correct position and orientation relative to the polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.

[0276] A "vector" is a composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into a cell. Numerous vectors are known, including, but not limited to, linear polynucleotides, polynucleotides bound to ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, etc.

[0277] As used herein, the term "specifically binds" means that an antibody or ligand recognizes and binds to its cognate binding partner protein present in a sample, but the antibody or ligand does not substantially recognize or bind to other molecules in the sample.

[0278] The term "stimulation" refers to a primary response elicited by the binding of a stimulatory molecule to its cognate ligand, thereby mediating a signal transduction event, such as, but not limited to, signal transduction through an appropriate NK receptor.

[0279] The term "xenogeneic" refers to a graft derived from an animal of a different species.

[0280] The term "specifically binds" refers to an antibody or ligand that recognizes and binds to a cognate binding partner protein present in a sample (e.g., a stimulatory and / or costimulatory molecule present on a T cell), but does not substantially recognize or bind to other molecules in the sample.

[0281] As used herein, the term "regulatable chimeric antigen receptor (RCAR)" refers to a set of polypeptides, generally two polypeptides in the simplest embodiment, that, when present in an immune effector cell, confers specificity for a target cell, generally a cancer cell, and controllable intracellular signal production to the cell. In some embodiments, an RCAR, as defined in the context of a CAR molecule, comprises at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") comprising a functional signaling domain derived from a stimulatory molecule and / or a costimulatory molecule. In some embodiments, the set of polypeptides in an RCAR are not contiguous with each other, e.g., in different polypeptide chains. In some embodiments, an RCAR comprises a dimerization switch that, in the presence of a dimerization molecule, can link the polypeptides to each other, e.g., link the antigen-binding domain to the intracellular signaling domain. In some embodiments, an RCAR is expressed in a cell (e.g., an immune effector cell), as described herein, e.g., an RCAR-expressing cell (also referred to herein as an "RCARX cell"). In some embodiments, the RCARX cell is a T cell and is referred to as an RCART cell. In some embodiments, the RCARX cells are NK cells and are referred to as RCARN cells. The RCAR provides the RCAR-expressing cells with specificity for target cells, generally cancer cells, and controllable intracellular signal production or proliferation that can optimize the immune effector properties of the RCAR-expressing cells. In some embodiments, the RCAR cells rely, at least in part, on the antigen-binding domain to provide specificity for target cells that contain the antigen bound by the antigen-binding domain.

[0282] As used herein, the term "membrane anchor" or "membrane tethering domain" refers to a polypeptide or moiety, e.g., a myristoyl group, sufficient to anchor an extracellular or intracellular domain to the plasma membrane.

[0283] As used herein, a "switch domain," e.g., when referring to an RCAR, refers to one that binds to another switch domain, typically a polypeptide-based one, in the presence of a dimerization molecule. Binding results in functional coupling of the first domain bound to, e.g., fused with, a first switch domain and the second domain bound to, e.g., fused with, a second switch domain. The first and second switch domains are collectively referred to as a dimerization switch. In some embodiments, the first and second switch domains are identical to one another, e.g., polypeptides having the same primary amino acid sequence, and are collectively referred to as a homodimerization switch. In some embodiments, the first and second switch domains are different from one another, e.g., polypeptides having different primary amino acid sequences, and are collectively referred to as a heterodimerization switch. In some embodiments, the switch is intracellular. In some embodiments, the switch is extracellular. In some embodiments, the switch domain is polypeptide-based, e.g., FKBP- or FRB-based, and the dimerization molecule is a small molecule, e.g., a rapalog. In some embodiments, the switch domain is polypeptide-based, e.g., an scFv that binds a myc peptide, and the dimerization molecule is a polypeptide, fragment thereof, or multimer of polypeptides, e.g., a myc ligand or multimer of myc ligands, that binds one or more myc scFvs. In some embodiments, the switch domain is polypeptide-based, e.g., a myc receptor, and the dimerization molecule is an antibody or fragment thereof, e.g., a myc antibody.

[0284] As used herein, the term "dimerization molecule," e.g., when referring to RCAR, refers to a molecule that promotes association of a first switch domain and a second switch domain. In some embodiments, the dimerization molecule does not naturally occur in a subject or does not occur at concentrations that result in significant dimerization. In some embodiments, the dimerization molecule is a small molecule, e.g., rapamycin or a rapalog, e.g., RAD001.

[0285] The term "bioequivalent" refers to the amount of an agent other than a reference compound (e.g., RAD001) required to produce an effect equivalent to that produced by a reference dose or amount of the reference compound (e.g., RAD001). In some embodiments, the effect is the level of mTOR inhibition, e.g., as assessed in an in vivo or in vitro assay, e.g., as measured by P70 S6 kinase inhibition, e.g., as measured in an assay described herein, e.g., the Boulay assay, or measurement of phosphorylated S6 levels by Western blot. In some embodiments, the effect is an alteration in the ratio of PD-1 positive / PD-1 negative immune effector cells, e.g., T cells or NK cells, as measured by cell sorting. In some embodiments, a bioequivalent amount or dose of an mTOR inhibitor is the amount or dose that achieves the same level of P70 S6 kinase inhibition as a reference dose or amount of a reference compound. In some embodiments, a bioequivalent amount or dose of an mTOR inhibitor is an amount or dose that achieves the same level of PD-1 positive / PD-1 negative immune effector cell, e.g., T cell or NK cell, ratio as a reference dose or reference amount of a reference compound.

[0286] The term "low immune-enhancing dose" refers to a dose of an mTOR inhibitor that, when used in combination with an mTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g., RAD001 or rapamycin, or a catalytic mTOR inhibitor, partially, but not completely, inhibits mTOR activity, e.g., as measured by inhibition of P70 S6 kinase activity. Methods for assessing mTOR activity, e.g., by inhibition of P70 S6 kinase, are described herein. This dose is insufficient to produce complete immune suppression, but is sufficient to enhance the immune response. In some embodiments, the low immune-enhancing dose of an mTOR inhibitor results in a decreased number of PD-1-positive immune effector cells, e.g., T cells or NK cells, and / or an increased number of PD-1-negative immune effector cells, e.g., T cells or NK cells, or an increased ratio of PD-1-negative T cells to PD-1-positive immune effector cells, e.g., T cells or NK cells.

[0287] In some embodiments, a low immune enhancing dose of an mTOR inhibitor results in an increase in the number of naive immune effector cells, e.g., T cells or NK cells. In some embodiments, a low immune enhancing dose of an mTOR inhibitor results in one or more of the following: Increased expression of one or more of the following markers: e.g., CD62L on memory T cells, e.g., memory T cell precursors 高 , CD127 高 , CD27 + and BCL2; Decreased expression of KLRG1, e.g., in memory T cells, e.g., memory T cell precursors; and Increased numbers of memory T cell precursors, e.g., cells, with any one or combination of the following characteristics: increased CD62L 高 , increased CD127 高 , increased CD27 + , decreased KLRG1 and increased BCL2; Here, any of the above changes occurs, for example, at least transiently, compared to, for example, an untreated subject.

[0288] As used herein, "refractory" refers to a disease, e.g., cancer, that does not respond to treatment. In some embodiments, a refractory cancer may be resistant to treatment before or at the start of treatment. In other embodiments, a refractory cancer may become resistant during treatment. A refractory cancer may also be referred to as a resistant cancer.

[0289] As used herein, "recurrent" or "relapse" refers to a reversal or reappearance of signs and symptoms of a disease (e.g., cancer) or cancer-like disease after a period of improvement or response, e.g., after treatment with a previous therapy, e.g., a cancer therapy. The initial period of response includes a decrease in the level of cancer cells below a certain threshold, e.g., below 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1%. Reappearance can include an increase in the level of cancer cells above a certain threshold, e.g., above 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1%. For example, in the context of B-ALL, reappearance can include the reappearance of blast cells in either the blood, bone marrow (>5%), or extramedullary sites, e.g., after a complete response. A complete response, in this context, can include <5% BM blasts. More generally, in some embodiments, a response (e.g., complete response or partial response) can include the absence of detectable MRD (minimal residual disease). In some embodiments, the initial period of response lasts for at least 1, 2, 3, 4, 5, or 6 days; at least 1, 2, 3, or 4 weeks; at least 1, 2, 3, 4, 6, 8, 10, or 12 months; or at least 1, 2, 3, 4, or 5 years.

[0290] In some embodiments, a therapy involving a CD19 inhibitor, e.g., CD19 CAR therapy, may result in relapse or refractory disease to treatment. Relapse or refractory disease may be caused by CD19 loss (e.g., antigen-loss mutation) or other CD19 alterations that reduce CD19 levels (e.g., by clonal selection of CD19-negative clones). Cancers carrying such CD19 loss or alterations are referred to herein as "CD19-negative cancers" or "CD19-negative recurrent cancers." It should be understood that CD19-negative cancers do not require 100% loss of CD19, but may be sufficiently reduced to reduce the effectiveness of CD19 therapy so that the cancer relapses or becomes refractory. In some embodiments, CD19-negative cancers result from CD19 CAR therapy.

[0291] Ranges: Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as a definitive limitation on the scope of the invention. Thus, the description of a range should be considered to include all specifically disclosed subranges as well as individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be construed to include specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity includes something with 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98%, and 98-99% identity. This applies regardless of the breadth of the scope.

[0292] NKR-CARS Disclosed herein are compositions and methods for controlling the specificity and activity of cytotoxic cells, such as T cells or NK cells, having, for example, non-naturally occurring chimeric antigen receptors (CARs). In some embodiments, the CAR is an NKR-CAR. An NKR-CAR is a CAR that shares functional and structural properties with NK cell immune function receptors (or NKRs). NKRs and NKR-CARs are described herein, for example, below. As described below, various NKRs can serve as the basis for an NKR-CAR.

[0293] NK cell immune function receptor (NKRS) and NK cells As used herein, NK cell immune function receptor (or NKR) refers to an endogenous, naturally occurring transmembrane protein expressed on NK cells that binds to a ligand on an antigen-presenting cell and can modulate an NK cell immune function response, e.g., modulate the cytolytic activity or cytokine secretion of an NK cell.

[0294] NK cells are mononuclear cells that arise in the bone marrow from lymphoid precursor cells. Their morphological and biological characteristics generally include expression of clusters of differentiation (CDs) CD16, CD56, and / or CD57; the absence of alpha / beta or gamma / delta TCR complexes on the cell surface; the ability to bind to and kill target cells that fail to express "self" major histocompatibility complex (MHC) / human leukocyte antigen (HLA) proteins; and the ability to kill tumor cells or other diseased cells that express ligands for activating NK receptors. NK cells are characterized by their ability to bind to and kill several types of tumor cell lines without the need for prior immunization or activation. NK cells can also release soluble proteins and cytokines that exert regulatory effects on the immune system; and can undergo multiple cell divisions to produce daughter cells with biological properties similar to those of the parent cell. Upon activation by interferon and / or cytokines, NK cells mediate the lysis of tumor cells and cells infected with intracellular pathogens by a mechanism that requires direct physical contact between the NK cell and the target cell. Lysis of target cells involves the release of cytotoxic granules from NK cells to the target surface and effector proteins such as perforin and granzyme B, which penetrate the target plasma membrane and induce apoptosis, or programmed cell death. Normal, healthy cells are protected from lysis by NK cells. NK cell activity is regulated by complex mechanisms involving both stimulatory and inhibitory signals.

[0295] In short, the lytic activity of NK cells is controlled by various cell surface receptors that transmit positive or negative intracellular signals upon interaction with ligands on target cells. The balance of positive and negative signals transmitted through these receptors determines whether a target cell is lysed (killed) by NK cells. NK cell stimulatory signals are mediated by natural cytotoxicity receptors (NCRs) such as NKp30, NKp44, and NKp46; as well as NKG2C receptors, NKG2D receptors, certain activating killer cell immunoglobulin-like receptors (KIRs), and other activating NK receptors (Lanier, Annual Review of Immunology 2005; 23:225-74). NK cell inhibitory signals can be mediated by receptors such as Ly49, CD94 / NKG2A, and certain inhibitory KIRs that recognize major histocompatibility complex (MHC) class I molecules (Karre et al., Nature 1986; 319:675-8; Ohlen et al, Science 1989; 246:666-8). These inhibitory receptors bind to polymorphic determinants of MHC class I molecules (including HLA class I) present on other cells and block NK cell-mediated lysis.

[0296] KIR-CARS Disclosed herein are chimeric antigen receptor (CAR) molecules (KIR-CARs) comprising an antigen-binding domain and a killer cell immunoglobulin-like receptor domain. In some embodiments, the KIR-CARs of the present invention are expressed on the surface of immune effector cells, such as T cells or NK cells.

[0297] NKCARS based on KIR-CAR Killer cell immunoglobulin-like receptors, or KIRs, have been characterized in humans and non-human primates, and polymorphic type 1 transmembrane molecules are present on certain subsets of lymphocytes, including NK cells and some T cells. KIRs interact with determinants in the alpha 1 and 2 domains of MHC class I molecules and, as described elsewhere herein, differ in whether they are stimulatory or inhibitory for NK cells.

[0298] The NKR-CARs described herein include KIR-CARs that share functional and structural properties with KIRs.

[0299] KIRs are a family of cell surface proteins found on NK cells. They regulate the killing function of these cells through interaction with MHC class I molecules, which are expressed on all cell types. This interaction enables them to detect virus-infected or tumor cells. Most KIRs are inhibitory, meaning that their recognition of MHC suppresses the cytotoxic activity of the NK cells that express them. Only a limited number of KIRs have the ability to activate cells.

[0300] The KIR gene family contains at least 15 loci (KIR2DL1, KIR2DL2 / L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1 / S1, KIR3DL2, KIR3DL3, and two pseudogenes, KIR2DP1 and KIR3DP1) encoded within a 100-200 Kb region of the leukocyte receptor complex (LRC) located on chromosome 19 (19q13.4). The LRC consists of a large, 1 Mb cluster of rapidly evolving immune genes, including genes encoding other cell surface molecules with distinctive Ig-like extracellular domains. Additionally, the extended LRC includes genes encoding the transmembrane adaptor molecules DAP10 and DAP12.

[0301] KIR genes vary in length from 4 to 16 Kb (complete genome sequence) and can contain 4 to 9 exons. KIR genes are classified as belonging to one of three groups based on their structural characteristics: (1) type I KIR2D genes, which encode two extracellular domain proteins with D1 and D2 conformations; (2) structurally divergent type II KIR2D genes, which encode two extracellular domain proteins with D0 and D2 conformations; and finally (3) KIR3D genes, which encode proteins with three extracellular Ig-like domains (D0, D1, and D2).

[0302] Type I KIR2D genes, including the pseudogene KIR2DP1 and the KIR2DL1-3 and KIR2DS1-5 genes, contain eight exons and a pseudoexon 3 sequence. This pseudoexon is inactivated in type I KIR2D. In some instances, this is due to a nucleotide substitution located at the intron 2-exon 3 splice site, where its nucleotide sequence shows a high degree of identity with the KIR3D exon 3 sequence and has a characteristic three-base deletion. In other instances, a premature stop codon initiates differential splicing of exon 3. Within the type I KIR2D family of genes, KIR2DL1 and KIR2DL2 share a common deletion in exon 7 that distinguishes these exons from all other KIRs, resulting in a short exon 7 sequence. Similarly, within type I KIR2D, KIR2DL1-3 differ from KIR2DS1-5 only by the length of the cytoplasmic tail encoding region in exon 9. The KIR2DP1 pseudogene structure differs from KIR2DL1-3 by the former having a short exon 4 sequence due to a single base pair deletion.

[0303] Type II KIR2D genes include KIR2DL4 and KIR2DL5. Unlike KIR3D and type I KIR2D, type II KIR2D characteristically lacks the region corresponding to exon 4 in all other KIRs. Furthermore, type II KIR2D genes differ from type I KIR2D genes in that the former process translated exon 3, whereas the latter possesses a nontranslated pseudoexon 3 sequence in its place. Within type II KIR2D genes, KIR2DL4 is further distinguished from KIR2DL5 (as well as other KIR genes) by the length of its exon 1 sequence. In KIR2DL4, exon 1 was found to be 6 nucleotides longer and to have a different start codon from those present in other KIR genes. This start codon matches the 'Kozak transcription initiation consensus sequence' better than the second potential start codon in KIR2DL4 that corresponds to the start codon present in other KIR genes.

[0304] The KIR3D gene contains nine exons and includes the structurally related KIR3DL1, KIR3DS1, KIR3DL2, and KIR3DL3 genes. The KIR3DL2 nucleotide sequence is the longest of all KIR genes, spanning 16,256 bp in the complete genomic sequence and 1,368 bp in the cDNA. Within the KIR3D family, four KIR genes differ in the length of the region encoding the cytoplasmic tail in exon 9. The length of the cytoplasmic tail of KIR proteins can vary from 14 amino acid residues (in some KIR3DS1 alleles) to 108 amino acid residues (in KIR2DL4 proteins). Furthermore, KIR3DS1 differs from KIR3DL1 or KIR3DL2 by the former possessing a short exon 8 sequence. KIR3DL3 differs from other KIR sequences by its complete lack of exon 6. The most extreme KIR gene structural differences observed were in KIR3DP1. This gene fragment completely lacks exons 6-9 and occasionally exon 2. The remaining part of the gene that is present (exons 1, 3, 4, and 5) shares a high level of sequence identity with other KIR3D sequences, particularly the KIR3DL3 sequence.

[0305] KIR proteins have a characteristic Ig-like domain in their extracellular region, which in some KIR proteins is involved in HLA class I ligand binding. They also have transmembrane and cytoplasmic regions that are functionally related in determining the type of signal transduced into NK cells. KIR proteins can have two or three Ig-like domains (hence KIR2D or KIR3D) and short or long cytoplasmic tails (designated KIR2DS or KIR2DL). Two-domain KIR proteins are subdivided into two types based on the origin of the membrane-distal Ig-like domain present. Type I KIR2D proteins (KIR2DL1, KIR2DL2, KIR2DL3, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, and KIR2DS5) have a membrane-distal Ig-like domain that is located at the origin of the KIR3D D1 Ig-like domain but lack the D0 domain. This D1 Ig-like domain is primarily encoded by exon 4 of the corresponding KIR gene. Type II KIR2D proteins, KIR2DL4 and KIR2DL5, have a membrane-distal Ig-like domain with a sequence similar to the DO domain present in KIR3D proteins; however, type II KIR2D lacks the D1 domain. Long cytoplasmic tails typically contain two immunotyrosine-based inhibitory motifs (ITIMs), which transmit inhibitory signals to NK cells. Short cytoplasmic tails contain positively charged amino acid residues in the transmembrane region, allowing them to bind to the DAP12 signaling molecule, which can generate activating signals. The exception to this is KIR2DL4, which contains only one N-terminal ITIM. Furthermore, KIR2DL4 also contains a charged residue (arginine) in its transmembrane domain, a feature that allows this receptor to elicit both inhibitory and activating signals. KIRs regulate human NK cell responses by delivering inhibitory or activating signals upon recognition of MHC class I ligands on the surface of potential target cells.

[0306] KIR proteins vary in length from 306 to 456 amino acid residues. The differences in protein length are largely a result of the number of Ig-like domains present, but variations in the length of the cytoplasmic domain are also an influencing factor. The leader peptide of most KIR proteins is 21 amino acid residues long. However, the presence of a different initiation codon produces a correspondingly longer leader peptide in the KIR2DL4 protein.

[0307] The D0 Ig-like domain is present in type II KIR2D proteins and approximately 96 amino acid residues long in KIR3D proteins. The D1 domain in type I KIR2D and KIR3D proteins is 102 amino acid residues long, while the D2 domain in all KIR proteins is 98 amino acid residues long. The length of the stem region varies from 24 amino acid residues in most KIR proteins to only 7 amino acid residues in the diverse KIR3DL3 proteins. The transmembrane region is 20 amino acid residues long in most KIR proteins but is one residue shorter in KIR2DL1 and KIR2DL2 proteins as a result of a 3-base-pair deletion in exon 7. Finally, the cytoplasmic region of KIR proteins shows a large variation in length, from 23 amino acid residues in some KIR3DS1 alleles to 96 amino acid residues in KIR3DL2 proteins.

[0308] The amino acid sequences of human KIR polypeptides (Homo sapiens) are available from the NCBI database, e.g., under accession numbers NP_037421.2 (GI: 134268644), NP_703144.2 (GI: 46488946), NP_001229796.1 (GI: 338968852), NP_001229796.1 (GI: 338968852), NP_006728.2 (GI: 134268642), NP_065396.1 (GI: 11968154), NP_001018091.1 (GI: 66267727), NP_001018091.1 (GI: 66267727), NP_001018091.1 (GI: 66267727), NP_001018091.1 (GI: 66268642 ... P_001077008.1(GI:134133244), NP_036444.1(GI:6912472), NP_055327.1(GI:7657277), NP_056952.2(GI:71143139), NP_036446.3(GI:11 6517309), NP_001074239.1(GI:124107610), NP_002246.5(GI:124107606), NP_001074241.1(GI:124107604), NP_036445.1(GI:6912474).

[0309] KIRs are named based on the number of extracellular domains (KIR2D and KIR3D have two and three extracellular Ig domains, respectively) and whether they have a long (KIR2DL or KIR3DL) or short (KIR2DS or KIR3DS) cytoplasmic tail. The presence or absence of a KIR varies from NK cell to NK cell within the NK population within a single individual. Within humans, there is also a relatively high level of polymorphism in KIR genes, with some KIR genes present in some individuals but absent in all. Expression of KIR alleles on NK cells is stochastically controlled, meaning that within a given individual, a given lymphocyte may express one, two, or more KIRs, depending on the individual's genotype. NK cells within a single individual typically express different combinations of KIRs, providing a repertoire of NK cells with different specificities for MHC class I molecules.

[0310] Certain KIR gene products, when bound to an appropriate ligand, cause stimulation of lymphocyte activity. Activating KIRs all have short cytoplasmic tails with charged transmembrane residues that bind to adaptor molecules containing immunoreceptor tyrosine-based activation motifs (ITAMs), which transmit stimulatory signals to NK cells. In contrast, inhibitory KIRs have long cytoplasmic tails containing immunoreceptor tyrosine-based inhibitory motifs (ITIMs), which transmit inhibitory signals to NK cells upon binding to MHC class I ligands. Known inhibitory KIRs include members of the KIR2DL and KIR3DL subfamilies. Inhibitory KIRs with two Ig domains (KIR2DL) include HLA-C allotypes: KIR2DL2 (previously designated p58.2) and the closely related allelic gene product KIR2DL3 both recognize "group 1" HLA-C allotypes (including HLA-Cw1, -3, -7, and -8), whereas KIR2DL1 (p58.1) recognizes "group 2" HLA-C allotypes (e.g., HLA-Cw2, -4, -5, and -6). Recognition by KIR2DL1 is dictated by the presence of a Lys residue at position 80 of the HLA-C allele. KIR2DL2 and KIR2DL3 recognition is dictated by the presence of an Asn residue at position 80 of HLA-C. Importantly, the majority of HLA-C alleles have either an Asn or Lys residue at position 80. Therefore, KIR2DL1, -2, and -3 collectively recognize essentially all of the HLA-C allotypes found in humans. One KIR with three Ig domains, KIR3DL1 (p70), recognizes an epitope shared by the HLA-Bw4 allele. Finally, KIR3DL2 (p140), a homodimer of molecules with three Ig domains, recognizes HLA-A3 and -A11.

[0311] However, the present invention should not be limited to inhibitory KIRs that contain an ITIM-containing cytoplasmic tail. Rather, any inhibitory protein that has a cytoplasmic domain associated with an inhibitory signal can be used to construct the CAR of the present invention. Non-limiting examples of inhibitory proteins include, but are not limited to, CTLA-4, PD-1, etc. These proteins are known to inhibit T cell activation.

[0312] Thus, the present invention provides KIR-CARs comprising an extracellular domain containing a target-specific binding element, otherwise referred to as an antigen-binding domain, fused to a KIR or a fragment thereof. In some embodiments, the KIR provides an activating KIR comprising a short cytoplasmic tail linked to an adaptor molecule having an immunoreceptor tyrosine-based activation motif (ITAM), which transmits stimulatory signals to NK cells (referred to elsewhere herein as an actKIR-CAR). In some embodiments, the KIR provides an inhibitory KIR comprising a long cytoplasmic tail containing an immunoreceptor tyrosine-based inhibitory motif (ITIM), which transmits inhibitory signals (referred to elsewhere herein as an inhKIR-CAR). In some instances, it is desirable to remove the hinge region for the activating KIR when constructing an actKIR-CAR. This is because the present invention is based, in part, on the discovery of an activated KIR CAR in which the KIR2DS2 hinge has been removed to produce a KIR2S CAR, which exhibits enhanced cytolytic activity compared to actKIR-CARs containing full-length wild-type KIR2DS2.

[0313] Nucleic acid sequences encoding the desired molecules of the invention can be obtained using recombinant methods known in the art, such as screening libraries from cells which express the gene, deriving the gene from a vector known to contain it, or isolating it directly from cells and tissues which contain it, using standard techniques. Alternatively, the gene of interest can be produced synthetically rather than cloned.

[0314] The present invention includes retroviral and lentiviral vector constructs expressing KIR-CAR that can be directly transduced into cells. The present invention also includes RNA constructs that can be directly transduced into cells. Methods for producing mRNA for use in gene transfer involve in vitro transcription (IVT) of a template with specially designed primers, followed by polyA enrichment, to produce a construct that contains 3' and 5' untranslated sequences ("UTRs"), a 5' cap and / or internal ribosome entry site (IRES), the gene to be expressed, and a polyA tail, typically 50-2000 bases in length. The RNA thus produced can be efficiently transduced into cells of different species. In some embodiments, the template contains the sequence of the KIR-CAR.

[0315] In some embodiments, the KIR-CAR comprises an antigen-binding domain and a KIR transmembrane domain. In some embodiments, the KIR-CAR comprises an antigen-binding domain and a KIR intracellular domain, e.g., an inhKIR intracellular domain.

[0316] As used herein, the term KIR D domain refers to the DO, D1 or D2 domain of a KIR.

[0317] As used herein, the term KIR D domain refers to a polypeptide domain having the structural and functional properties of a KIR D domain.

[0318] As used herein, the term KIR D0 domain refers to the D0 domain of a KIR. In some embodiments, the KIR D0 domain of a KIR-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology to a reference sequence, e.g., a naturally occurring KIR D0 domain or a KIR D0 domain described herein. In some embodiments, the KIR D0 domain of a KIR-CAR does not differ in residues by more than 15%, 10%, 5%, 2%, or 1% from a reference sequence, e.g., a naturally occurring KIR D0 domain or a KIR D0 domain described herein. In some embodiments, the KIR D0 domain of a KIR-CAR does not differ in residues by more than 5, 4, 3, 2, or 1 from a reference sequence, e.g., a naturally occurring KIR D0 domain or a KIR D0 domain described herein. In some embodiments, the KIR D0 domain of a KIR-CAR does not differ from or shares 100% homology with a reference sequence, e.g., a naturally occurring KIR D0 domain or a KIR D0 domain described herein.

[0319] As used herein, the term KIR D1 domain refers to a polypeptide domain having the structural and functional properties of a KIR D1 domain. In some embodiments, the KIR D1 domain of a KIR-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology with a reference sequence, e.g., a naturally occurring KIR D1 domain or a KIR D1 domain described herein. In some embodiments, the KIR D1 domain of a KIR-CAR does not differ in residues by more than 15%, 10%, 5%, 2%, or 1% from a reference sequence, e.g., a naturally occurring KIR D1 domain or a KIR D1 domain described herein. In some embodiments, the KIR D1 domain of a KIR-CAR does not differ in residues by more than 5, 4, 3, 2, or 1 from a reference sequence, e.g., a naturally occurring KIR D0 domain or a KIR D1 domain described herein. In some embodiments, the KIR D1 domain of the KIR-CAR shares no difference or 100% homology with a reference sequence, e.g., a naturally occurring KIR D1 domain or a KIR D1 domain described herein.

[0320] As used herein, the term KIR D2 domain refers to a polypeptide domain having the structural and functional properties of a KIR D2 domain. In some embodiments, the KIR D2 domain of a KIR-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology with a reference sequence, e.g., a naturally occurring KIR D2 domain or a KIR D2 domain described herein. In some embodiments, the KIR D2 domain of a KIR-CAR does not differ in residues by more than 15%, 10%, 5%, 2%, or 1% from a reference sequence, e.g., a naturally occurring KIR D2 domain or a KIR D2 domain described herein. In some embodiments, the KIR D2 domain of a KIR-CAR does not differ in residues by more than 5, 4, 3, 2, or 1 from a reference sequence, e.g., a naturally occurring KIR D2 domain or a KIR D2 domain described herein. In certain embodiments, the KIR D2 domain of the KIR-CAR shares no difference or 100% homology with a reference sequence, e.g., a naturally occurring KIR D2 domain or a KIR D2 domain described herein.

[0321] As used herein, the term KIR hinge or stem domain refers to a polypeptide domain having the structural and functional properties of a KIR hinge or stem domain. In some embodiments, the KIR hinge or stem domain of a KIR-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology with a reference sequence, e.g., a naturally occurring KIR hinge or stem domain or a KIR hinge or stem domain described herein. In some embodiments, the KIR hinge or stem domain of a KIR-CAR does not differ in residues by more than 15%, 10%, 5%, 2%, or 1% from a reference sequence, e.g., a naturally occurring KIR hinge or stem domain or a KIR hinge or stem domain described herein. In some embodiments, the KIR hinge or stem domain of a KIR-CAR does not differ in residues by more than 5, 4, 3, 2, or 1 from a reference sequence, e.g., a naturally occurring KIR hinge or stem domain or a KIR hinge or stem domain described herein. In some embodiments, the KIR hinge or stem domain of the KIR-CAR does not differ from or shares 100% homology with a reference sequence, e.g., a naturally occurring KIR hinge or stem domain or a KIR hinge or stem domain described herein.

[0322] As used herein, the term KIR transmembrane domain refers to a polypeptide domain having the structural and functional properties of a KIR transmembrane domain. In some embodiments, the KIR transmembrane domain of a KIR-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology with a reference sequence, e.g., a naturally occurring KIR transmembrane domain or a KIR transmembrane domain described herein. In some embodiments, the KIR transmembrane domain of a KIR-CAR does not differ in residues from a reference sequence, e.g., a naturally occurring KIR transmembrane domain or a KIR transmembrane domain described herein, by more than 15%, 10%, 5%, 2%, or 1%. In some embodiments, the KIR transmembrane domain of a KIR-CAR does not differ in residues from a reference sequence, e.g., a naturally occurring KIR transmembrane domain or a KIR transmembrane domain described herein, by more than 5, 4, 3, 2, or 1 residue. In some embodiments, the KIR transmembrane domain of the KIR-CAR does not differ from or shares 100% homology with a reference sequence, e.g., a naturally occurring KIR transmembrane domain or a KIR transmembrane domain described herein.

[0323] As used herein, a KIR intracellular domain refers to a polypeptide domain having the structural and functional properties of a KIR intracellular domain. The KIR intracellular domain includes an inhibitory KIR intracellular domain (referred to herein as an inhKIR intracellular domain) and an activating KIR intracellular domain (referred to herein as an actKIR intracellular domain). In some embodiments, the inhKIR intracellular domain comprises an ITIM sequence. In some embodiments, the KIR intracellular domain of a KIR-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology with a reference sequence, e.g., a naturally occurring KIR intracellular domain or a KIR intracellular domain described herein. In some embodiments, the KIR intracellular domain of a KIR-CAR does not differ in residues by more than 15%, 10%, 5%, 2%, or 1% from a reference sequence, e.g., a naturally occurring KIR intracellular domain or a KIR intracellular domain described herein. In some embodiments, the KIR intracellular domain of the KIR-CAR differs from a reference sequence, e.g., a naturally occurring KIR intracellular domain or a KIR intracellular domain described herein, by no more than 5, 4, 3, 2, or 1 residue. In some embodiments, the KIR intracellular domain of the KIR-CAR does not differ from or shares 100% homology with a reference sequence, e.g., a naturally occurring KIR intracellular domain or a KIR intracellular domain described herein.

[0324] NCR The NKR-CARs described herein include NCR-CARs, which share functional and structural properties with NCRs.

[0325] Natural killer (NK) cells are cytotoxic lymphoid cells specialized for the destruction of tumor and virus-infected cells. Unlike cytotoxic T lymphocytes, NK cells do not express antigen-specific receptors. Recognition of transformed cells occurs through the binding of numerous cell surface receptors to target cell surface markers. NK cell surface receptors can be distinguished by whether they activate or inhibit NK cell-mediated cytotoxicity. Numerous interactions between different receptors appear to lead to the formation of a synapse between the NK and target cells. The integration of activating and inhibitory signals at the synapse determines whether the NK cell should exert its cytolytic function against the target cell. Among activating receptors, a family of Ig-like molecules is called natural cytotoxicity receptors (NCRs). These natural cytotoxicity receptors include NKp30, NKp44, and NKp46 molecules. NCRs are key activating receptors for NK cells in tumor cell recognition. All three NCRs are involved in the elimination of both tumor and virus-infected cells. In the latter, antiviral activity is initiated by the interaction of NKp44 with influenza virus or Sendai virus hemagglutinin. NKp46 targets virus-infected cells by binding to influenza virus hemagglutinin or Sendai virus hemagglutinin-neuraminidase. In contrast, NK cell-mediated cytotoxicity is inhibited by the binding of NKp30 to the human cytomegalovirus protein pp65 (see, e.g., Arnon, et al., Nat. Immunol. (2005) 6:515-523).

[0326] The amino acid sequence of the human NCR polypeptide (Homo sapiens) is available from the NCBI database, see, e.g., accession numbers NP_004819.2 (GI:153945782), O14931.1 (GI:47605770), O95944.2 (GI:251757303), O76036.1 (GI:47605775), NP_001138939.1 (GI:224586865) and / or NP_001138938.1 (GI:224586860).

[0327] In some embodiments, the NCR-CAR comprises an antigen-binding domain and an NCR transmembrane domain. In some embodiments, the KIR-CAR comprises an antigen-binding domain and an NCR intracellular domain.

[0328] As used herein, an NCR extracellular domain refers to a polypeptide having the structural and functional properties of an NCR extracellular domain. In some embodiments, the NCR extracellular domain of an NCR-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology with a reference sequence, such as a naturally occurring NCR extracellular domain or an NCR extracellular domain described herein. In some embodiments, the NCR extracellular domain of an NCR-CAR does not differ in residues from a reference sequence, such as a naturally occurring NCR extracellular domain or an NCR extracellular domain described herein, by more than 15%, 10%, 5%, 2%, or 1%. In some embodiments, the NCR extracellular domain of an NCR-CAR does not differ in residues from a reference sequence, such as a naturally occurring NCR extracellular domain or an NCR extracellular domain described herein, by more than 5, 4, 3, 2, or 1 residue. In some embodiments, the NCR extracellular domain of the NCR-CAR does not differ from or shares 100% homology with a reference sequence, e.g., a naturally occurring NCR extracellular domain or an NCR extracellular domain described herein.

[0329] As used herein, the term NCR hinge or stem domain refers to a polypeptide having the structural and functional properties of the hinge or stem domain of NCR. In some embodiments, the NCR hinge or stem domain of NCR-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology with a reference sequence, such as a naturally occurring NCR hinge or stem domain or an NCR hinge or stem domain described herein. In some embodiments, the NCR hinge or stem domain of NCR-CAR does not differ in residues from a reference sequence, such as a naturally occurring NCR hinge or stem domain or an NCR hinge or stem domain described herein, by more than 15%, 10%, 5%, 2%, or 1%. In some embodiments, the NCR hinge or stem domain of NCR-CAR does not differ in residues from a reference sequence, such as a naturally occurring NCR hinge or stem domain or an NCR hinge or stem domain described herein, by more than 5, 4, 3, 2, or 1 residue. In some embodiments, the NCR hinge or stem domain of the NCR-CAR does not differ from or shares 100% homology with a reference sequence, e.g., a naturally occurring NCR hinge or stem domain or an NCR hinge or stem domain described herein.

[0330] As used herein, the term NCR transmembrane domain refers to a polypeptide having the structural and functional properties of an NCR transmembrane domain. In some embodiments, the NCR transmembrane domain of an NCR-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology with a reference sequence, such as a naturally occurring NCR transmembrane domain or an NCR transmembrane domain described herein. In some embodiments, the NCR transmembrane domain of an NCR-CAR does not differ in residues from a reference sequence, such as a naturally occurring NCR transmembrane domain or an NCR transmembrane domain described herein, by more than 15%, 10%, 5%, 2%, or 1%. In some embodiments, the NCR transmembrane domain of an NCR-CAR does not differ in residues from a reference sequence, such as a naturally occurring NCR transmembrane domain or an NCR transmembrane domain described herein, by more than 5, 4, 3, 2, or 1 residue. In some embodiments, the NCR transmembrane domain of the NCR-CAR does not differ from or shares 100% homology with a reference sequence, e.g., a naturally occurring NCR transmembrane domain or an NCR transmembrane domain described herein.

[0331] As used herein, the term NCR intracellular domain refers to a polypeptide having the structural and functional properties of an NCR intracellular domain. In some embodiments, the NCR intracellular domain of an NCR-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology with a reference sequence, e.g., a naturally occurring NCR intracellular domain or an NCR intracellular domain described herein. In some embodiments, the NCR intracellular domain of an NCR-CAR does not differ in residues by more than 15%, 10%, 5%, 2%, or 1% from a reference sequence, e.g., a naturally occurring NCR intracellular domain or an NCR intracellular domain described herein. In some embodiments, the NCR intracellular domain of an NCR-CAR does not differ in residues by more than 5, 4, 3, 2, or 1 from a reference sequence, e.g., a naturally occurring NCR intracellular domain or an NCR intracellular domain described herein. In some embodiments, the NCR intracellular domain of an NCR-CAR does not differ from or shares 100% homology with a reference sequence, e.g., a naturally occurring NCR intracellular domain or an NCR intracellular domain described herein.

[0332] SLAM receptor The NKR-CARs described herein include SLAMF-CARs, which share functional and structural properties with SLAMFs.

[0333] The signaling lymphocyte activation molecule (SLAM) family of immune cell receptors is closely related to the CD2 family of molecules in the immunoglobulin (Ig) superfamily. The SLAM family (SLAMF) currently contains nine members, designated SLAM, CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, and CD2F-10. Generally, SLAM molecules contain two to four extracellular Ig domains, a transmembrane segment, and an intracellular tyrosine-rich region. These molecules are differentially expressed in various immune cell types. Some are self-ligands, and SLAM has been identified as the human measles virus receptor. Several SH2-containing adaptor proteins are known to bind to the intracellular domains of SLAM family members and mediate receptor signaling, including SH2D1A (also known as SLAM-associated protein [SAP]) and SH2D1B (also known as EAT2). For example, in T cells and NK cells, activated SLAM family receptors become tyrosine phosphorylated and recruit the adaptor SAP and subsequently the Src kinase Fyn. The subsequent signaling cascades influence the outcome of T cell-antigen-presenting cell and NK cell-target cell interactions.

[0334] Amino acid sequence of the human SLAM receptor polypeptide (Homo sapiens) are available from the NCBI database, see, for example, accession numbers NP_057466.1 (GI:7706529), NP_067004.3 (GI:19923572), NP_003028.1 (GI:4506969), NP_001171808.1 (GI:296434285), NP_001171643.1 (GI:296040491), NP_001769.2 (GI:21361571), NP_254273.2 (GI:226342990), NP_064510.1 (GI:9910342) and / or NP_002339.2 (GI:55925578).

[0335] In some embodiments, the SLAMF-CAR comprises an antigen-binding domain and a SLAMF transmembrane domain. In some embodiments, the SLAMF-CAR comprises an antigen-binding domain and a SLAMF intracellular domain.

[0336] As used herein, the term SLAMF extracellular domain refers to a polypeptide having the structural and functional properties of the extracellular domain of an SLAMF. In some embodiments, the SLAMF extracellular domain of an SLAMF-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology to a reference sequence, e.g., a naturally occurring SLAMF extracellular domain or an SLAMF extracellular domain described herein. In some embodiments, the SLAMF extracellular domain of an SLAMF-CAR does not differ in more than 15%, 10%, 5%, 2%, or 1% of its residues from a reference sequence, e.g., a naturally occurring SLAMF extracellular domain or an SLAMF extracellular domain described herein. In some embodiments, the SLAMF extracellular domain of an SLAMF-CAR does not differ in more than 5, 4, 3, 2, or 1 residue from a reference sequence, e.g., a naturally occurring SLAMF extracellular domain or an SLAMF extracellular domain described herein. In some embodiments, the SLAMF extracellular domain of the SLAMF-CAR does not differ from or shares 100% homology with a reference sequence, e.g., a naturally occurring SLAMF extracellular domain or a SLAMF extracellular domain described herein.

[0337] As used herein, the term SLAMF hinge or stem domain refers to a polypeptide having the structural and functional properties of a SLAMF hinge or stem domain. In some embodiments, the SLAMF hinge or stem domain of an SLAMF-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology to a reference sequence, e.g., a naturally occurring SLAMF hinge or stem domain or an SLAMF hinge or stem domain described herein. In some embodiments, the SLAMF hinge or stem domain of an SLAMF-CAR does not differ in residues by more than 15%, 10%, 5%, 2%, or 1% from a reference sequence, e.g., a naturally occurring SLAMF hinge or stem domain or an SLAMF hinge or stem domain described herein. In some embodiments, the SLAMF hinge or stem domain of the SLAMF-CAR does not differ by more than 5, 4, 3, 2, or 1 residue from a reference sequence, e.g., a naturally occurring SLAMF hinge or stem domain or a SLAMF hinge or stem domain described herein. In some embodiments, the SLAMF hinge or stem domain of the SLAMF-CAR does not differ from or shares 100% homology with a reference sequence, e.g., a naturally occurring SLAMF hinge or stem domain or a SLAMF hinge or stem domain described herein.

[0338] As used herein, the term SLAMF transmembrane domain refers to a polypeptide having the structural and functional properties of a transmembrane domain of an SLAMF. In some embodiments, the SLAMF transmembrane domain of an SLAMF-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology to a reference sequence, e.g., a naturally occurring SLAMF transmembrane domain or an SLAMF transmembrane domain described herein. In some embodiments, the SLAMF transmembrane domain of an SLAMF-CAR does not differ in residues by more than 15%, 10%, 5%, 2%, or 1% from a reference sequence, e.g., a naturally occurring SLAMF transmembrane domain or an SLAMF transmembrane domain described herein. In some embodiments, the SLAMF transmembrane domain of an SLAMF-CAR does not differ in residues by more than 5, 4, 3, 2, or 1 from a reference sequence, e.g., a naturally occurring SLAMF transmembrane domain or an SLAMF transmembrane domain described herein. In some embodiments, the SLAMF transmembrane domain of the SLAMF-CAR does not differ from or shares 100% homology with a reference sequence, e.g., a naturally occurring SLAMF transmembrane domain or a SLAMF transmembrane domain described herein.

[0339] As used herein, the term SLAMF intracellular domain refers to a polypeptide having the structural and functional properties of an intracellular domain of an SLAMF. In some embodiments, the SLAMF intracellular domain of an SLAMF-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology to a reference sequence, e.g., a naturally occurring SLAMF intracellular domain or an SLAMF intracellular domain described herein. In some embodiments, the SLAMF intracellular domain of an SLAMF-CAR does not differ in residues by more than 15%, 10%, 5%, 2%, or 1% from a reference sequence, e.g., a naturally occurring SLAMF intracellular domain or an SLAMF intracellular domain described herein. In some embodiments, the SLAMF intracellular domain of an SLAMF-CAR does not differ in residues by more than 5, 4, 3, 2, or 1 from a reference sequence, e.g., a naturally occurring SLAMF intracellular domain or an SLAMF intracellular domain described herein. In some embodiments, the SLAMF intracellular domain of the SLAMF-CAR does not differ from or shares 100% homology with a reference sequence, e.g., a naturally occurring SLAMF intracellular domain or a SLAMF intracellular domain described herein.

[0340] Fc-binding receptors The NKR-CARs described herein include CARs based on Fc receptors, FcR-CARs, e.g., CD16-CARs and CD64-CARs, which share functional and structural properties with CD16 and CD64.

[0341] Upon activation, NK cells produce abundant cytokines and chemokines while simultaneously exerting potent cytolytic activity. NK cell activation can occur via direct binding of the NK cell receptor to a ligand on the target cell, as seen in direct tumor cell killing, or via cross-linking of the Fc receptor (CD16; FcγRIII) by binding of the Fc portion of an antibody bound to an antigen-bearing cell. This CD16 binding (CD16 cross-linking) initiates the NK cell response via intracellular signaling that produces one or both of the CD16-associated adaptor chains, FcRγ and CD3ζ. CD16 engagement leads to phosphorylation of the γ or ζ chain, which subsequently recruits the tyrosine kinases Syk and ZAP-70, initiating a signaling cascade that leads to rapid and potent effector function. The most prominent effector function is the release of cytoplasmic granules carrying toxic proteins that kill nearby target cells via the process of antibody-dependent cellular cytotoxicity. CD16 cross-linking also leads to the production of cytokines and chemokines, which in turn activate and orchestrate a range of immune responses.

[0342] However, unlike T and B lymphocytes, NK cells were thought to have only a limited capacity for target recognition using germline-encoded activating receptors (Bottino et al., Curr Top Microbiol Immunol. 298:175-182 (2006); Stewart et al., Curr Top Microbiol Immunol. 298:1-21 (2006)). NK cells express the activating Fc receptor CD16, which recognizes IgG-coated target cells, thereby extending target recognition (Ravetch & Bolland, Annu Rev Immunol. 19:275-290 (2001); Lanier Nat. Immunol. 9(5):495-502 (2008); Bryceson & Long, Curr Opin Immunol. 20(3):344-352 (2008)). The expression and signaling activity of several NK cell-activating receptors requires physically associated adaptors, which transmit signals via immunoreceptor tyrosine-based activation motifs (ITAMs). Among these adaptors, the FcRγ and CD3ζ chains can associate with CD16 and natural cytotoxicity receptors (NCRs) as disulfide-linked homodimers or heterodimers; these chains are thought to be expressed by all mature NK cells.

[0343] The amino acid sequence of CD16 (Homo sapiens) is available from the NCBI database, see for example accession numbers NP_000560.5 (GI:50726979), NP_001231682.1 (GI:348041254).

[0344] In some embodiments, the FcR-CAR comprises an antigen-binding domain and an FcR transmembrane domain. In some embodiments, the FcR-CAR comprises an antigen-binding domain and an FcR intracellular domain.

[0345] As used herein, a CD16 extracellular domain refers to a polypeptide having the structural and functional properties of the extracellular domain of CD16. In one embodiment, the CD16 extracellular domain of a CD16-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology with a reference sequence, e.g., a naturally occurring CD16 extracellular domain or a CD16 extracellular domain described herein. In one embodiment, the CD16 extracellular domain of a CD16-CAR does not differ in residues by more than 15%, 10%, 5%, 2%, or 1% from a reference sequence, e.g., a naturally occurring CD16 extracellular domain or a CD16 extracellular domain described herein. In one embodiment, the CD16 extracellular domain of a CD16-CAR does not differ in residues by more than 5, 4, 3, 2, or 1 from a reference sequence, e.g., a naturally occurring CD16 extracellular domain or a CD16 extracellular domain described herein. In one embodiment, the CD16 extracellular domain of the CD16-CAR does not differ from or shares 100% homology with a reference sequence, e.g., a naturally occurring CD16 extracellular domain or a CD16 extracellular domain described herein.

[0346] As used herein, the term CD16 hinge or stem domain refers to a polypeptide having the structural and functional properties of the hinge or stem domain of CD16. In one embodiment, the CD16 hinge or stem domain of a CD16-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology with a reference sequence, for example, a naturally occurring CD16 hinge or stem domain or a CD16 hinge or stem domain described herein. In one embodiment, the CD16 hinge or stem domain of a CD16-CAR does not differ in residues by more than 15%, 10%, 5%, 2%, or 1% from a reference sequence, for example, a naturally occurring CD16 hinge or stem domain or a CD16 hinge or stem domain described herein. In one embodiment, the CD16 hinge or stem domain of the CD16-CAR does not differ by more than 5, 4, 3, 2, or 1 residue from a reference sequence, e.g., a naturally occurring CD16 hinge or stem domain or a CD16 hinge or stem domain described herein. In one embodiment, the CD16 hinge or stem domain of the CD16-CAR does not differ from or shares 100% homology with a reference sequence, e.g., a naturally occurring CD16 hinge or stem domain or a CD16 hinge or stem domain described herein.

[0347] As used herein, the term CD16 transmembrane domain refers to a polypeptide having the structural and functional properties of the transmembrane domain of CD16. In one embodiment, the CD16 transmembrane domain of CD16-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology with a reference sequence, e.g., a naturally occurring CD16 transmembrane domain or a CD16 transmembrane domain described herein. In one embodiment, the CD16 transmembrane domain of CD16-CAR does not differ in residues from a reference sequence, e.g., a naturally occurring CD16 transmembrane domain or a CD16 transmembrane domain described herein, by more than 15%, 10%, 5%, 2%, or 1%. In one embodiment, the CD16 transmembrane domain of CD16-CAR does not differ in residues from a reference sequence, e.g., a naturally occurring CD16 transmembrane domain or a CD16 transmembrane domain described herein, by more than 5, 4, 3, 2, or 1 residue. In one embodiment, the CD16 transmembrane domain of the CD16-CAR does not differ from or shares 100% homology with a reference sequence, e.g., a naturally occurring CD16 transmembrane domain or a CD16 transmembrane domain described herein.

[0348] As used herein, the term CD16 intracellular domain refers to a polypeptide having the structural and functional properties of the intracellular domain of CD16. In one embodiment, the CD16 intracellular domain of a CD16-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology with a reference sequence, e.g., a naturally occurring CD16 intracellular domain or a CD16 intracellular domain described herein. In one embodiment, the CD16 intracellular domain of a CD16-CAR does not differ in residues by more than 15%, 10%, 5%, 2%, or 1% from a reference sequence, e.g., a naturally occurring CD16 intracellular domain or a CD16 intracellular domain described herein. In one embodiment, the CD16 intracellular domain of a CD16-CAR does not differ in residues by more than 5, 4, 3, 2, or 1 from a reference sequence, e.g., a naturally occurring CD16 intracellular domain or a CD16 intracellular domain described herein. In one embodiment, the CD16 intracellular domain of the CD16-CAR does not differ from or shares 100% homology with a reference sequence, e.g., a naturally occurring CD16 intracellular domain or a CD16 intracellular domain described herein.

[0349] As used herein, the term CD64 extracellular domain refers to a polypeptide having the structural and functional properties of the extracellular domain of CD64. In one embodiment, the CD64 extracellular domain of a CD64-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology with a reference sequence, e.g., a naturally occurring CD64 extracellular domain or a CD64 extracellular domain described herein. In one embodiment, the CD64 extracellular domain of a CD64-CAR does not differ in residues by more than 15%, 10%, 5%, 2%, or 1% from a reference sequence, e.g., a naturally occurring CD64 extracellular domain or a CD64 extracellular domain described herein. In one embodiment, the CD64 extracellular domain of a CD64-CAR does not differ in residues by more than 5, 4, 3, 2, or 1 from a reference sequence, e.g., a naturally occurring CD64 extracellular domain or a CD64 extracellular domain described herein. In one embodiment, the CD64 extracellular domain of the CD64-CAR does not differ from or shares 100% homology with a reference sequence, e.g., a naturally occurring CD64 extracellular domain or a CD64 extracellular domain described herein.

[0350] As used herein, the term CD64 hinge or stem domain refers to a polypeptide having the structural and functional properties of the hinge or stem domain of CD64. In one embodiment, the CD64 hinge or stem domain of a CD64-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology with a reference sequence, e.g., a naturally occurring CD64 hinge or stem domain or a CD64 hinge or stem domain described herein. In one embodiment, the CD64 hinge or stem domain of a CD64-CAR does not differ in residues by more than 15%, 10%, 5%, 2%, or 1% from a reference sequence, e.g., a naturally occurring CD64 hinge or stem domain or a CD64 hinge or stem domain described herein. In one embodiment, the CD64 hinge or stem domain of the CD64-CAR does not differ by more than 5, 4, 3, 2, or 1 residue from a reference sequence, e.g., a naturally occurring CD64 hinge or stem domain or a CD64 hinge or stem domain described herein. In one embodiment, the CD64 hinge or stem domain of the CD64-CAR does not differ from or shares 100% homology with a reference sequence, e.g., a naturally occurring CD64 hinge or stem domain or a CD64 hinge or stem domain described herein.

[0351] As used herein, the term CD64 transmembrane domain refers to a polypeptide having the structural and functional properties of the transmembrane domain of CD64. In one embodiment, the CD64 transmembrane domain of CD64-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology with a reference sequence, e.g., a naturally occurring CD64 transmembrane domain or a CD64 transmembrane domain described herein. In one embodiment, the CD64 transmembrane domain of CD64-CAR does not differ in residues by more than 15%, 10%, 5%, 2%, or 1% from a reference sequence, e.g., a naturally occurring CD64 transmembrane domain or a CD64 transmembrane domain described herein. In one embodiment, the CD64 transmembrane domain of CD64-CAR does not differ in residues by more than 5, 4, 3, 2, or 1 from a reference sequence, e.g., a naturally occurring CD64 transmembrane domain or a CD64 transmembrane domain described herein. In one embodiment, the CD64 transmembrane domain of the CD64-CAR does not differ from or shares 100% homology with a reference sequence, e.g., a naturally occurring CD64 transmembrane domain or a CD64 transmembrane domain described herein.

[0352] As used herein, the term CD64 intracellular domain refers to a polypeptide having the structural and functional properties of the intracellular domain of CD64. In one embodiment, the CD64 intracellular domain of a CD64-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology with a reference sequence, e.g., a naturally occurring CD64 intracellular domain or a CD64 intracellular domain described herein. In one embodiment, the CD64 intracellular domain of a CD64-CAR does not differ in residues by more than 15%, 10%, 5%, 2%, or 1% from a reference sequence, e.g., a naturally occurring CD64 intracellular domain or a CD64 intracellular domain described herein. In one embodiment, the CD64 intracellular domain of a CD64-CAR does not differ in residues by more than 5, 4, 3, 2, or 1 from a reference sequence, e.g., a naturally occurring CD64 intracellular domain or a CD64 intracellular domain described herein. In one embodiment, the CD64 intracellular domain of the CD64-CAR does not differ from or shares 100% homology with a reference sequence, e.g., a naturally occurring CD64 intracellular domain or a CD64 intracellular domain described herein.

[0353] Ly49 and related killer cell lectin-like receptors The NKR-CARs described herein include Ly49-CARs, which share functional and structural properties with Ly49.

[0354] Ly49 receptors are derived from at least 23 identified genes in mice (Ly49A-W). Despite their distinct structures (type II integral membrane proteins of the C-type lectin superfamily), these receptors largely share the same roles in mouse NK cells and T cells as those played by KIRs in humans, and they also contain a considerable degree of genetic variation, like human KIRs. The striking functional similarities between Ly49 and KIR receptors suggest that these groups of receptors evolved independently, but convergently, to perform the same physiological functions in NK cells and T cells.

[0355] Similar to human KIRs, different Ly49 receptors recognize different MHC class I alleles and are differentially expressed on subsets of NK cells. The original prototypic Ly49 receptors, Ly49A and Ly49C, have cytoplasmic domains bearing two immunotyrosine-based inhibitory motifs (ITIMs), similar to inhibitory KIRs such as KIR2DL3. These domains recruit the phosphatase SHP-1 and, like inhibitory KIRs, function in NK cell activation and T cell restriction. In addition to inhibitory Ly49 molecules, several family members, such as Ly49D and Ly49H, lack the ITIM-containing domain and instead possess the ability to interact with the signaling adaptor molecule DAP12, similar to activating KIRs such as human KIR2DS2.

[0356] The amino acid sequences of Ly49 family members are available from the NCBI database, see, for example, accession numbers AAF82184.1 (GI:9230810), AAF99547.1 (GI:9801837), NP_034778.2 (GI:133922593), NP_034779.1 (GI:6754462), NP_001095090.1 (GI:197333718), NP_034776.1 (GI:21327665), AAK11559.1 (GI:13021834) and / or NP_038822.3 (GI:9256549).

[0357] In some embodiments, the Ly49-CAR comprises an antigen-binding domain and a Ly49 transmembrane domain. In some embodiments, the Ly49-CAR comprises an antigen-binding domain and a Ly49 intracellular domain.

[0358] As used herein, the term LY49 extracellular domain refers to a polypeptide having the structural and functional properties of the extracellular domain of LY49. In some embodiments, the LY49 extracellular domain of LY49-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology with a reference sequence, e.g., a naturally occurring LY49 extracellular domain or an LY49 extracellular domain described herein. In some embodiments, the LY49 extracellular domain of LY49-CAR does not differ in residues by more than 15%, 10%, 5%, 2%, or 1% from a reference sequence, e.g., a naturally occurring LY49 extracellular domain or an LY49 extracellular domain described herein. In some embodiments, the LY49 extracellular domain of LY49-CAR does not differ in residues by more than 5, 4, 3, 2, or 1 from a reference sequence, e.g., a naturally occurring LY49 extracellular domain or an LY49 extracellular domain described herein. In some embodiments, the LY49 extracellular domain of LY49-CAR does not differ from or shares 100% homology with a reference sequence, e.g., a naturally occurring LY49 extracellular domain or an LY49 extracellular domain described herein.

[0359] As used herein, the term LY49 hinge or stem domain refers to a polypeptide having the structural and functional properties of the hinge or stem domain of LY49. In one embodiment, the LY49 hinge or stem domain of LY49-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology with a reference sequence, for example, a naturally occurring LY49 hinge or stem domain or an LY49 hinge or stem domain described herein. In one embodiment, the LY49 hinge or stem domain of LY49-CAR does not differ in residues by more than 15%, 10%, 5%, 2%, or 1% from a reference sequence, for example, a naturally occurring LY49 hinge or stem domain or an LY49 hinge or stem domain described herein. In some embodiments, the LY49 hinge or stem domain of LY49-CAR does not differ by more than 5, 4, 3, 2, or 1 residue from a reference sequence, e.g., a naturally occurring LY49 hinge or stem domain or an LY49 hinge or stem domain described herein. In some embodiments, the LY49 hinge or stem domain of LY49-CAR does not differ from or shares 100% homology with a reference sequence, e.g., a naturally occurring LY49 hinge or stem domain or an LY49 hinge or stem domain described herein.

[0360] As used herein, the term LY49 transmembrane domain refers to a polypeptide having the structural and functional properties of the transmembrane domain of LY49. In some embodiments, the LY49 transmembrane domain of LY49-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology with a reference sequence, e.g., a naturally occurring LY49 transmembrane domain or an LY49 transmembrane domain described herein. In some embodiments, the LY49 transmembrane domain of LY49-CAR does not differ in residues by more than 15%, 10%, 5%, 2%, or 1% from a reference sequence, e.g., a naturally occurring LY49 transmembrane domain or an LY49 transmembrane domain described herein. In some embodiments, the LY49 transmembrane domain of LY49-CAR does not differ in residues by more than 5, 4, 3, 2, or 1 from a reference sequence, e.g., a naturally occurring LY49 transmembrane domain or an LY49 transmembrane domain described herein. In some embodiments, the LY49 transmembrane domain of LY49-CAR does not differ from or shares 100% homology with a reference sequence, e.g., a naturally occurring LY49 transmembrane domain or an LY49 transmembrane domain described herein.

[0361] As used herein, the term LY49 intracellular domain refers to a polypeptide having the structural and functional properties of the intracellular domain of LY49. In some embodiments, the LY49 intracellular domain of LY49-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology with a reference sequence, e.g., a naturally occurring LY49 intracellular domain or an LY49 intracellular domain described herein. In some embodiments, the LY49 intracellular domain of LY49-CAR does not differ in residues by more than 15%, 10%, 5%, 2%, or 1% from a reference sequence, e.g., a naturally occurring LY49 intracellular domain or an LY49 intracellular domain described herein. In some embodiments, the LY49 intracellular domain of LY49-CAR does not differ in residues by more than 5, 4, 3, 2, or 1 from a reference sequence, e.g., a naturally occurring LY49 intracellular domain or an LY49 intracellular domain described herein. In one embodiment, the LY49 intracellular domain of LY49-CAR does not differ from or shares 100% homology with a reference sequence, e.g., a naturally occurring LY49 intracellular domain or an LY49 intracellular domain described herein.

[0362] Intracellular signaling domains or adapter molecules, e.g., DAP12 Some NKR-CARs interact with other molecules, e.g., molecules that contain an intracellular signaling domain, e.g., an ITAM. In some embodiments, the intracellular signaling domain is DAP12.

[0363] DAP12 is so named due to its structural characteristics and hypothesized function. Some cell surface receptors lack intrinsic functionality, suggesting that it is a 12 kD protein that can hypothetically interact with other protein partners. The signaling mechanism may involve ITAM signaling.

[0364] DAP12 was identified from sequence databases based on its hypothesized relationship to CD3 (see Olcese, et al. (1997) J. Immunol. 158:5083-5086), the presence of an ITAM sequence (see Thomas (1995) J. Exp. Med. 181:1953-1956), certain size predictions (see Olcese; and Takase, et al. (1997) J. Immunol. 159:741-747), and other properties. In particular, the transmembrane domain is hypothesized to contain charged residues, which would allow salt cross-linking with the corresponding transmembrane segment of its putative receptor partner, the KIR CD94 protein, and possibly other similar proteins. See Daeron, et al. (1995) Immunity 3:635-646.

[0365] Indeed, many of the known KIR, MIR, ILT, and CD94 / NKG2 receptor molecules can actually function with accessory proteins that are part of the functional receptor complex. See Olcese, et al. (1997) J. Immunol. 158:5083-5086; and Takase, et al. (1997) J. Immunol. 159:741-747.

[0366] As used herein, the term DAP12 domain refers to a polypeptide having the structural and functional properties of the cytoplasmic domain of DAP12, generally including an ITAM domain. In some embodiments, the DAP12 domain of a KIR-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology with a reference sequence, e.g., naturally occurring DAP12 or DAP12 described herein. In some embodiments, the DAP12 domain of a KIR-CAR does not differ in residues from a reference sequence, e.g., naturally occurring DAP12 or DAP12 described herein, by more than 15%, 10%, 5%, 2%, or 1%. In some embodiments, the DAP12 domain of a KIR-CAR does not differ in residues from a reference sequence, e.g., naturally occurring DAP12 or DAP12 described herein, by more than 5, 4, 3, 2, or 1. In some embodiments, the DAP12 domain of the KIR-CAR shares no difference or 100% homology with a reference sequence, e.g., a naturally occurring DAP12 or a DAP12 described herein.

[0367] DAP10 was identified in part by its homology with DAP12 and other properties. In particular, in contrast to DAP12, which displays ITAM activating motifs, DAP10 displays ITIM inhibitory motifs. MDL-1 was identified by its functional relationship with DAP12.

[0368] For example, functional interaction of DAP12 or DAP10 with its accessory receptors may allow for the use of structural combinations in the receptor that are not normally seen in truncated receptors. Therefore, signaling mechanisms via accessory proteins such as DAP12 and DAP10 allow for interesting manipulation of other KIR-like receptor complexes, such as KIR, MIR, ILT, and CD94 NKG2-type receptors. Truncated forms of the complete receptor that interact with DAP12 or DAP10 to form functional signaling complexes can be constructed.

[0369] The primate nucleotide sequence of DAP12 corresponds to nucleotides 1-339 of SEQ ID NO:332, and the amino acid sequence corresponds to amino acids 1-113 of SEQ ID NO:333. The signal sequence appears to extend from met(-26) to gln(-1) or ala1, and the mature protein is approximately ala1 (or gln2). The extracellular domain extends from ala1 to pro14; the extracellular domain contains two cysteines at positions 7 and 9, which may allow disulfide bonding to additional homologous or heterologous accessory proteins; the transmembrane region extends from approximately gly15 or val16 to approximately gly39; and the ITAM motif extends from tyr65 to leu79 (YxxL-6 / 8x-YxxL) (SEQ ID NO:341). The LVA03A EST was identified and used to extract other overlapping sequences. See also Genbank Human ESTs that are part of human DAP12; some, but not all, inclusive Genbank Accession #s AA481924; H39980; W60940; N41026; R49793; W60864; W92376; H12338; T52100; AA480109; H12392; W74783; and T55959.

[0370] Inhibitory NKR-CARS The present invention provides compositions and methods for limiting the depletion of non-cancerous cells by certain CAR T cell therapies. As disclosed herein, certain CAR T cell therapies comprise NK receptors, including, but not limited to, activating and inhibitory receptors for NK cells, known as killer cell immunoglobulin-like receptors (KIRs). Thus, the present invention provides compositions and methods of use comprising NKR-CARs, e.g., KIR-CARs, including, but not limited to, activating NKR-CARs (actNKR-CARs), e.g., activating KIR-CARs (actKIR-CARs), and inhibitory NKR-CARs (inhNKR-CARs), e.g., inhibitory KIR-CARs (inhKIR-CARs).

[0371] In some embodiments, the KIR of the InhKIR-CAR comprises an inhibitory KIR that includes a long cytoplasmic tail containing an immunoreceptor tyrosine-based inhibitory motif (ITIM) that transmits an inhibitory signal (referred to elsewhere herein as inhKIR-CAR).

[0372] In some embodiments, the inhKIR-CAR comprises the cytoplasmic domain of an inhibitory molecule other than KIR. These inhibitory molecules may, in some embodiments, reduce the ability of a cell to mount an immune effector response. The cytoplasmic domain of the inhibitory molecule may be linked to the transmembrane domain of KIR, for example, by fusion. Examples of inhibitory molecules are shown in Table 1. [Table 1]

[0373] In some embodiments, the inhKIR-CAR comprises a PD1 cytoplasmic domain. As used herein, the term PD1 cytoplasmic domain refers to a polypeptide having the structural and functional properties of the cytoplasmic domain of PD1. In some embodiments, the PD1 cytoplasmic domain of the KIR-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology to a reference sequence, e.g., a naturally occurring PD1 cytoplasmic domain or a PD1 cytoplasmic domain described herein (SEQ ID NO: 338). In some embodiments, the PD1 cytoplasmic domain of the KIR-CAR does not differ in residues by more than 15%, 10%, 5%, 2%, or 1% from a reference sequence, e.g., a naturally occurring PD1 cytoplasmic domain or a PD1 cytoplasmic domain described herein. In some embodiments, the PD1 cytoplasmic domain of the KIR-CAR does not differ in residues by more than 5, 4, 3, 2, or 1 from a reference sequence, e.g., a naturally occurring PD1 cytoplasmic domain or a PD1 cytoplasmic domain described herein. In some embodiments, the PD1 cytoplasmic domain of the KIR-CAR does not differ from or shares 100% homology with a reference sequence, e.g., a naturally occurring PD1 cytoplasmic domain or a PD1 cytoplasmic domain described herein.

[0374] In some embodiments, the inhKIR-CAR comprises a CTLA-4 cytoplasmic domain. As used herein, the term CTLA-4 cytoplasmic domain refers to a polypeptide having the structural and functional properties of the cytoplasmic domain of CTLA-4. In some embodiments, the CTLA-4 cytoplasmic domain of the KIR-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology to a reference sequence, e.g., a naturally occurring CTLA-4 cytoplasmic domain or a CTLA-4 cytoplasmic domain described herein (SEQ ID NO: 339). In some embodiments, the CTLA-4 cytoplasmic domain of the KIR-CAR does not differ in residues by more than 15%, 10%, 5%, 2%, or 1% from a reference sequence, e.g., a naturally occurring CTLA-4 cytoplasmic domain or a CTLA-4 cytoplasmic domain described herein. In some embodiments, the CTLA-4 cytoplasmic domain of the KIR-CAR differs from a reference sequence, e.g., a naturally occurring CTLA-4 cytoplasmic domain or a CTLA-4 cytoplasmic domain described herein, by no more than 5, 4, 3, 2, or 1 residue. In some embodiments, the CTLA-4 cytoplasmic domain of the KIR-CAR does not differ from or shares 100% homology with a reference sequence, e.g., a naturally occurring CTLA-4 cytoplasmic domain or a CTLA-4 cytoplasmic domain described herein.

[0375] In some embodiments, the inhNKR-CAR, e.g., inhKIR-CAR, inactivates cytotoxic cells containing the inhNKR-CAR by binding to an antigen on a non-target or bystander cell. While much of the following relates to inhKIR-CARs, the invention also encompasses similar applications of other inhNKR-CARs.

[0376] In some embodiments, T cells expressing an actKIR-CAR exert anti-tumor properties upon binding to their target, while T cells expressing an inhKIR-CAR result in inhibition of cellular activity when the inhKIR-CAR binds to its target.

[0377] Regardless of the type of KIR-CAR, the KIR-CAR is engineered to contain an extracellular domain with an antigen-binding domain fused to a cytoplasmic domain. In some embodiments, when expressed in T cells, KIR-CARs can redirect antigen recognition based on antigen specificity. An example of an antigen is CD19, because this antigen is expressed on B-cell lymphomas. However, CD19 is also expressed on normal B cells, and therefore, CARs containing anti-CD19 domains can lead to the depletion of normal B cells. Depletion of normal B cells renders the treated subject susceptible to infection, as B cells normally assist T cells in infection control. The present invention provides compositions and methods for limiting the depletion of normal tissues during KIR-CAR T-cell therapy. In some embodiments, the present invention provides methods for treating cancer and other disorders using KIR-CAR T-cell therapy while limiting the depletion of healthy bystander cells.

[0378] In some embodiments, the present invention involves modulation or control of KIR-CAR T cell activity. In some embodiments, the present invention includes compositions and methods involving T cells genetically modified to express multiple types of KIR-CARs, where KIR-CAR T cell activation is dependent on the binding of multiple types of KIR-CARs to their target receptors. The reliance on binding of multiple types of KIR-CARs improves the specificity of the lytic activity of KIR-CAR T cells, thereby reducing the likelihood of depleting normal healthy tissue.

[0379] In other embodiments, the invention includes compositions and methods relating to genetically modified T cells harboring an inhibitory KIR-CAR. In some embodiments, the inhibitory KIR-CAR comprises an extracellular antigen-binding domain that recognizes an antigen that binds to a normal, non-cancerous, cell and an inhibitory cytoplasmic domain.

[0380] In some embodiments, the present invention provides dual KIR-CARs in which T cells are genetically modified to express an inhKIR-CAR and an actKIR-CAR. In some embodiments, binding of the inhKIR-CAR to normal, non-cancerous cells results in inhibition of the dual KIR-CAR T cells. For example, in some embodiments, binding of the inhKIR-CAR to normal, non-cancerous cells results in the death of the dual KIR-CAR T cells. In other embodiments, binding of the inhKIR-CAR to normal, non-cancerous cells results in inhibition of actKIR-CAR signaling. In yet other embodiments, binding of the inhKIR-CAR to normal, non-cancerous cells results in the induction of a signaling signal that prevents the actKIR-CAR T cells from exerting anti-tumor activity. Thus, the dual KIR-CARs of the present invention, comprising at least one inhKIR-CAR and at least one actKIR-CAR, provide a mechanism for controlling the activity of dual KIR-CAR T cells.

[0381] In some embodiments, the present invention provides methods for using KIR-CAR T cell therapy to treat cancer and other disorders while minimizing the depletion of normal healthy tissue.Cancer can be a hematopoietic tumor, a solid tumor, a primary tumor, or a metastatic tumor.Other diseases that can be treated using the compositions and methods of the present invention include viral, bacterial, and parasitic infections, as well as autoimmune diseases.

[0382] Extracellular hinge domain As used herein, the term extracellular hinge domain refers to a polypeptide sequence of an NKR-CAR located between the transmembrane domain and the antigen-binding domain. In some embodiments, the extracellular hinge domain allows sufficient distance between the outer surface of the cell and the antigen-binding domain, as well as flexibility to minimize steric hindrance between the cell and the antigen-binding domain. In some embodiments, the extracellular hinge domain is sufficiently short or flexible so as not to interfere with binding of a cell comprising an NKR-CAR to an antigen-bearing cell, e.g., a target cell. In some embodiments, the extracellular hinge domain is 2-20, 5-15, 7-12, or 8-10 amino acids in length. In some embodiments, the hinge domain comprises at least 50, 20, or 10 residues. In some embodiments, the hinge is 10-300, 10-250, or 10-200 residues in length. In some embodiments, the distance the hinge extends from the cell is sufficiently short so as not to interfere with binding to the surface of a target cell. In some embodiments, the hinge extends less than 20, 15, or 10 nanometers from the cytotoxic cell surface. Therefore, the compatibility of the hinge can be affected by the linear length, number of amino acid residues, and flexibility of the hinge. IgG4 hinges can be as long as 200 amino acids, but the distance they extend from the cytotoxic cell surface is smaller due to Ig domain folding. The CD8 alpha hinge, which is about 43 amino acids, is rather linear, about 8 nm long. In contrast, IgG4 C2 and C3 hinges are about 200 amino acids long, but have a distance from the cytotoxic cell surface comparable to that of the CD8 alpha hinge. Without wishing to be bound by theory, the similarity of extension is affected by flexibility.

[0383] In some instances, the extracellular hinge domain is, for example, a hinge from a human protein, a fragment thereof, or a short oligo- or polypeptide linker.

[0384] In some embodiments, the hinge is an artificial sequence. In some embodiments, the hinge is a short oligopeptide linker comprising a glycine-serine doublet.

[0385] In some embodiments, the hinge is a naturally occurring sequence. In some embodiments, the hinge can be a human Ig (immunoglobulin) hinge or a fragment thereof. In some embodiments, for example, the hinge comprises (e.g., consists of) the amino acid sequence of an IgG4 hinge (SEQ ID NO: 3). In some embodiments, for example, the hinge comprises (e.g., consists of) the amino acid sequence of an IgD hinge (SEQ ID NO: 4). In some embodiments, the hinge can be a human CD8 hinge or a fragment thereof. In some embodiments, for example, the hinge comprises (e.g., consists of) the amino acid sequence of a CD8 hinge (SEQ ID NO: 2). Additional example sequences for hinge domains are provided in Table 5.

[0386] TCARS In some embodiments, the CAR cell therapy of the present invention comprises an NKR-CAR in combination with a TCAR. In some embodiments, the CAR cell therapy of the present invention comprises an NKR-CAR-expressing cell described herein further comprising, e.g., expressing, a TCAR. In another embodiment, the CAR cell therapy of the present invention comprises a first cell expressing an NKR-CAR and a second cell expressing a TCAR described herein.

[0387] In some embodiments, a TCAR comprises an antigen-binding domain fused to an intracellular domain, e.g., a cytoplasmic domain. In some embodiments, the cytoplasmic domain comprises an intracellular signaling domain, which generates an intracellular signal when the extracellular domain, e.g., the antigen-binding domain, to which it is fused, binds to a counterligand. The intracellular signaling domain may comprise a primary intracellular signaling domain and a costimulatory signaling domain. In some embodiments, a TCAR molecule may be constructed for expression in immune effector cells, e.g., T cells or NK cells, such that the TCAR molecule comprises domains derived from polypeptides commonly associated with immune cells, e.g., a primary intracellular signaling domain, a costimulatory signaling domain, an inhibitory domain, etc. For example, a TCAR for expression in immune effector cells, e.g., T cells or NK cells, may comprise a 4-1BB domain and a CD3 zeta domain. In this case, both the 4-1BB and CD3 zeta domains are derived from polypeptides associated with immune effector cells, e.g., T cells or NK cells. In other embodiments, TCAR molecules can be constructed for expression in immune effector cells, such as T cells or NK cells, such that the TCAR molecule contains domains derived from polypeptides not typically associated with immune effector cells. Alternatively, a TCAR for expression in NK cells can contain the 4-1BB domain and CD3 zeta domain from T cells (see, e.g., WO2013 / 033626, incorporated herein by reference).

[0388] The next section relates to the construction and expression of the NKR-CARs and TCARs described herein and methods of their use.

[0389] antigen-binding domain The CARs described herein, e.g., the KIR-CARs and TCARs described herein, comprise an antigen-binding domain in the extracellular region. As used herein, the term "antigen-binding domain" refers to a molecule that has affinity for a target antigen, generally an antigen on a target cell, e.g., a cancer cell. Examples of antigen-binding domains include polypeptides, e.g., antibody molecules (including antibodies and antigen-binding fragments thereof, e.g., immunoglobulins, single-domain antibodies (sdAbs), and scFvs), or non-antibody scaffolds, e.g., fibronectin. In some embodiments, the antigen-binding domain is a single polypeptide. In some embodiments, the antigen-binding domain comprises one, two, or more polypeptides.

[0390] The selection of the antigen-binding domain depends on the type and number of ligands or receptors that define the target cell surface. For example, the antigen-binding domain can be selected to recognize a ligand or receptor that serves as a cell surface marker for target cells associated with a particular disease state. Examples of cell surface markers that can serve as ligands or receptors include cell surface markers associated with particular disease states, such as viral diseases, bacterial diseases, parasitic infections, autoimmune diseases, and disorders associated with unwanted cell proliferation, such as cancer, for example, the cancers described herein.

[0391] In the context of this disclosure, a "tumor antigen" or "proliferative disorder antigen" or "antigen associated with a proliferative disorder" refers to an antigen that is common to a particular proliferative disorder. In certain aspects, the proliferative disorder antigens of the present invention are derived from cancers including, but not limited to, primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer (e.g., NSCLC or SCLC), liver cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia, multiple myeloma, glioblastoma, neuroblastoma, uterine cancer, cervical cancer, renal cancer, thyroid cancer, bladder cancer, kidney cancer and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colon cancer, and the like. In some embodiments, the cancer is one or more chronic leukemias, including but not limited to B-cell acute lymphoid leukemia ("BALL"), T-cell acute lymphoid leukemia ("TALL"), acute lymphoid leukemia (ALL), acute myeloid leukemia (AML); chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL); B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma. , hairy cell leukemia, small cell- or large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndromes, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's hypergammaglobulinemia, and additional blood cancers or hematological conditions, including, but not limited to, idiopathic leukemia ...

[0392] In some embodiments, the tumor antigen comprises one or more antigenic cancer epitopes that are immunologically recognized by tumor-infiltrating lymphocytes (TILs) derived from a mammalian cancer tumor.

[0393] Tumor antigens are proteins produced by tumor cells that provoke an immune response, particularly a T cell-mediated immune response. The selection of the antigen-binding domain of the present invention depends on the specific type of cancer to be treated. Tumor antigens are well known in the art and include, for example, the tumor antigens described in International Application No. PCT / US2015 / 020606. In some embodiments, the tumor antigen is a glioma-associated antigen, carcinoembryonic antigen (CEA), EGFRvIII, interleukin-11 receptor alpha (IL-11Ra), interleukin-13 receptor subunit alpha-2 (IL-13Ra or CD213A2), epidermal growth factor receptor (EGFR), B7H3 (CD276), Kit (CD117), carbonic anhydrase (CA-IX), CS-1 (also known as CD2 subset 1), mucin 1, cell surface associated (MUC1), BCMA, the oncogene fusion protein bcr-abl consisting of a cleavage-prone region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl), receptor tyrosine-protein kinase ERBB2 (HER2 / neu), β-human chorionic gonadotropin, alpha-fetoprotein (AFP), anaplastic lymphoma kinase (A LK), CD19, CD123, cyclin B1, lectin-reactive AFP, Fos-related antigen 1, adrenoceptor beta 3 (ADRB3), thyroglobulin, tyrosinase; ephrin type A receptor 2 (EphA2), receptor for advanced glycation end products (RAGE-1), renal ubiquitous 1 (RU1), renal ubiquitous 2 (RU2), synovial sarcoma, X-breakpoint 2 (SSX2), A kinase anchoring protein 4 (AKAP-4), lymphocyte-specific protein tyrosine kinase (LCK), proacrosin-binding protein p32 (OY-TES1), paired box protein Pax-5 (PAX5), squamous cell carcinoma antigen 3 recognized by T cells (SART3), C-type lectin-like molecule-1 (CLL-1 or CLECL1), fucosyl GM1, hexasaccharide moiety of globoH glycoceramide (GloboH), MN-CA IX, epithelial cell adhesion molecule (EPCAM), EVT6-AML, transglutaminase 5 (TGS5), human telomerase reverse transcriptase (hTERT), polysialic acid, placenta-specific 1 (PLAC1), intestinal carboxylesterase, Lewis Y antigen, sialyl Lewis adhesion molecule (sLe), lymphocyte antigen 6 complex, locus K9 (LY6K), heat shock protein 70-2 mutant (mut hsp70-2), M-CSF, v-myc avian myelomatous myeloablative viral oncogene neuroblastoma-derived homolog (MYCN), Ras homolog family member C (RhoC), tyrosinase-related protein 2 (TRP-2), cytochrome P450 1B1 (CYP1B1), CCCTC-binding factor (zinc finger protein)-like (BORIS or Brother of the regulator of imprinted sites), prostase, prostate-specific antigen (PSA), paired box protein Pax-3 (PAX3), prostatic acid phosphatase (PAP), cancer / testis antigen 1 (NY-ESO-1), cancer / testis antigen 2 (LAGE-1a), LMP2, neural cell adhesion molecule (NCAM), tumor protein p53 (p53), p53 mutant, rat sarcoma (Ras) mutant, glycoprotein 100 (gp100), prostein, OR51E2, pannexin 3 (PANX3), prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), high-molecular-weight melanoma-associated antigen (HMWMAA), hepatitis A virus cellular receptor 1 (HAVCR1), vascular endothelial growth factor receptor 2 (VEGFR2), platelet-derived growth factor receptor beta (PDGFR-beta), legumain, human papilloma virus E6 (HPV E6), human papilloma virus E7 (HPV E7), survivin, telomerase, sperm protein 17 (SPA17), stage-specific embryonic antigen-4 (SSEA-4), tyrosinase, TCR gamma alternate reading frame protein (TARP), Wilms tumor protein (WT1), prostate-cancer tumor antigen-1 (PCTA-1), melanoma inhibitor of apoptosis (ML-IAP), MAGE, melanoma-associated antigen 1 (MAGE-A1), melanoma cancer-testis antigen-1 (MAD-CT-1), melanoma cancer-testis antigen-2 (MAD-CT-2), melanoma antigen 1 recognized by T cells (MelanA / MART1), X antigen family, member 1A (XAGE1), elongation factor 2 mutated (ELF2M), ERG (TMPRSS2)ETS fusion gene), N-acetylglucosaminyltransferase V (NA17), neutrophil elastase, sarcoma translocation breakpoint, mammary differentiation antigen (NY-BR-1), ephrin B2, CD20, CD22, CD24, CD30, CD33, CD38, CD44v6, CD97, CD171, CD179a, androgen receptor, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, ganglioside GD2 (GD2), o-acetyl-GD2 ganglioside (OAcGD2), ganglioside GD3 (aNeu5Ac(2-8) aNeu5Ac(2-3) bDGalp(1-4)bDGlcp(1-1)Cer), ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer), G protein-coupled receptor class C group 5, member D (GPRC5D), G protein-coupled receptor 20 (GPR20), chromosome X open reading frame 61 (CXORF61), folate receptor (FRa), folate receptor beta, receptor tyrosine kinase-like orphan receptor 1 (ROR1), Fms-like tyrosine kinase 3 (Flt3), tumor-associated glycoprotein 72 (TAG72), Tn antigen (TN Ag or (GalNAcα-Ser / Thr)), angiopoietin-binding cell surface receptor 2 (Tie2), tumor endothelial marker 1 (TEM1 or CD248), tumor endothelial marker 7-related (TEM7R), claudin 6 (CLDN6), thyroid-stimulating hormone receptor (TSHR), uroplakin 2 (UPK2), mesothelin, protease serine 21 (Testisin or PRSS21), epidermal growth factor receptor (EGFR), fibroblast activation protein alpha (FAP), olfactory receptor 51E2 (OR51E2), ETS translocation variant gene 6, located on chromosome 12p (ETV6-AML), CD79a;CD79b;CD72; leukocyte-associated immunoglobulin-like receptor 1 ( LAIR1); Fc fragment of IgA receptor (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1). In a preferred embodiment, the tumor antigen is selected from the group consisting of folate receptor (FRa), mesothelin, EGFRvIII, IL-13Ra, CD123, CD19, CD33, BCMA, GD2, CLL-1, CA-IX, MUC1, HER2, and any combination thereof.

[0394] In some embodiments, the tumor antigen comprises one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express numerous proteins that can serve as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase, and GP100 in melanoma and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target antigens include transformation-related molecules such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens includes carcinoembryonic antigens such as carcinoembryonic antigen (CEA). In B-cell lymphomas, tumor-specific idiotypic immunoglobulins consist of true tumor-specific immunoglobulin antigens unique to an individual's tumor. B-cell differentiation antigens such as CD19, CD20, and CD37 are other candidate target antigens in B-cell lymphomas.

[0395] Non-limiting examples of tumor antigens include: differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, and p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, and HER-2 / neu; unique tumor antigens resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, and MYL-RAR; and viral antigens such as the Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 / CA 27.29 / BCAA, CA 195, CA 242, CA-50, CAM43, CD68 / P1, CO-029, FGF-5, G250, Ga733 / EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 / Mac-2 binding protein / cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0396] Depending on the desired antigen to be targeted, the CAR of the present invention can be engineered to contain an appropriate antigen-binding domain specific for the desired antigen target.

[0397] Antigen-binding domains derived from antibody molecules The antigen-binding domain can be derived from an antibody molecule, such as one or more monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, single-domain antibodies, for example, heavy chain variable domains (VH), light chain variable domains (VL), and variable domains (VHH), of human or camelid origin. In some instances, it is beneficial for the antigen-binding domain to be derived from the same species as the CAR will ultimately be used in; for example, for human use, it may be beneficial for the antigen-binding domain of a CAR, such as a KIR-CAR described herein, to comprise a human or humanized antigen-binding domain. Antibodies can be obtained using techniques known in the art.

[0398] In one aspect, the scFv is contiguous with and in the same reading frame as the leader sequence, hi one aspect, the leader sequence is the amino acid sequence provided as SEQ ID NO:1.

[0399] In one aspect, the antigen binding domain is a fragment, e.g., a single-chain variable fragment (scFv). In one aspect, the antigen binding domain is an Fv, Fab, (Fab')2, or a bifunctional (e.g., bispecific) hybrid antibody (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)). In one aspect, the antibodies and fragments thereof of the present invention bind to a tumor antigen protein or fragment thereof with wild-type or enhanced affinity.

[0400] In certain instances, scFvs can be produced by methods known in the art (see, e.g., Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). As described above and elsewhere, ScFv molecules can be produced by linking VH and VL domains together using a flexible polypeptide linker. The scFv molecule contains a linker (e.g., a Ser-Gly linker) with optimal length and / or amino acid composition. The length of the flexible polypeptide linker can greatly affect how the variable regions of an scFv fold and interact. In fact, if a short polypeptide linker is used (e.g., 5-10 amino acids), intrachain folding is prevented. Intrachain folding is also required for the two variable regions to join together to form a functional epitope-binding site. For examples of linker orientations and sizes, see, e.g., Hollinger et al. 1993 Proc Natl Acad. Sci. USA 90:...

Claims

1. An isolated nucleic acid molecule encoding a natural killer cell immune function receptor-chimeric antigen receptor (NKR-CAR), wherein the encoded NKR-CAR is A non-mouse antigen-binding domain that binds extracellular mesothelin and A transmembrane domain, e.g., a NKR transmembrane domain; or Cytoplasmic domains, e.g., NKR cytoplasmic domains including one or both of the following: An isolated nucleic acid molecule.

2. 2. The isolated nucleic acid molecule of claim 1, wherein the encoded NKR-CAR comprises an antigen-binding domain that binds extracellular non-mouse mesothelin, a transmembrane domain, and an NKR cytoplasmic domain.

3. 3. The isolated nucleic acid molecule of claim 1 or 2, wherein the antigen-binding domain that binds extracellular non-mouse mesothelin is an antigen-binding domain that binds human or humanized mesothelin.

4. 4. The isolated nucleic acid molecule of claim 3, wherein the encoded human antigen-binding domain that binds to mesothelin comprises heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3) of any of the human anti-mesothelin heavy chain amino acid sequences listed in Table 4; and / or light chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2), and light chain complementarity determining region 3 (LC CDR3) of any of the human anti-mesothelin light chain amino acid sequences listed in Table 4.

5. A human antigen-binding domain that binds to mesothelin: i) any of the amino acid sequences of the human anti-mesothelin heavy chain variable regions listed in Table 4; ii) an amino acid sequence having at least one, two or three modifications, but not more than 30, 20 or 10 modifications, in any of the amino acid sequences of a human anti-mesothelin heavy chain variable region listed in Table 4; or iii) an amino acid sequence having 95 to 99% identity with any of the amino acid sequences of the human anti-mesothelin heavy chain variable regions listed in Table 4; a heavy chain variable region comprising: and / or i) any of the amino acid sequences of the human anti-mesothelin light chain variable regions listed in Table 4; ii) an amino acid sequence having at least one, two or three modifications, but not more than 30, 20 or 10 modifications, in any of the amino acid sequences of a human anti-mesothelin light chain variable region listed in Table 4; or iii) an amino acid sequence having 95 to 99% identity with any of the amino acid sequences of the human anti-mesothelin light chain variable regions listed in Table 4; A light chain variable region comprising 5. The isolated nucleic acid molecule of claim 3 or 4, comprising:

6. The human antigen-binding domain that binds to mesothelin i) any of the amino acid sequences of SEQ ID NOs: 234, 240, 230-233, 235-239 and 241-253; ii) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications in any of SEQ ID NOs: 234, 240, 230-233, 235-239 and 241-253; or iii) an amino acid sequence having 95 to 99% identity to any one of SEQ ID NOs: 234, 240, 230 to 233, 235 to 239, and 241 to 253; The isolated nucleic acid molecule according to any one of claims 3 to 5, comprising:

7. The encoded NKR-CAR is a killer cell immunoglobulin-like receptor chimeric antigen receptor (KIR-CAR), wherein the KIR-CAR comprises either or both of a transmembrane domain from a KIR (KIR transmembrane domain) or a cytoplasmic domain containing a functional signaling domain from a KIR (KIR cytoplasmic domain); a natural cytotoxicity receptor-chimeric antigen receptor (NCR-CAR), wherein the NCR-CAR comprises one or both of a transmembrane domain from NCR (NCR transmembrane domain) or a cytoplasmic domain containing a functional signaling domain from NCR (NCR cytoplasmic domain); a signaling lymphocyte activation molecule family chimeric antigen receptor (SLAMF-CAR), wherein the SLAMF-CAR comprises one or both of a transmembrane domain from SLAMF (SLAMF transmembrane domain) or a cytoplasmic domain containing a functional signaling domain from SLAMF (SLAMF cytoplasmic domain); An Fc receptor-chimeric antigen receptor (FcR-CAR), wherein the FcR-CAR comprises one or both of a transmembrane domain from an FcR selected from CD16 or CD64 or a cytoplasmic domain comprising a functional signaling domain from an FcR selected from CD16 or CD64; or A Ly49 receptor-chimeric antigen receptor (Ly49-CAR), wherein the Ly49-CAR comprises either a transmembrane domain from Ly49 (Ly49 transmembrane domain) or a cytoplasmic domain that comprises a functional signaling domain from Ly49 (Ly49 cytoplasmic domain), or both. The isolated nucleic acid molecule according to any one of claims 1 to 6, comprising:

8. 8. The isolated nucleic acid molecule of claim 7, wherein the KIR transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of KIR2DS2, KIR2DL3, KIR2DL1, KIR2DL2, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DS1, KIR3DL2, KIR3DL3, KIR2DP1 and KIR3DP1.

9. 8. The isolated nucleic acid molecule of claim 7, wherein the KIR cytoplasmic domain comprises a functional signaling domain of a protein selected from the group consisting of KIR2DS2, KIR2DL3, KIR2DL1, KIR2DL2, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DS1, KIR3DL2, KIR3DL3, KIR2DP1 and KIR3DP1.

10. The isolated nucleic acid molecule of any one of claims 7 to 9, wherein the KIR-CAR further comprises one or more of a KIR D0 domain, a KIR D1 domain and / or a KIR D2 domain.

11. 8. The isolated nucleic acid molecule of claim 7, wherein the NCR transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of NKp46, NKp30 and NKp44.

12. 8. The isolated nucleic acid molecule of claim 7, wherein the NCR cytoplasmic domain comprises a functional signaling domain of a protein selected from the group consisting of NKp46, NKp30 and NKp44.

13. 8. The isolated nucleic acid molecule of claim 7, wherein the SLAMF transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of SLAM, CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME and CD2F-10.

14. 8. The isolated nucleic acid molecule of claim 7, wherein the SLAMF cytoplasmic domain comprises a functional signaling domain of a protein selected from the group consisting of SLAM, CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME and CD2F-10.

15. 8. The isolated nucleic acid molecule of claim 7, wherein the Ly49 transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of Ly49A, Ly49C, Ly49H and Ly49D.

16. 8. The isolated nucleic acid molecule of claim 7, wherein the Ly49 cytoplasmic domain comprises a functional signaling domain of a protein selected from the group consisting of Ly49A, Ly49C, Ly49H and Ly49D.

17. The isolated nucleic acid molecule of any one of claims 1 to 6, wherein the encoded transmembrane domain comprises an NKR transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of KIR2DS2, KIR2DL3, NKp46, killer cell immunoglobulin-like receptor (KIR), natural cytotoxicity receptor (NCR), signaling lymphocyte activation molecule family (SLAMF), Fc receptor (FcR) and Ly49 receptor (Ly49).

18. i) the encoded transmembrane domain comprises the amino acid sequence of SEQ ID NO: 357, 358 or 359; an amino acid sequence of SEQ ID NO: 357, 358 or 359 containing at least one, two or three modifications but not more than five modifications; or an amino acid sequence having 95-99% sequence identity to SEQ ID NO: 357, 358 or 359; or ii) comprising a nucleic acid sequence encoding a transmembrane domain comprising nucleotides 771-830 of SEQ ID NO: 343, nucleotides 773-832 of SEQ ID NO: 345, or nucleotides 803-875 of SEQ ID NO: 347, or a nucleic acid sequence having 95-99% sequence identity thereto, or a nucleic acid sequence having 95-99% sequence identity thereto; 18. An isolated nucleic acid molecule according to claim 17.

19. 19. The isolated nucleic acid molecule of any of claims 1-6 and 17-18, wherein the encoded cytoplasmic domain comprises an NKR cytoplasmic domain that comprises one or more functional signaling domains of a protein selected from the group consisting of KIR2DS2, KIR2DL3, NKp46, DAP12, KIR, NCR, SLAMF, FcR and Ly49.

20. i) the encoded cytoplasmic domain comprises the amino acid sequence of SEQ ID NO: 360, 361 or 362; an amino acid sequence of SEQ ID NO: 360, 361 or 362 that contains at least one, two or three modifications but not more than 20, 10 or 5 modifications; or an amino acid sequence having 95-99% sequence identity to SEQ ID NO: 360, 361 or 362; or ii) comprising a nucleic acid sequence encoding a cytoplasmic domain comprising nucleotides 831-947 of SEQ ID NO: 343, nucleotides 833-1060 of SEQ ID NO: 345, or nucleotides 876-949 of SEQ ID NO: 347, or a nucleic acid sequence having 95-99% sequence identity thereto; 20. The isolated nucleic acid molecule of claim 19.

21. 21. The isolated nucleic acid molecule of any one of claims 1 to 20, further comprising a leader sequence encoding the amino acid sequence of SEQ ID NO:

1.

22. 22. The isolated nucleic acid molecule of any one of claims 1 to 21, wherein an antigen-binding domain that binds extracellular non-mouse mesothelin is linked to the transmembrane domain by a hinge domain.

23. 23. The isolated nucleic acid molecule of claim 22, wherein the encoded hinge domain is selected from the group consisting of a CD8 hinge, a GS hinge, an IgG4 hinge, an IgD hinge, a KIR2DS2 hinge, a KIR hinge, an NCR hinge, a SLAMF hinge, an FcR hinge and an LY49 hinge.

24. i) the encoded hinge domain comprises the amino acid sequence of SEQ ID NO:5, 2, 3 or 4; an amino acid sequence having at least one, two or three modifications but not more than five modifications of the amino acid sequence of SEQ ID NO:5, 2, 3 or 4; or an amino acid sequence having 95-99% identity to the amino acid sequence of SEQ ID NO:5, 2, 3 or 4; or ii) the nucleic acid sequence encoding the hinge domain comprises the nucleic acid sequence of SEQ ID NO: 356, 16, 13, 14 or 15 or a nucleic acid sequence having 95-99% identity thereto; 24. The isolated nucleic acid molecule of claim 23.

25. i) the encoded transmembrane and cytoplasmic domains collectively the amino acid sequence of amino acids 413 to 487 of SEQ ID NO:333 or amino acids 386 to 454 of SEQ ID NO:335; an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications in amino acids 413-487 of SEQ ID NO:333 or amino acids 386-454 of SEQ ID NO:335; or or comprising an amino acid sequence having 95-99% identity to amino acids 413-487 of SEQ ID NO:333 or amino acids 386-454 of SEQ ID NO:335; or ii) the nucleic acid sequence encoding the transmembrane and cytoplasmic domains comprises nucleotides 1237-1464 of SEQ ID NO: 332 or nucleotides 1156-1365 of SEQ ID NO: 334 or a sequence having 95-99% identity thereto; 7. An isolated nucleic acid molecule according to claim 6.

26. 26. The isolated nucleic acid molecule of any one of claims 1 to 25, further comprising a nucleic acid sequence encoding an adapter molecule.

27. 27. The isolated nucleic acid molecule of claim 26, wherein the encoded adapter molecule comprises a functional signaling domain of DAP12 or Fc epsilon receptor gamma (FcεRγ).

28. i) the encoded adapter molecule has the amino acid sequence of amino acids 1 to 113 of SEQ ID NO:333 or amino acids 1 to 86 of SEQ ID NO:335; an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications in amino acids 1 to 113 of SEQ ID NO:333 or amino acids 1 to 86 of SEQ ID NO:335; or or comprising an amino acid sequence having 95-99% identity to amino acids 1-113 of SEQ ID NO:333 or amino acids 1-86 of SEQ ID NO:335; or ii) the nucleic acid sequence encoding the adapter molecule comprises nucleotides including nucleotides 1 to 339 of SEQ ID NO: 332 or nucleotides 1 to 258 of SEQ ID NO: 334; 28. An isolated nucleic acid molecule according to claim 26 or 27.

29. 29. The isolated nucleic acid molecule of any of claims 26 to 28, further comprising a nucleic acid sequence encoding a peptide cleavage site selected from the group consisting of T2A, P2A, E2A and F2A, wherein the nucleic acid encoding the peptide cleavage site links the nucleic acid sequence encoding the NKR-CAR to a nucleic acid sequence encoding an adapter molecule.

30. 30. The isolated nucleic acid molecule of claim 29, wherein the nucleic acid sequence encoding the peptide cleavage site encodes an amino acid sequence of SEQ ID NO: 57, 58, 59 or 60; or an amino acid sequence having 95-99% sequence identity thereto.

31. 31. The isolated nucleic acid molecule of any of claims 1 to 30, further comprising a nucleic acid sequence encoding a TCAR or a second NKR-CAR.

32. 32. The isolated nucleic acid molecule of claim 31, wherein the encoded TCAR or second NKR-CAR comprises an antigen-binding domain that binds to a target antigen that is not mesothelin.

33. A purified or non-naturally occurring polypeptide encoded by a nucleic acid molecule according to any one of claims 1 to 32.

34. A non-mouse antigen-binding domain that binds extracellular mesothelin and A transmembrane domain, e.g., a NKR transmembrane domain; or Cytoplasmic domains, e.g., NKR cytoplasmic domains Either or both 1. An isolated NKR-CAR polypeptide comprising:

35. 35. The isolated NKR-CAR polypeptide of claim 34, wherein the NKR-CAR comprises an antigen-binding domain that binds to extracellular non-mouse mesothelin; a transmembrane domain; and an NKR cytoplasmic domain.

36. The isolated NKR-CAR polypeptide of claim 33 or 34, wherein the antigen-binding domain that binds extracellular non-mouse mesothelin is an antigen-binding domain that binds human or humanized mesothelin.

37. 37. The isolated NKR-CAR polypeptide of claim 36, wherein the human antigen-binding domain that binds to mesothelin comprises heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3) of any of the human anti-mesothelin heavy chain amino acid sequences listed in Table 4; and / or light chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2), and light chain complementarity determining region 3 (LC CDR3) of any of the human anti-mesothelin light chain amino acid sequences listed in Table 4.

38. The human antigen-binding domain that binds to mesothelin i) any of the amino acid sequences of the human anti-mesothelin heavy chain variable regions listed in Table 4; ii) an amino acid sequence having at least one, two or three modifications, but not more than 30, 20 or 10 modifications, in any of the amino acid sequences of a human anti-mesothelin heavy chain variable region listed in Table 4; or iii) an amino acid sequence having 95 to 99% identity to any of the amino acid sequences of the human anti-mesothelin heavy chain variable regions listed in Table 4; a heavy chain variable region comprising: and / or i) any of the amino acid sequences of the human anti-mesothelin light chain variable regions listed in Table 4; ii) an amino acid sequence having at least one, two or three modifications, but not more than 30, 20 or 10 modifications, in any of the amino acid sequences of a human anti-mesothelin light chain variable region listed in Table 4; or iii) an amino acid sequence having 95 to 99% identity with any of the amino acid sequences of the human anti-mesothelin light chain variable regions listed in Table 4; A light chain variable region comprising 38. The isolated NKR-CAR polypeptide of claim 36 or 37, comprising:

39. The human antigen-binding domain that binds to mesothelin i) any of the amino acid sequences of SEQ ID NOs: 234, 240, 230-233, 235-239 and 241-253; ii) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications in any of SEQ ID NOs: 234, 240, 230-233, 235-239 and 241-253; or iii) an amino acid sequence having 95 to 99% identity to any one of SEQ ID NOs: 234, 240, 230 to 233, 235 to 239, and 241 to 253; The isolated NKR-CAR polypeptide according to any one of claims 36 to 38, comprising:

40. NKR-CAR a KIR-CAR, the KIR-CAR comprising either or both of a transmembrane domain from a KIR (KIR transmembrane domain) or a cytoplasmic domain comprising a functional signaling domain from a KIR (KIR cytoplasmic domain); an NCR-CAR, wherein the NCR-CAR comprises one or both of a transmembrane domain from an NCR (NCR transmembrane domain) or a cytoplasmic domain comprising a functional signaling domain from an NCR (NCR cytoplasmic domain); an SLAMF-CAR, wherein the SLAMF-CAR comprises one or both of a transmembrane domain from SLAMF (SLAMF transmembrane domain) or a cytoplasmic domain comprising a functional signaling domain from SLAMF (SLAMF cytoplasmic domain); an FcR-CAR, wherein the FcR-CAR comprises one or both of a transmembrane domain from an FcR selected from CD16 or CD64 or a cytoplasmic domain comprising a functional signaling domain from an FcR selected from CD16 or CD64; or Ly49-CAR, wherein the Ly49-CAR comprises one or both of a transmembrane domain from Ly49 (Ly49 transmembrane domain) and a cytoplasmic domain comprising a functional signaling domain from Ly49 (Ly49 cytoplasmic domain). The isolated NKR-CAR polypeptide of any one of claims 34 to 39, comprising:

41. 41. The isolated NKR-CAR polypeptide of claim 40, wherein the KIR transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of KIR2DS2, KIR2DL3, KIR2DL1, KIR2DL2, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DS1, KIR3DL2, KIR3DL3, KIR2DP1 and KIR3DP1.

42. 41. The isolated NKR-CAR polypeptide of claim 40, wherein the KIR cytoplasmic domain comprises a functional signaling domain of a protein selected from the group consisting of KIR2DS2, KIR2DL3, KIR2DL1, KIR2DL2, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DS1, KIR3DL2, KIR3DL3, KIR2DP1 and KIR3DP1.

43. The isolated NKR-CAR polypeptide of any of claims 40 to 42, wherein the KIR-CAR further comprises one or more of a KIR D0 domain, a KIR D1 domain and / or a KIR D2 domain.

44. 41. The isolated NKR-CAR polypeptide of claim 40, wherein the NCR transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of NKp46, NKp30 and NKp44.

45. 41. The isolated NKR-CAR polypeptide of claim 40, wherein the NCR cytoplasmic domain comprises a functional signaling domain of a protein selected from the group consisting of NKp46, NKp30 and NKp44.

46. 41. The isolated NKR-CAR polypeptide of claim 40, wherein the SLAMF transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of SLAM, CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME and CD2F-10.

47. 41. The isolated NKR-CAR polypeptide of claim 40, wherein the SLAMF cytoplasmic domain comprises a functional signaling domain of a protein selected from the group consisting of SLAM, CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME and CD2F-10.

48. 41. The isolated NKR-CAR polypeptide of claim 40, wherein the Ly49 transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of Ly49A, Ly49C, Ly49H and Ly49D.

49. 41. The isolated NKR-CAR polypeptide of claim 40, wherein the Ly49 cytoplasmic domain comprises a functional signaling domain of a protein selected from the group consisting of Ly49A, Ly49C, Ly49H and Ly49D.

50. 40. The isolated NKR-CAR polypeptide of any of claims 34 to 39, wherein the transmembrane domain comprises an NKR transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of KIR2DS2, KIR2DL3, NKp46, KIR, NCR, SLAMF, FcR and Ly49.

51. The transmembrane domain i) the amino acid sequence of SEQ ID NO: 357, 358 or 359; ii) an amino acid sequence containing at least one, two or three modifications but not more than five modifications of the amino acid sequence of SEQ ID NO: 357, 358 or 359; or iii) an amino acid sequence having 95-99% sequence identity to SEQ ID NO: 357, 358 or 359 51. The isolated NKR-CAR polypeptide of claim 50, comprising:

52. The isolated NKR-CAR polypeptide according to any one of claims 34 to 39 and 50 to 51, wherein the cytoplasmic domain comprises an NKR cytoplasmic domain comprising one or more functional signaling domains of a protein selected from the group consisting of KIR2DS2, KIR2DL3, NKp46, DAP12, KIR, NCR, SLAMF, FcR and Ly49.

53. The cytoplasmic domain i) the amino acid sequence of SEQ ID NO: 360, 361 or 362; ii) an amino acid sequence of SEQ ID NO: 360, 361 or 362 containing at least one, two or three modifications but not more than 20, 10 or 5 modifications; or iii) an amino acid sequence having 95-99% sequence identity to SEQ ID NO: 360, 361 or 362 53. The isolated NKR-CAR polypeptide of claim 52, comprising:

54. The isolated NKR-CAR polypeptide of any of claims 34 to 53, further comprising a leader sequence comprising the amino acid sequence of SEQ ID NO:

1.

55. The isolated NKR-CAR polypeptide of any one of claims 34 to 54, wherein an antigen-binding domain that binds to extracellular non-mouse mesothelin is linked to a transmembrane domain by a hinge domain.

56. 56. The isolated NKR-CAR polypeptide of claim 55, wherein the hinge domain is selected from the group consisting of a CD8 hinge, a GS hinge, an IgG4 hinge, an IgD hinge, a KIR2DS2 hinge, a KIR hinge, an NCR hinge, a SLAMF hinge, a CD16 hinge, a CD64 hinge, and a LY49 hinge.

57. The hinge domain i) the amino acid sequence of SEQ ID NO: 5, 2, 3 or 4; ii) an amino acid sequence having at least one, two or three modifications but not more than five modifications of the amino acid sequence of SEQ ID NO: 5, 2, 3 or 4; or iii) an amino acid sequence having 95 to 99% identity with the amino acid sequence of SEQ ID NO: 5, 2, 3 or 4 57. The isolated NKR-CAR polypeptide of claim 56, comprising:

58. The transmembrane and cytoplasmic domains collectively i) the amino acid sequence of amino acids 413 to 487 of SEQ ID NO: 333 or amino acids 386 to 454 of SEQ ID NO: 335; ii) an amino acid sequence having at least one, two or three modifications but not more than 30, 20 or 10 modifications in amino acids 413 to 487 of SEQ ID NO: 333 or amino acids 386 to 454 of SEQ ID NO: 335; or iii) an amino acid sequence having 95-99% identity to amino acids 413-487 of SEQ ID NO: 333 or amino acids 386-454 of SEQ ID NO: 335 40. The isolated NKR-CAR polypeptide of claim 39, comprising:

59. An NKR-CAR complex comprising an NKR-CAR encoded by the nucleic acid according to any one of claims 1 to 32, or the NKR-CAR according to any one of claims 33 to 58, and an adaptor molecule.

60. The NKR-CAR complex of claim 59, wherein the adaptor molecule is hsDAP12 or FcεRγ.

61. The NKR-CAR complex of claim 59 or 60, wherein the NKR-CAR interacts with an adaptor molecule by binding of the extracellular non-mouse antigen-binding domain of the NKR-CAR to mesothelin.

62. 33. The nucleic acid molecule according to any one of claims 1 to 32, which is a DNA molecule, an RNA molecule, such as an mRNA molecule, or a combination thereof.

63. A vector comprising the nucleic acid molecule according to any one of claims 1 to 32, which is a DNA vector or an RNA vector.

64. 64. The vector of claim 63, which is selected from the group consisting of a plasmid, a lentiviral vector, an adenoviral vector and a retroviral vector.

65. 65. The vector of claim 63 or 64, further comprising an EF-1 promoter comprising the sequence of SEQ ID NO:

11.

66. A cell, for example an immune effector cell, comprising a nucleic acid molecule according to any one of claims 1 to 32, an NKR-CAR polypeptide according to any one of claims 33 to 58, a vector according to any one of claims 63 to 65, or an NKR-CAR complex according to any one of claims 59 to 61.

67. 66. The cell of claim 65, which is a cytotoxic cell, e.g., a T cell or an NK cell.

68. A method for producing a cell, e.g. an immune effector cell, comprising introducing into an immune effector cell a nucleic acid molecule according to any one of claims 1 to 32 or a vector according to any one of claims 63 to 65.

69. A method for providing anti-tumor immunity in a mammal, comprising administering to the mammal an effective amount of a nucleic acid molecule according to any one of claims 1 to 32, a NKR-CAR polypeptide according to any one of claims 33 to 58, a vector according to any one of claims 63 to 65, or a cell comprising the NKR-CAR complex according to any one of claims 59 to 61.

70. A method for treating a mammal having a disease or disorder, comprising administering to the mammal an effective amount of a nucleic acid molecule according to any one of claims 1 to 32, an NKR-CAR polypeptide according to any one of claims 33 to 58, a vector according to any one of claims 63 to 65, or a population of cells, e.g., immune effector cells, comprising the NKR-CAR complex according to any one of claims 59 to 61.

71. 71. The method of claim 70, wherein the disease or disorder is associated with expression of mesothelin.

72. 72. The method of claim 70 or 71, wherein the disease or disorder is selected from the group consisting of mesothelioma, malignant pleural mesothelioma, non-small cell lung cancer, small cell lung cancer, squamous cell lung cancer, large cell lung cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, pancreatic metastases, ovarian cancer, colorectal cancer and bladder cancer or any combination thereof.

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