EA040145B1 - Targeting Cytotoxic Cells with Chimeric Receptors for Adoptive Immunotherapy - Google Patents

Description

Related Applications

This patent application claims priority of PCT/CN2014/086694, filed September 17, 2014, and PCT/CN2014/090578, filed November 7, 2014. The entire contents of each of these applications are incorporated herein by reference.

State of the art prior to the invention

Using gene transfer technologies, T cells can be genetically modified to stably express antibody binding domains on their surface that confer specificities to T cells that are independent of major histocompatibility complex (MHC) restrictions. Chimeric antigen receptors (CARs) are synthetic proteins expressed by T cells (CART cells) in which an antigen recognition antibody fragment (eg, scFv or single chain variable region fragment) is fused to the intracellular domain of the CD3 zeta chain. When interacting with a target cell expressing a cognate scFv antigen, CARs expressed by T cells can initiate T cell activation resulting in cytolysis of the target cell (also referred to as target cell lysis). When combined with additional costimulatory signals, such as the intracellular domain of CD137 or CD28, these receptors can also lead to proliferation. However, some of this proliferation appears to be antigen-independent, in contrast to normal T cell receptor (TCR) responses (Milone et al., 2009, Mol. Ther., 17(8):1453-64). Artificial receptors do not fully reproduce the intracellular signaling resulting from the binding of natural TCRs to an antigenic peptide in complex with MHC molecules (Brocker, 2000, Blood, 96(5):1999-2001). Defects in the signaling cascade may limit long-term survival of CART cells upon adoptive transfer in the absence of high levels of cytokines such as IL-2 (Lo et al., 2010, Clin. Cancer Res., 16(10):2769-80). They are also characterized by dysregulation, which may be beneficial in some applications against cancer (Loskog et al., 2006, Leukemia, 20(10):181928), but such dysregulation also led to possible problems in controlling their side activity against normal tissues. , which also express the antigen, even at very low levels. Such side effects are a major limitation for CAR-based therapeutics, and have resulted in the eventual death of CAR-modified T cells during early phase I evaluation (Morgan et al., 2010, Mol. Ther., 18(4):843-51 ).

Thus, there is a need in the art for alternative CAR design approaches that overcome the limitations of currently existing CAR-based therapeutics. The present invention addresses this unmet need in the art.

The essence of the invention

In a first aspect, the 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 an extracellular antigen-binding domain, a transmembrane domain, e.g., an NKR transmembrane domain, and a cytoplasmic domain, e.g., cytoplasmic domain of NKR. In one embodiment, the NKR-CAR polypeptide comprises an extracellular antigen-binding domain and one or both of: a transmembrane domain, eg, an NKR transmembrane domain, or a cytoplasmic domain, eg, a NKR cytoplasmic domain. In one embodiment, said NKR-CAR polypeptide comprises an extracellular antigen-binding domain; transmembrane domain and cytoplasmic domain of NKR. In one embodiment, the NKR-CAR polypeptide comprises an extracellular antigen-binding domain, an NKR transmembrane domain, and a cytoplasmic domain. In one embodiment, the NKRCAR polypeptide comprises an extracellular antigen binding domain, an NKR transmembrane domain, and a cytoplasmic domain.

In one embodiment, said NKR-CAR polypeptide comprises KIR-CAR, such as actKIR-CAR or inhKIR-CAR, NCR-CAR, such as actNCR-CAR, SLAMF-CAR, such as inhSLAMF-CAR, FcR-CAR, such as CD16-CAR , such as actCD16-CAR, or CD64-CAR, such as actCD64-CAR, or Ly49-CAR, such as actLy49-CAR or inhLy49-CAR.

In one embodiment, the NKR-CAR polypeptide comprises an immunoglobulin-like killer cell receptor-chimeric antigen receptor (KIR-CAR), wherein the KIR-CAR comprises one or both of a transmembrane domain from the KIR (KIR transmembrane domain) or a cytoplasmic domain containing a functional signaling domain from KIR (cytoplasmic domain of KIR). In one embodiment, 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 one embodiment, the cytoplasmic domain of KIR 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 one of the embodiments

- 1 040145

The KIR-CAR further comprises one or more of a KIR D0 domain, a KIR D1 domain, and/or a D2 domain

K.I.R.

In one embodiment, 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 NCR (NCR transmembrane domain) or a cytoplasmic domain containing a functional signaling domain from NCR (cytoplasmic domain of NCR). In one embodiment, the NCR transmembrane domain comprises the transmembrane domain of a protein selected from the group consisting of NKp46, NKp30 and NKp44. In one embodiment, where the cytoplasmic domain of the NCR contains a functional signaling domain of a protein selected from the group consisting of NKp46, NKp30 and NKp44.

In one embodiment, the NKR-CAR polypeptide comprises a chimeric antigen receptor of the lymphocyte-activating signaling molecule (SLAMF-CAR) family, 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). In one embodiment, the transmembrane domain of SLAMF 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 one embodiment, the cytoplasmic domain of SLAMF 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.

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 containing a functional signaling a domain from an FcR selected from CD16 or CD64.

In one embodiment, the NKR-CAR polypeptide comprises a Ly49 chimeric antigen receptor (Ly49-CAR), where Ly49-CAR contains one or both of a transmembrane domain from Ly49 (Ly49 transmembrane domain) or a cytoplasmic domain containing a functional signaling domain from Ly49 ( cytoplasmic domain of Ly49). In one embodiment, the transmembrane domain of Ly49 comprises a transmembrane domain of a protein selected from the group consisting of Ly49A, Ly49C, Ly49H, and Ly49D. In one embodiment, the cytoplasmic domain of Ly49 contains a functional signaling domain of a protein selected from the group consisting of Ly49A, Ly49C, Ly49H, and Ly49D.

In one embodiment, the transmembrane domain of the NKR-CAR polypeptide is an NKR transmembrane domain, wherein the NKR transmembrane domain comprises the transmembrane domain of a protein selected from the group consisting of KIR2DS2, KIR2DL3, NKp46, KIR, NCR, SLAMF, FcR, and Ly49. In one embodiment, the implementation of the transmembrane domain contains: 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 5 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.

In one embodiment, the encoded transmembrane domain of a NKRCAR polypeptide is a transmembrane domain, e.g., a TCAR transmembrane domain as described herein, comprising a transmembrane domain of a protein selected from the group consisting of 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, T cell receptor alpha, beta, or zeta chain, MHC class I molecule, TNF receptor protein , immunoglobulin-like protein, cytokine receptor, integrin, NK cell-activating receptor, BTLA, Toll-like 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, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7- H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, N Kp46, 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), 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 a ligand that specifically binds to CD83, or any combination thereof.

In one embodiment, the cytoplasmic domain of the NKR-CAR polypeptide is an NKR cytoplasmic domain containing one or more functional protein signaling domains selected from the group consisting of KIR2DS2, KIR2DL3, NKp46, KIR, NCR, SLAMF, FcR, and Ly49. In one embodiment, the cytoplasmic domain comprises: i) the amino acid sequence of SEQ ID NO: 360, 361, or 362; ii) amino acid sequence,

- 2 040145 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 NOs: 360, 361.

In one embodiment, the cytoplasmic domain of the NKR-CAR polypeptide contains an intracellular signaling domain or adapter molecule, such as DAP12. In one embodiment, the encoded cytoplasmic domain of the NKR-CAR polypeptide contains an adapter molecule, for example, DAP12 or FcεRγ. In one embodiment, the cytoplasmic domain comprises: i) an encoded adapter molecule comprising 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 containing at least one, two or three modifications, but not more than 20, 10 or 5 modifications of 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 with amino acids 1-113 of SEQ ID NO: 333 or amino acids 1-86 of SEQ ID NO: 335. a CD8 domain; and a cytoplasmic domain containing DAP12.

In one embodiment, the cytoplasmic domain of a NKR-CAR polypeptide is a cytoplasmic domain, e.g., TCAR, as described herein, containing one or more functional protein signaling domains selected from the group consisting of 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, T cell receptor alpha, beta or zeta chains, MHC class I molecules, TNF receptor protein, immunoglobulin-like protein, cytokine receptor, NK cell-activating integrin receptor, BTLA, Toll-like 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, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137) , B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, 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), 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 a ligand that specifically binds to CD83, or any combination thereof.

In one embodiment, the NKR-CAR polypeptide further comprises a leader sequence or a signal sequence, for example, containing the amino acid sequence of SEQ ID NO: 1.

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

In one embodiment, the transmembrane domain and the cytoplasmic domain together comprise: i) an 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 containing at least one, two or three modifications but not more than 30, 20 or 10 modifications 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 ; or iii) an amino acid sequence with 95-99% identity with 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 any of the above embodiments, the NKR-CAR is an activating NKR-CAR, and the extracellular antigen-binding domain is the antigen-binding domain described in the present invention, for example, in table. 4. In any of the above embodiments, the extracellular antigen binding domain is a non-mouse antigen binding domain that binds to mesothelin. In one embodiment, the non-mouse extracellular antigen binding domain that binds to mesothelin contains a human or humanized antigen binding domain that binds to mesothelin. In one embodiment, a human-owned or humanized antigen-binding domain that binds to mesothelin is provided in Table 1. 4.

In one embodiment, the human antigen binding domain that binds to mesothelin comprises: heavy chain complementarity determining region 1 (CDR1 HC), heavy chain complementarity determining region 2 (CDR2 HC), and heavy chain complementarity determining region 3 (CDR3 HC) of any amino acid heavy chain sequence of human antibodies against mesothelin given in table. 4; and/or light chain complementarity determining region 1 (CDR1 LC), light chain complementarity determining region 2 (CDR2 LC), and light chain complementarity determining region 3 (CDR3 LC) of any human anti-mesothelin antibody light chain amino acid sequence shown in Table 1. 4. In one embodiment, the human antigen-binding domain that binds to mesothelin comprises: a heavy chain variable region comprising: i) the amino acid sequence of any heavy chain variable region of a human anti-mesothelin antibody shown in Table 1. 4; ii) an amino acid sequence containing at least one, two or three modifications, but not more than 30, 20 or 10 modifications of the amino acid sequence of any heavy chain variable region of the human anti-mesothelin antibody shown in table. 4; or iii) an amino acid sequence with 95-99% identity with the amino acid sequence of any heavy chain variable region of the human anti-mesothelin antibody shown in Table. 4; and/or a light chain variable region comprising: i) the amino acid sequence of any light chain variable region of a human mesothelin antibody shown in Table 1. 4; ii) an amino acid sequence containing at least one, two or three modifications, but not more than 30, 20 or 10 modifications of the amino acid sequence of any light chain variable region of the human anti-mesothelin antibody shown in table. 4; or iii) an amino acid sequence with 95-99% identity with the amino acid sequence of any light chain variable region of a human anti-mesothelin antibody shown in Table. 4. In one embodiment, the human antigen-binding domain that binds to mesothelin comprises: i) the amino acid sequence of any of SEQ ID NOs: 230-253; ii) an amino acid sequence containing at least one, two or three modifications, but not more than 30, 20 or 10 modifications of any of SEQ ID NO: 230-253; or iii) an amino acid sequence with 95-99% identity with any of SEQ ID NOs: 230-253. In such embodiments, the NKR-CAR further comprises, for example, a transmembrane and cytoplasmic domain jointly containing, the amino acid sequence of SEQ ID NO: 371, an amino acid sequence containing 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 having at least 95-99% sequence identity with SEQ ID NO: 371.

In a first aspect, the 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 as described herein, for example, comprising one, two or all of an extracellular antigen-binding domain, a transmembrane domain, for example, the transmembrane domain of NKR, and the cytoplasmic domain, for example, the cytoplasmic domain of NKR. In one embodiment, the nucleic acid molecule encoding the NKR-CAR comprises an extracellular antigen-binding domain and one or both of a transmembrane domain, eg, the NKR transmembrane domain, or a cytoplasmic domain, eg, the NKR cytoplasmic domain. In one embodiment, the nucleic acid molecule encoding the NKR-CAR contains an extracellular antigen-binding domain; transmembrane domain and cytoplasmic domain of NKR. In one embodiment, the nucleic acid molecule encoding the NKR-CAR contains an extracellular antigen-binding domain, an NKR transmembrane domain, and a cytoplasmic domain. In one embodiment, the nucleic acid molecule encoding the NKR-CAR comprises an extracellular antigen-binding domain, an NKR transmembrane domain, and a cytoplasmic domain.

In one embodiment, the nucleic acid molecule encodes an NKR-CAR containing a KIR-CAR, such as actKIR-CAR or inhKIR-CAR, NCR-CAR, such as actNCR-CAR, SLAMFCAR, such as inhSLAMF-CAR, FcR-CAR, such as CD16 -CAR, such as actCD16-CAR, or CD64CAR, such as actCD64-CAR, or Ly49-CAR, such as actLy49-CAR or inhLy49-CAR.

In one embodiment, the encoded KIR-CAR comprises one or both of a transmembrane domain from KIR (transmembrane domain of KIR) or a cytoplasmic domain containing a functional signaling domain from KIR (cytoplasmic domain of KIR). In one embodiment, the KIR transmembrane domain comprises a protein transmembrane domain 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 one embodiment, the cytoplasmic domain of KIR contains 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 one embodiment, the KIR-CAR further comprises one or more of a KIR D0 domain, a KIR D1 domain, and/or a KIR D2 domain.

In one embodiment, the encoded 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). In one embodiment, the NCR transmembrane domain comprises the transmembrane domain of a protein selected from the group consisting of NKp46, NKp30 and NKp44. In one embodiment, the cytoplasmic domain of the NCR contains a functional signaling domain of a protein selected from the group consisting of NKp46, NKp30 and NKp44.

In one embodiment, the encoded 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). In one embodiment, the transmembrane domain of SLAMF 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 one embodiment, the cytoplasmic domain of SLAMF 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.

In one embodiment, the encoded FcR-CAR comprises one or both of a transmembrane domain from an FcR selected from CD16 or CD64 or a cytoplasmic domain containing a functional signaling domain from an FcR selected from CD16 or CD64.

In one embodiment, the encoded Ly49-CAR comprises one or both of a transmembrane domain from Ly49 (Ly49 transmembrane domain) or a cytoplasmic domain containing a functional signaling domain from Ly49 (Ly49 cytoplasmic domain). In one embodiment, the transmembrane domain of Ly49 comprises a transmembrane domain of a protein selected from the group consisting of Ly49A, Ly49C, Ly49H, and Ly49D. In one embodiment, the cytoplasmic domain of Ly49 contains a functional signaling domain of a protein selected from the group consisting of Ly49A, Ly49C, Ly49H, and Ly49D.

In one embodiment, the encoded NKR-CAR transmembrane domain is an NKR transmembrane domain, wherein the NKR transmembrane domain comprises a protein transmembrane domain selected from the group consisting of KIR2DS2, KIR2DL3, NKp46, KIR, NCR, SLAMF, FcR, and Ly49. In one embodiment, the encoded transmembrane domain of 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 5 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 one embodiment, the nucleic acid molecule encoding NKR-CAR comprises a nucleic acid sequence containing nucleotides 803-875 of SEQ ID NO: 347 , or a nucleic acid sequence with 95-99% sequence identity with it, which encodes a transmembrane domain.

In one embodiment, the encoded transmembrane domain of a NKRCAR polypeptide is a transmembrane domain, e.g., TCAR as described herein, comprising a transmembrane domain of a protein selected from the group consisting of 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, T cell receptor alpha, beta or zeta chains, MHC class I molecule, TNF receptor protein , immunoglobulin-like protein, cytokine receptor, integrin, NK cell-activating receptor, BTLA, Toll-like 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, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, 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), 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 a ligand that specifically binds to CD83, or any combination thereof.

In one embodiment, the encoded NKR-CAR cytoplasmic domain is an NKR cytoplasmic domain, wherein the NKR cytoplasmic domain contains one or more functional signaling domains of a protein selected from the group consisting of KIR2DS2,

- 5 040145

KIR2DL3, NKp46, KIR, NCR, SLAMF, FcR and Ly49. In one embodiment, 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 one embodiment, the nucleic acid molecule encoding 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 with 95-99% sequence identity thereto that encodes a cytoplasmic domain.

In one embodiment, the encoded cytoplasmic domain of the NKRCAR polypeptide contains an intracellular signaling domain or adapter molecule, such as DAP12. In one embodiment, the encoded cytoplasmic domain of the NKR-CAR polypeptide contains an adapter molecule, for example, DAP12 or FcεRγ. In one embodiment, the encoded cytoplasmic domain comprises: i) an encoded adapter molecule comprising 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 containing at least one, two or three modifications, but not more than 20, 10 or 5 modifications of 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 with amino acids 1-113 of SEQ ID NO: 333 or amino acids 1-86 of SEQ ID NO: 335. In one embodiment, the nucleic acid molecule encoding NKR-CAR comprises the nucleic acid sequence , containing nucleotides, contains nucleotides 1-339 of SEQ ID NO: 332 or nucleotides 1258 of SEQ ID NO: 334, or a nucleic acid sequence with 95-99% identity with it, which encodes an adapter molecule. In one embodiment, the NKR-CAR comprises an antigen-binding domain of the present invention, a transmembrane domain of CD8, and a cytoplasmic domain containing DAP12.

In one embodiment, the encoded cytoplasmic domain of a NKRCAR polypeptide is a cytoplasmic domain, e.g., TCAR, as described herein, containing one or more functional protein signaling domains selected from the group consisting of 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, T-cell receptor alpha, beta or zeta chain, MHC class I molecule, receptor TNF protein, immunoglobulin-like protein, cytokine receptor, integrin, NK cell-activating receptor, BTLA, Toll-like 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, CDS, ICAM-1, LFA-1 (CD11a/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp4 4, 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), 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 a ligand that specifically binds to CD83, or any combination thereof.

In one embodiment, the nucleic acid molecule encoding the NKR-CAR further comprises a leader sequence or a signal sequence that encodes the amino acid sequence of SEQ ID NO: 1.

In one embodiment, the encoded NKR-CAR comprises an extracellular antigen-binding domain linked to the transmembrane domain by a hinge domain. In one embodiment, the hinge domain is selected from the group consisting of GS hinge, CD8 hinge, IgG4 hinge, IgD hinge, KIR2DS2 hinge, KIR hinge, NCR hinge, SLAMF hinge, CD16 hinge, CD64 hinge, and LY49 hinge. In one embodiment, the implementation of the encoded hinge domain contains: i) the amino acid sequence of SEQ ID NO: 5, 2, 3 or 4; ii) an amino acid sequence containing at least one, two or three modifications, but not more than 5 modifications of the amino acid sequence of SEQ ID NO: 5, 2, 3 or 4; or iii) an amino acid sequence with 95-99% identity with 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 with 95-99% identity with it, which encodes a hinge domain.

In one embodiment, the encoded transmembrane domain and the encoded cyto

- 6 040145 plasma domain together contain: i) the amino acid sequence of amino acids 413487 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 containing at least one, two or three modifications but not more than 30, 20 or 10 modifications 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; or iii) an amino acid sequence with 95-99% identity with 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 one embodiment, a nucleic acid molecule encoding NKR- CAR contains a nucleic acid sequence containing nucleotides 1237-1464 of SEQ ID NO: 332 or nucleotides 1156-1365 of SEQ ID NO: 334, or a sequence with 95-99% identity with it, which encodes both a transmembrane and cytoplasmic domain.

In another aspect, the invention relates to a nucleic acid molecule, for example, a purified or non-natural nucleic acid, for example, a nucleic acid containing a DNA or RNA sequence, for example mRNA, containing a sequence encoding the NKR-CAR described in the present invention.

In one embodiment, 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 one embodiment, the encoded adapter molecule contains a functional DAP12 or FcεRγ signaling domain. In one embodiment, the encoded adapter molecule comprises an 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 containing at least one, two or three modifications, but not more than 20, 10 or 5 modifications of 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 with amino acids 1-113 of SEQ ID NO: 333 or amino acids 1-86 of SEQ ID NO: 335. In one embodiment, the nucleic acid molecule encoding NKR-CAR comprises a nucleic acid sequence encoding nucleotides 1-339 of SEQ ID NO: 332 or nucleotides 1-258 of SEQ ID NO: 334, or a nucleic acid sequence having 95-99% identity with it, which encodes an adapter molecule.

In one embodiment, the nucleic acid molecule further comprises a nucleic acid sequence encoding a TCAR or a second NKR-CAR. In one embodiment, the encoded TCAR or encoded second NKR-CAR contains an antigen-binding domain that binds to a target antigen that is not mesothelin.

In one embodiment, the nucleic acid molecule encoding the 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 binds a nucleic acid sequence acid encoding NKR-CAR with a second nucleic acid sequence, for example, a nucleic acid sequence encoding an adapter molecule. In one embodiment, the nucleic acid sequence encodes a peptoid 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 with it.

In one embodiment, the nucleic acid encodes an activating NKR-CAR, and the encoded extracellular antigen-binding domain is the antigen-binding domain described in the present invention, for example, in table. 4. In any of the above embodiments, the encoded extracellular antigen-binding domain is a non-mouse antigen-binding domain that binds to mesothelin. In one embodiment, the encoded non-mouse extracellular antigen binding domain that binds to mesothelin comprises a human or humanized antigen binding domain that binds to mesothelin. In one embodiment, a human-owned or humanized antigen-binding domain that binds to mesothelin is provided in Table 1. 4.

In one embodiment, the encoded human antigen-binding domain that binds to mesothelin comprises: a heavy chain complementarity determining region 1 (CDR1 HC), a heavy chain complementarity determining region 2 (CDR2 HC), and a heavy chain complementarity determining region 3 (CDR3 HC) of any the amino acid sequence of the heavy chain of human antibodies against mesothelin given in table. 4; and/or light chain complementarity determining region 1 (CDR1 LC), light chain complementarity determining region 2 (CDR2 LC), and light chain complementarity determining region 3 (CDR3 LC) of any human anti-mesothelin antibody light chain amino acid sequence shown in Table 1. 4. In one embodiment, the encoded human antigen binding domain that binds to mesothelin comprises: a heavy chain variable region comprising: i) the amino acid sequence of any heavy chain variable region of a human anti-mesote antibody

- 7 040145 line given in table. 4; ii) an amino acid sequence containing at least one, two or three modifications, but not more than 30, 20 or 10 modifications of the amino acid sequence of any heavy chain variable region of the human anti-mesothelin antibody shown in table. 4; or iii) an amino acid sequence with 95-99% identity with the amino acid sequence of any heavy chain variable region of the human anti-mesothelin antibody shown in Table. 4; and/or a light chain variable region comprising: i) the amino acid sequence of any light chain variable region of a human mesothelin antibody shown in Table 1. 4; ii) an amino acid sequence containing at least one, two or three modifications, but not more than 30, 20 or 10 modifications of the amino acid sequence of any light chain variable region of the human anti-mesothelin antibody shown in table. 4; or iii) an amino acid sequence with 95-99% identity with the amino acid sequence of any light chain variable region of a human anti-mesothelin antibody shown in Table. 4. In one embodiment, the encoded human antigen-binding domain that binds to mesothelin comprises: i) the amino acid sequence of any of SEQ ID NO: 230253; ii) an amino acid sequence containing at least one, two or three modifications, but not more than 30, 20 or 10 modifications of any of SEQ ID NO: 230-253; or iii) an amino acid sequence with 95-99% identity with any of SEQ ID NOs: 230-253. In such embodiments, the NKR-CAR further comprises, for example, a transmembrane and cytoplasmic domain, together containing, the amino acid sequence of SEQ ID NO: 371, an amino acid sequence containing 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 having at least 95-99% sequence identity with SEQ ID NO: 371.

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

In another aspect, the invention relates to a cell, for example, an effector cell of the immune system, for example, a cytotoxic cell, for example, a natural or non-natural T cell, an NK cell or a cytotoxic T cell line or NK cell line containing the NKR-CAR described in of the present invention, a nucleic acid molecule encoding an NKR-CAR of the present invention, a vector of the present invention, or an NKR-CAR complex of the present invention.

In one embodiment, the cytotoxic cell further comprises an adapter molecule or an intracellular signaling domain that interacts with said NKR-CAR.

In another aspect, the invention relates to a method for producing a cell described in the present invention, for example, an effector cell of the immune system, for example, a cytotoxic cell, for example, a natural or non-natural T cell, NK cell or cytotoxic T cell line or NK cell, containing the NKR-CAR described in the present invention, which includes introducing into the cytotoxic cell a nucleic acid, for example, an mRNA containing a sequence encoding the NKR-CAR described in the present invention. In one of the embodiments, the method further includes obtaining NKR-CAR, described in the present description, in a cytotoxic cell. In one embodiment, the method includes obtaining a population of cells described in the present invention, for example, a population of effector cells of the immune system containing NKR-CAR described in the present invention.

In another aspect, the invention relates to a method of treating an individual, for example, a method of providing antitumor immunity in a mammal, comprising administering to the mammal an effective amount of a cell described in the present invention, or a population of cells described in the present description, for example, a cytotoxic cell, for example, natural or non-natural T cells, NK cells, or cytotoxic T cell or NK cell lines containing the NKRCAR of the present invention.

In another aspect, the invention relates to a method of treating an individual suffering from a disease associated with the expression of a tumor antigen, e.g., a tumor antigen described herein, (e.g. antigen) comprising administering to an individual an effective amount of a cell containing an NKR-CAR, for example as described herein. In one embodiment, the NKR-CAR is an activating NKR-CAR and the extracellular antigen binding domain is

- 8 040145 is an antigen-binding domain described in the present invention, for example, in table. 4. In one embodiment, the method further comprises administering a cell containing a TCAR, for example, as described herein.

In one embodiment, the disease associated with the expression of the tumor antigen is a malignant tumor, for example, a malignant tumor described in the present description. In one embodiment, the cancer is a solid tumor, such as the solid tumor described herein.

In one embodiment, 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 ( for example, ductal adenocarcinoma of the pancreas), ovarian cancer, colorectal cancer, and bladder cancer, or any combination thereof. In one embodiment, the disease is pancreatic cancer, eg, metastatic pancreatic ductal adenocarcinoma (PDA), eg, in an individual who has experienced disease progression during at least one previous standard therapy. In one embodiment, the disease is a mesothelioma (eg, malignant pleural mesothelioma), for example, in an individual who has experienced disease progression during at least one previous standard therapy. In one embodiment, the disease is ovarian cancer, eg, malignant serous epithelial ovarian tumor, eg, in an individual who has experienced progression of the disease following at least one previous standard therapy regimen.

Additional features and embodiments of the above compositions and methods include one or more of the following.

In another aspect, the invention provides a purified or unnatural KIR-CAR comprising an extracellular antigen binding domain and a transmembrane domain, eg a KIR transmembrane domain, or a cytoplasmic domain, eg 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.

In one of the embodiments, the implementation of the specified transmembrane domain can interact, for example, contact the transmembrane domain of DAP12. In one embodiment, said transmembrane domain contains a positively charged group, eg an amino acid residue containing a positively charged group, eg a side chain. In one embodiment, said transmembrane domain comprises a KIR transmembrane domain.

In one embodiment, said KIR-CAR is an activating KIRCAR. In one embodiment, said KIR-CAR contains a KIR transmembrane domain. In one embodiment, said KIR-CAR is an inhibitory KIRCAR. In one embodiment, said KIR-CAR contains the cytoplasmic domain of KIR. In one embodiment, said KIR-CAR comprises an extracellular antigen binding domain and a transmembrane domain, e.g., a transmembrane domain containing a positively charged group, e.g., an amino acid residue containing a positively charged group, e.g., a side chain, or a transmembrane domain of the KIR.

In one embodiment, the implementation of the KIR-CAR described in the present invention contains an antigen-binding domain containing scFv. In one embodiment, said antigen-binding domain comprises a single VH domain, such as a single camel, shark, or lamprey VH domain, or a single VH domain derived from a human or mouse sequence, or a non-antibody backbone, such as fibronectin, such as an antibody-like type III fibronectin molecules. In one embodiment, said antigen-binding domain comprises a nanobody. In one embodiment, said antigen-binding domain comprises a camelid VHH domain.

In one embodiment, the KIR-CAR is an activating KIR-CAR, and the extracellular antigen-binding domain is the antigen-binding domain described in the present invention, for example, in table. 4.

In one embodiment, the implementation of the KIR-CAR described in the present invention contains an extracellular hinge domain. In one embodiment, the extracellular hinge domain is other than the KIR hinge domain, eg, other than the KIR2DS2 hinge domain. In one embodiment, the extracellular hinge domain is derived from a natural molecule. In one embodiment, the extracellular hinge domain is derived from a natural molecule other than KIR. In one embodiment, the extracellular hinge domain contains a non-natural polypeptide sequence. In one embodiment, the extracellular hinge domain comprises an extracellular hinge from human CD8 alpha. In one

- 9 040145 of the embodiments, the extracellular hinge domain contains a synthetic extracellular hinge. In one embodiment, the extracellular hinge domain is less than 50, 20, or 10 amino acids in length. In one embodiment, the extracellular hinge domain contains fewer amino acids than the KIR2DS2 hinge domain.

In one embodiment, the implementation of the KIR-CAR described in the present invention is actKIR-CAR. In one embodiment, said actKIR-CAR comprises a transmembrane domain containing a positively charged group, eg an amino acid residue containing a positively charged group, eg a positively charged side chain or an actKIR transmembrane domain. In one embodiment, said actKIR-CAR may interact with and promote signal transduction from an ITAM-containing polypeptide or adapter molecule. In one embodiment, said actKIR-CAR may interact with and promote signal transduction from a DAP12 polypeptide. In one embodiment, said actKIR-CAR contains the D domain of the KIR. In one embodiment, said actKIR-CAR contains the D1 domain of the KIR. In one embodiment, said actKIR-CAR contains a D2 KIR domain. In one embodiment, said actKIR-CAR does not contain the D domain of the KIR. In one embodiment, said actKIR-CAR contains the KIR2DS2 transmembrane domain. In one embodiment, said actKIR-CAR further comprises a cytoplasmic domain of KIR2DS2. In one embodiment, said actKIR-CAR does not contain the D domain of the KIR.

In one embodiment, the antigen-binding domain of the KIR-CAR described herein binds to an antigen presented on a target cell, such as a cancer cell. In one embodiment, said antigen-binding domain binds to an antigen that is more highly expressed on a target cell, such as a cancer cell, than on a non-target cell, such as a non-cancerous cell, such as a non-cancerous cell of the same type as target cell. In one embodiment, said antigen-binding domain binds to an antigen described herein, such as a tumor antigen as described herein. In one embodiment, the tumor antigen is expressed by a solid tumor, e.g., a solid tumor as 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 (eg, pancreatic ductal adenocarcinoma), ovarian cancer, colorectal cancer, and bladder cancer, or any combination thereof.

In one embodiment, the implementation of the KIR-CAR described in the present invention is an inhKIR-CAR. In one embodiment, the inhKIR-CAR contains the inhKIR transmembrane domain. In one embodiment, the inhKIR-CAR contains an ITIM-containing cytoplasmic domain, eg, an inhKIR cytoplasmic domain, eg, a KIR2DL or KIR3DL cytoplasmic domain. In one embodiment, the inhKIR-CAR comprises a transmembrane domain other than the KIR transmembrane domain, e.g., a transmembrane domain from PD-1 receptors, CTLA4 or ITIM-containing receptors from ILT (CD85), Siglec, LMIR (CD300), and/or the receptor family SLAM genes. In one embodiment, the inhKIR-CAR comprises a cytoplasmic domain from an inhibitory receptor other than the KIR receptor, e.g., from PD-1, CTLA4, or ITIM-containing receptors from ILT (CD85), Siglec, LMIR (CD300), and/or the receptor family SLAM genes. In one embodiment, the inhKIR-CAR comprises a transmembrane and cytoplasmic domain from an inhibitory receptor other than the KIR receptor, e.g., a transmembrane and cytoplasmic domain, independently, e.g., from PD-1 receptors, CTLA4 or ITIM containing receptors from ILT (CD85), Siglec , LMIR (CD300), and/or SLAM gene family receptors. In one embodiment, said cytoplasmic domain contains ITIM. In one embodiment, the inhKIR-CAR contains the D domain of the KIR. In one embodiment, the inhKIR-CAR contains the D0 domain of the KIR. In one embodiment, the inhKIR-CAR contains the KIR D1 domain. In one embodiment, the inhKIR-CAR contains the D2 domain of the KIR. In one embodiment, the inhKIR-CAR does not contain the D domain of the KIR.

In one embodiment, the antigen-binding domain of the inhKIR-CARs described herein binds to an antigen not present on a target cell, such as a cancer cell. In one embodiment, said antigen-binding domain binds to an antigen that is highly expressed on a non-target cell, such as a non-cancerous cell other than the target cell, such as a cancer cell, such as a cancer cell of the same type as the cell target. In one embodiment, said antigen-binding domain binds to desmoglein 1/3 (DSG1/3). In one embodiment, inhCAR, such as inhTCAR or inhNKR-CAR, such as inhKIRCAR, and actCAR, such as actTCAR or actNKR-CAR, such as actKIR-CAR, are provided, wherein the inhCAR contains an antigen binding domain that targets desmoglein 1/3 (DSG1 /3), and act- 10 040145

CAR contains an antigen binding domain that targets an antigen other than DSG1/3, such as EGFR. In one embodiment, the pair is used to treat an EGFR-expressing cancer, such as adenocarcinoma of the lung or colon. In one embodiment, cancer cells express less DSG1/3 than non-cancerous cells. In one embodiment, such a combination may minimize CAR-mediated attack on skin cells or squamous cells in the GI tract (ie, oral mucosa). In one embodiment, said antigen-binding domain binds to an aphrin receptor or claudin.

In another aspect, the invention relates to a nucleic acid, e.g., a purified or non-natural nucleic acid, e.g., a nucleic acid, containing a DNA or RNA sequence, e.g., an mRNA, containing (a) a sequence encoding a KIR-CAR, e.g., the first KIR-CAR described in the present invention. In one embodiment, said KIR-CAR, such as said first KIR-CAR, is an actKIR-CAR, such as actKIRCAR, as described in the present invention. In one embodiment, the nucleic acid encodes actKIR-CAR and the encoded extracellular antigen-binding domain is the antigen-binding domain described in the present invention, for example, in table. 4. In one embodiment, said KIR-CAR, such as said first KIR-CAR, is an inhKIR-CAR, such as an inhKIR-CAR, as described in the present invention.

In one embodiment, said nucleic acid contains a DNA sequence. In one embodiment, said nucleic acid contains an RNA sequence, such as an mRNA sequence.

In one embodiment, the implementation of the specified nucleic acid contains a sequence encoding KIR-CAR, such as actKIR-CAR, and a sequence encoding an inhibitory molecule containing the cytoplasmic domain inhKIR; a transmembrane domain, eg, a KIR transmembrane domain; and an inhibitory cytoplasmic domain, eg the ITIM domain, eg the inhKIR ITIM domain. In one embodiment, the inhibitory molecule is a natural inhKIR or a sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% homology to the natural inhKIR, or which differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 residues from native inhKIR.

In one embodiment, the implementation of the specified nucleic acid contains a sequence encoding KIR-CAR, such as actKIR-CAR, and a sequence encoding an inhibitory molecule containing the cytoplasmic domain of the SLAM family; a transmembrane domain, for example, a transmembrane domain of the SLAM family; and an inhibitory cytoplasmic domain, eg, the SLAM family domain, eg, the ITIM domain of the SLAM family. In one embodiment, the inhibitory molecule is a naturally occurring member of the SLAM family, or a sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% homology to a naturally occurring member of the SLAM family, or which differs by no more than, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 residues from a natural member of the SLAM family.

In one embodiment, said nucleic acid as described herein further comprises (b) a sequence encoding a second KIR-CAR as described herein, e.g., a second KIR-CAR that is different from said first KIR-CAR. In one embodiment, (a) and (b) are located on the same nucleic acid molecule, eg the same vector, eg the same viral vector, eg a lentiviral vector. In one embodiment, one of (a) and (b) is located on a first nucleic acid molecule, such as a first vector, such as a viral vector, such as a lentiviral vector, and the other is located on a second nucleic acid molecule, such as a second vector, eg a viral vector, eg a lentiviral vector. In one embodiment, said first KIR-CAR and said second KIR-CAR are actKIR-CAR. In one embodiment, the participation of one actKIR-CAR is insufficient to induce significant levels of activation. In one embodiment, the participation of the first and second actKIRCAR provides an additive or synergistic level of activation. In one embodiment, said first KIR-CAR and said second KIR-CAR are inhKIR-CAR. In one embodiment, one of said first KIR-CAR and said second KIRCAR is actKIR-CAR and the other is inhKIR-CAR. In one embodiment, said actKIR-CAR is an actKIR-CAR as described in the present invention. In one embodiment, said inhKIR-CAR is an inhKIRCAR as described in the present invention. In one embodiment, the implementation of the nucleic acid described in the present description contains actKIR-CAR described in the present invention, and inhKIR-CAR described in the present invention.

In one embodiment, the nucleic sequence further comprises (c) a sequence encoding an intracellular signaling domain, such as an adapter molecule that can produce an activating signal. In one of the embodiments, the

- 11 040145 The intracellular signaling domain contains the ITAM motif. In one embodiment, said sequence encodes a DAP 12 polypeptide comprising a DAP 12 intracellular signaling domain. In one embodiment, said DAP 12 polypeptide further comprises a transmembrane domain. In one embodiment, said DAP 12 polypeptide further comprises an extracellular domain. In one embodiment, each of (a), (b) and (c) is contained in the same nucleic acid molecule, eg a vector, eg a viral vector, eg a lentiviral vector. In one embodiment, one of (a), (b), and (c) is encoded in 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) encoded in a second nucleic acid molecule, eg a vector, eg a viral vector, eg a lentiviral vector. In one embodiment, (a) is contained in a first nucleic acid molecule, such as a vector, such as a viral vector, such as a lentiviral vector, and (b) and (c) is contained in a second nucleic acid molecule, such as a vector, such as a viral vector, such as a lentiviral vector. In another embodiment, (b) is contained in a first nucleic acid molecule, such as a vector, such as a viral vector, such as a lentiviral vector, and (a) and (c) are contained in a second nucleic acid molecule, such as a vector, such as a viral vector, for example, a lentiviral vector. In one embodiment, (c) is contained in a first nucleic acid molecule, such as a vector, such as a viral vector, such as a lentiviral vector, and (b) and (a) are contained in a second nucleic acid molecule, such as a vector, such as a viral vector, such as a lentiviral vector. In one embodiment, each of (a), (b) and (c) is contained in a different nucleic acid molecule, eg different vectors, eg viral vectors, eg lentiviral vectors.

In one embodiment, (i) the antigen-binding domain of one of said first KIR-CAR and said second KIR-CAR does not contain a light chain variable domain and a heavy chain variable domain, (ii) an antigen-binding domain of one of said first KIR-CAR and said second KIR-CAR is a scFv and the other is an antigen-binding domain other than scFv, (iii) when present on the cell surface, the antigen-binding domains of said first KIR-CAR and said second KIR-CAR interact with each other to a lesser extent than if would both be scFv antigen-binding domains, (iv) wherein, when present on the cell surface, the binding of the antigen-binding domain of said first KIR-CAR to its cognate antigen is not substantially reduced by the presence of said second KIR-CAR, (v) the antigen-binding domain of one of said of the first KIR-CAR and the specified second KIR-CAR contains a single VH domain, for example, a single VH domain ver dishes, sharks or lampreys, or a single VH domain derived from a human or mouse sequence, or a non-antibody backbone, e.g., fibronectin, e.g., a type III fibronectin antibody-like molecule, (vi) an antigen-binding domain of one of said first KIR-CAR and said second KIR -CAR is a scFv and the other contains a single VH domain, such as a single camel, shark, or lamprey VH domain, or a single VH domain derived from a human or mouse sequence, or a non-antibody backbone, such as fibronectin, such as an antibody-like type III fibronectin molecules, (vii) the antigen-binding domain of one of said first KIRCAR and said second KIR-CAR is scFv and the other contains a nanobody, or (viii) the antigen-binding domain of one of said first KIR-CAR and said second KIR-CAR is scFv and the other contains the camel VHH domain.

In one embodiment, the antigen binding domain of one of said first KIR-CAR and said second KIR-CAR does not contain a light chain variable domain and a heavy chain variable domain. In one embodiment, the antigen-binding domain of one of said first KIR-CAR and said second KIR-CAR is an scFv and the other is an antigen-binding domain other than scFv.

In one embodiment, when present on the cell surface, the antigen-binding domains of said first KIR-CAR and said second KIR-CAR interact with each other to a lesser extent than if both were scFv antigen-binding domains. In one embodiment, when present on the cell surface, the binding of the antigen-binding domain of said first KIR-CAR to its cognate antigen is not substantially reduced by the presence of said second KIR-CAR. In one embodiment, the antigen-binding domain of one of said first KIR-CAR and said second KIR-CAR comprises a single VH domain, such as a single camel, shark, or lamprey VH domain, or a single VH domain derived from a human or mouse sequence, or not a scaffold antibody, eg fibronectin, eg a type III fibronectin antibody-like molecule. In one embodiment, the antigen binding domain of one of said first KIR-CAR and said second KIR-CAR is scFv and the other contains a single VH domain, such as a single camel, shark, or lamprey VH domain, or a single VH domain derived from a human or mouse sequence, or a non-antibody backbone, eg fibronectin, eg, an antibody-like molecule of type III fibronectin. In one embodiment, the antigen-binding domain of one of said first KIR-CAR and said second KIR-CAR is scFv and the other contains a nanobody. In one embodiment, the antigen binding domain of one of said first KIR-CAR and said second KIR-CAR is scFv and the other contains a camelid VHH domain.

In one embodiment, the nucleic acid contains a sequence encoding a TCAR. In one embodiment, said TCAR comprises an antigen binding domain and an activating cytoplasmic domain from a T cell receptor complex with CD3, e.g. CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta. In one embodiment, said TCAR contains a costimulatory domain from a costimulatory receptor, such as CD28, CD137, CD27, ICOS, or OX40.

In one embodiment, (i) the antigen-binding domain of one of said KIRCAR and said TCAR does not contain a light chain variable domain and a heavy chain variable domain, (ii) the antigen-binding domain of one of said KIR-CAR and said TCAR is scFv and the other is is an antigen-binding domain other than scFv, (iii) when present on the cell surface, the antigen-binding domains of said KIR-CAR and said TCAR interact with each other to a lesser extent than if both were antigen-binding domains of scFv, (iv) when present on the cell surface, the binding of the antigen-binding domain of said KIR-CAR to its cognate antigen is not substantially reduced by the presence of said second TCAR, (v) the antigen-binding domain of one of said KIR-CAR and said TCAR contains a single VH domain, e.g. a single VH- camel, shark, or lamprey domain, or a single VH domain derived from a sequence of people sheep or mouse, or a non-antibody scaffold, e.g., fibronectin, e.g., a type III fibronectin antibody-like molecule, (vi) the antigen-binding domain of one of said KIR-CAR and said TCAR is scFv and the other contains a single VH domain, e.g. Camelid, shark, or lamprey VH domain, or a single VH domain derived from a human or mouse sequence, or a non-antibody backbone, e.g., fibronectin, e.g., a type III fibronectin antibody-like molecule, (vii) an antigen-binding domain of one of the specified KIR-CAR and said TCAR is cFv and the other contains a nanobody, or (viii), where, the antigen binding domain of one of said KIR-CAR and said TCAR is scFv and the other contains a camelid VHH domain. In one embodiment, the antigen binding domain of one of said KIR-CAR and said TCAR does not contain a light chain variable domain and a heavy chain variable domain.

In one embodiment, the antigen-binding domain of one of said KIR-CAR and said TCAR is scFv and the other is an antigen-binding domain other than scFv. In one embodiment, when present on the cell surface, the antigen-binding domains of said KIR-CAR and said TCAR interact with each other to a lesser extent than if both were scFv antigen-binding domains. In one embodiment, when present on the cell surface, the binding of the antigen-binding domain of said KIR-CAR to its cognate antigen is not substantially reduced by the presence of said second TCAR. In one embodiment, the antigen-binding domain of one of the specified KIR-CAR of the specified TCAR contains a single VH domain, for example, a single VH domain of a camel, shark or lamprey, or a single VH domain derived from a human or mouse sequence, or a framework not belonging to an antibody, for example, fibronectin, for example, an antibody-like molecule of type III fibronectin. In one embodiment, the antigen binding domain of one of said KIR-CAR and said TCAR is scFv and the other contains a single VH domain, such as a single camel, shark, or lamprey VH domain, or a single VH domain derived from a human or mouse sequence, or a non-antibody backbone, such as fibronectin, such as a type III fibronectin antibody-like molecule. In one embodiment, the antigen binding domain of one of said KIR-CAR and said TCAR is scFv and the other contains a nanobody. In one embodiment, the antigen binding domain of one of said KIR-CAR and said TCAR is scFv and the other contains a camelid VHH domain.

In one embodiment, the antigen-binding domain of one of said KIR-CAR and said 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 those the same stromal cells, eg FAP, a target other than mesothelin on prostate cancer cells, eg the androgen receptor, OR51E2, PSMA, PSCA, PDGRF-β, TARP, GloboH, MAD-CT-1 or MAD-CT-2; target other than mesothelin on ovarian cancer cells, eg Tn, PRSS21, CD171, Lewis Y, folate receptor α, claudin 6, GloboH, or sperm protein 17).

- 13 040145

In another aspect, the invention provides a cytotoxic cell, e.g., a natural or non-natural T cell, an NK cell, or a cytotoxic T cell, or an NK cell line, e.g., NK92, comprising (a) a first KIR-CAR as described herein . In one embodiment, said cytotoxic cell is a T cell. In one embodiment, said cytotoxic cell is an NK cell. In one embodiment, said cytotoxic cell is from an NK cell line, such as an NK92 cell. In one embodiment, said first KIR-CAR is an actKIR-CAR as described in the present invention. In one embodiment, said first KIR-CAR is an inhKIR-CAR as described in the present invention.

In one embodiment, the implementation of the specified cytotoxic cell contains KIR-CAR, such as actKIR-CAR, and an inhibitory molecule containing the cytoplasmic domain inhKIR; a transmembrane domain, eg the KIR transmembrane domain; and an inhibitory cytoplasmic domain, eg the ITIM domain, eg the inhKIR ITIM domain. In one embodiment, the inhibitory molecule is a native inhKIR, or a sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% homology to the native inhKIR, or which differs by no more than 1, 2 , 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 residues from natural inhKIR.

In one embodiment, the implementation of the specified cytotoxic cell contains KIR-CAR, such as actKIR-CAR, and an inhibitory molecule containing the cytoplasmic domain of the SLAM family; a transmembrane domain, eg the SLAM family transmembrane domain; and an inhibitory cytoplasmic domain, eg the SLAM family domain, eg the SLAM family ITIM domain. In one embodiment, the inhibitory molecule is a naturally occurring member of the SLAM family, or a sequence having at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% homology to a naturally occurring member of the SLAM family, or which differs by no more than, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 residues from a natural member of the SLAM family.

In one embodiment, the cytotoxic cell further comprises (b) a second KIR-CAR as described herein, eg a second KIR-CAR that is different from said first KIR-CAR. In one embodiment, one of said KIR-CAR and said second KIR-CAR is actKIR-CAR and the other is inhKIR-CAR. In one embodiment, said actKIR-CAR is an actKIR-CAR as described in the present invention. In one embodiment, one specified inhKIR-CAR is the inhKIR-CAR described in the present invention. In one embodiment, the implementation of the cytotoxic cell described in the present description contains actKIR-CAR, described in the present invention, and inhKIR-CAR, described in the present invention.

In one embodiment, the cytotoxic cell further comprises an intracellular signaling domain, eg, an adapter molecule that can produce an activating signal, eg that is exogenous to said cell that can produce an activating signal. In one embodiment, said intracellular signaling domain contains an ITAM motif. In one embodiment, said intracellular signaling domain comprises a DAP 12 polypeptide comprising a DAP 12 intracellular signaling domain. In one embodiment, said DAP 12 polypeptide further comprises a transmembrane domain. In one embodiment, said DAP 12 polypeptide further comprises an extracellular domain.

In one embodiment, the cytotoxic cell comprises a first and a second KIR-CAR of the present invention, wherein (i) the antigen-binding domain of one of said first KIR-CAR and said second KIR-CAR does not contain a light chain variable domain and a heavy chain variable domain , (ii) the antigen-binding domain of one of said first KIR-CAR and said second KIR-CAR is scFv, and the other is an antigen-binding domain other than scFv, (iii) when present on the cell surface, antigen-binding domains of said first KIR-CAR, and said second KIR-CAR interact with each other to a lesser extent than if both were scFv antigen-binding domains, (iv) when present on the cell surface, the binding of the antigen-binding domain of said first KIR-CAR to its cognate antigen is not substantially reduced as a result the presence of said second KIR-CAR, (v) an antigen-binding domain of one of said p The first KIR-CAR and said second KIR-CAR contain a single VH domain, e.g., a single camel, shark, or lamprey VH domain, or a single VH domain derived from a human or mouse sequence, or a non-antibody backbone, e.g., fibronectin, e.g. , an antibody-like molecule of type III fibronectin, (vi) the antigen-binding domain of one of said first KIR-CAR and said second KIR-CAR contains scFv, and the other contains a single VH domain, for example, a single camel, shark, or lamprey VH domain, or a single VH- a domain derived from a human or mouse sequence or a non-antibody backbone, e.g., fibronectin, e.g., a type III fibronectin antibody-like molecule, (vii), where, the antigen-binding domain of one of said first KIRCAR and said second KIR-CAR contains scFv, and the other contains a nanobody, or (viii) the antigen-binding domain of one of said first KIR-CAR and said second KIR-CAR contains sc Fv and the other contains the camel VHH domain.

In one embodiment, the antigen binding domain of one of said first KIR-CAR and said second KIR-CAR does not contain a light chain variable domain and a heavy chain variable domain. In one embodiment, the antigen-binding domain of one of said first KIR-CAR and said second KIR-CAR is scFv and the other is an antigen-binding domain other than scFv. In one embodiment, when present on the cell surface, the antigen-binding domains of said first KIR-CAR and said second KIR-CAR interact with each other to a lesser extent than if both were scFv antigen-binding domains. In one embodiment, when present on the cell surface, the binding of the antigen-binding domain of said first KIR-CAR to its cognate antigen is not substantially reduced by the presence of said second KIR-CAR. In one embodiment, the antigen binding domain of one of said first KIR-CAR and said second KIR-CAR comprises a single VH domain, such as a single camel, shark, or lamprey VH domain, or a single VH domain derived from a human or mouse sequence, or a non-antibody backbone, such as fibronectin, such as a type III fibronectin antibody-like molecule. In one embodiment, the antigen binding domain of one of said first KIR-CAR and said second KIR-CAR contains scFv and the other contains a single VH domain, such as a single camel, shark, or lamprey VH domain, or a single VH domain derived from the sequence human or mouse, or a non-antibody scaffold, eg fibronectin, eg a type III fibronectin antibody-like molecule. In one embodiment, the antigen-binding domain of one of said first KIR-CAR and said second KIR-CAR contains scFv and the other contains a nanobody. In one embodiment, the antigen binding domain of one of said first KIR-CAR and said second KIRCAR contains scFv and the other contains a camelid VHH domain.

In one embodiment, the antigen-binding domain of one of said first KIR-CAR and said second KIR-CAR 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 from mesothelin, on stromal cells, e.g. FAP; a different target from mesothelin on prostate cancer cells, e.g. androgen receptor, OR51E2, PSMA, PSCA, PDGRF-β, TARP, GloboH, MAD-CT-1 or MAD-CT -2; target other than mesothelin on ovarian cancer cells, eg Tn, PRSS21, CD171, Lewis Y, folate receptor α, claudin 6, GloboH, or sperm protein 17).

In one embodiment, the cytotoxic cell contains a KIR-CAR, as described herein, and additionally contains a TCAR. In one embodiment, said TCAR comprises an antigen-binding domain and a primary stimulatory domain. In one embodiment, said TCAR contains a costimulatory domain.

In one embodiment, the cytotoxic cell, such as a natural or non-natural T cell, NK cell, or a cytotoxic T cell or NK cell line, such as an NK92 cell, contains a nucleic acid as described herein, or a KIR-CAR encoded by the nucleic acid described in the present invention. In one embodiment, said cytotoxic cell is a T cell. In one embodiment, said cytotoxic cell is an NK cell. In one embodiment, said cytotoxic cell is from an NK cell line, such as NK92.

In another aspect, the invention relates to methods for producing a cell described in the present invention, including the introduction of a cytotoxic cell nucleic acid described in the present invention, in the specified cell. In one of the embodiments, the implementation of the specified method includes the formation in the cytotoxic cell KIR-CAR, described in the present description.

In another aspect, the invention relates to methods of treating a subject, for example, a method of providing anti-tumor immunity in a mammal, comprising administering to the mammal an effective amount of a cell described in the present invention. In one embodiment, said cell is autologous. In one embodiment, said cell is allogeneic. In one embodiment, the cell is a T cell, such as an autologous T cell. In one embodiment, the cell is an allogeneic T cell. In one embodiment, the cell is an NK cell, such as an autologous NK cell. In one embodiment, the cell is an allogeneic NK cell. In one embodiment, the cell is a cell from an NK cell line, such as NK92. In one embodiment, said mammal is a human. In one embodiment, the method further comprises evaluating said mammal, such as a human, for side effects of said treatment. In one embodiment, said side effect includes acute respiratory distress syndrome, febrile neutropenia, hypotension, encephalopathy, hepatic transaminitis, seizures, or macrophage activation syndrome. In one embodiment, the method further comprises treating said individual suffering from the side effect with an agent of the present invention, e.g., an anti-cytokine agent, e.g., a tumor necrosis factor antagonist, e.g. m, for example, an IL-6 receptor antagonist, such as an anti-IL6 receptor antibody, such as tocilizumab, or a corticosteroid. In one embodiment, the treatment comprises administering an anti-IL6 receptor antibody to said individual.

In one embodiment, the disease associated with the expression of the tumor antigen is a malignant tumor, for example, a malignant tumor described in the present description. In one embodiment, the cancer is a solid tumor, such as the solid tumor described herein, such as 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). lung cancer), pancreatic cancer (eg, pancreatic ductal adenocarcinoma), ovarian cancer, colorectal cancer, and bladder cancer, or any combination thereof. In one embodiment, the disease is pancreatic cancer, eg, metastatic pancreatic ductal adenocarcinoma (PDA), eg, in an individual who has experienced disease progression during at least one previous standard therapy. In one embodiment, the disease is a mesothelioma (eg, malignant pleural mesothelioma), for example, in an individual who has experienced disease progression during at least one previous standard therapy. In one embodiment, the disease is ovarian cancer, eg, malignant serous epithelial ovarian tumor, eg, in an individual who has progressed following at least one previous standard therapy regimen.

In one embodiment, the method includes treating a mammal, such as a human, suffering from a disease associated with the expression of mesothelin or CD19. In one embodiment, the method includes treating a mammal, such as a human, suffering from an unwanted cell proliferation disorder, such as a cancer. In one embodiment, said disorder is pancreatic carcinoma, mesothelioma, lung carcinoma, ovarian carcinoma, leukemia, or lymphoma.

In another aspect, the invention relates to a purified or non-natural NCR-CAR, e.g., an activating NCR-CAR, containing an extracellular antigen-binding domain, a transmembrane domain, e.g. a charged side chain or transmembrane domain of NCR, and a cytoplasmic domain, for example, the cytoplasmic domain of NCR.

In one embodiment, said NCR-CAR comprises a transmembrane domain containing a positively charged group, such as an amino acid residue containing a positively charged group, such as a positively charged side chain, such as an NCR transmembrane domain, such as a cytoplasmic domain of NKp30, NKp44, or NKp46. In one embodiment, said NCR-CAR contains a cytoplasmic domain that can interact with an adapter or intracellular signaling molecule containing, for example, a cytoplasmic domain of DAP12, FcRy, or CD3Z. In one embodiment, said NCR-CAR, eg NKp30-CAR, contains a transmembrane domain that can interact with an adapter or intracellular signaling molecule, eg DAP12. In one embodiment, said NCR-CAR contains a NKp46-CAR. In one embodiment, said NKp46-CAR contains a transmembrane domain containing a positively charged group, such as an amino acid residue containing a positively charged group, such as a positively charged side chain or, for example, an NCR transmembrane domain that can interact with an adapter molecule or an intracellular molecule. signaling, for example, a molecule containing the cytoplasmic domain of FcRy or CD3Z. In one embodiment, said NCR-CAR of the present invention further comprises a hinge domain located between said transmembrane domain and said extracellular antigen-binding domain.

In one embodiment, the NCR-CAR is an activating NCR-CAR, and the extracellular antigen-binding domain is the antigen-binding domain described in the present invention, for example, in table. 4.

In another aspect, the invention relates to a nucleic acid, for example, a purified or non-natural nucleic acid, for example, a nucleic acid containing a DNA or RNA sequence, for example, an mRNA, containing a sequence encoding an NCR-CAR described in

- 16 040145 of the present invention. In one embodiment, the nucleic acid contains a sequence encoding NKp30-CAR and optionally an adapter or intracellular signaling molecule, such as DAP12. In one embodiment, said NCR-CAR, e.g., NKp46-CAR, contains a transmembrane domain containing a positively charged group, e.g., an amino acid residue containing a positively charged group, e.g., a positively charged side chain, or an NCR transmembrane domain that can interact with an adapter molecule or an intracellular signaling molecule, such as an FcRy or CD3Z molecule. In one embodiment, the nucleic acid further comprises a sequence encoding an adapter or intracellular signaling molecule, which, for example, contains DAP12, FcRy, or CD3Z.

In one embodiment, the nucleic acid encodes an actNCR-CAR and the encoded extracellular antigen-binding domain is the antigen-binding domain described in the present invention, for example, in Table. 4.

In another aspect, the invention relates to a cytotoxic cell, for example, a natural or non-natural T cell, NK cell or a line of cytotoxic T cells or NK cells containing NCRCAR described in the present invention. In one embodiment, the cytotoxic cell further comprises an adapter or intracellular signaling molecule, which, for example, contains DAP12, an FcRy cytoplasmic domain, or CD3Z. In one embodiment, the cytotoxic cell contains NKp30-CAR and optionally an adapter or intracellular signaling molecule, such as DAP12. In one embodiment, said NKp46-CAR contains a transmembrane domain that can interact with an adapter or intracellular signaling molecule, such as an FcRy or CD3Z molecule.

In another aspect, the invention relates to a method for producing a cell described in the present invention, including introducing into a cytotoxic cell, a nucleic acid containing a sequence encoding an NCR-CAR described in the present invention. In one embodiment, the implementation of the method includes the formation in the cytotoxic cell NCR-CAR, described in the present description.

In another aspect, the invention relates to a method of treating an individual, for example, a method of providing antitumor immunity in a mammal, comprising administering to the mammal an effective amount of a cell described in the present invention, for example, a cell according to item described herein, containing an NCR-CAR described herein invention.

In another aspect, the invention relates to a method of treating an individual suffering from a disease associated with the expression of a tumor antigen, for example, a tumor antigen described in the present description (for example, a proliferative disease, a precancerous condition and a non-malignant tumor associated indication associated with the expression of a tumor antigen) comprising administering to an individual an effective amount of a cell containing an NCR-CAR, for example as described herein. In one embodiment, the NCR-CAR is an activating NCR-CAR, and the extracellular antigen-binding domain is the antigen-binding domain described in the present invention, for example, in table. 4.

In one embodiment, the disease associated with the expression of the tumor antigen is a malignant tumor, for example, a malignant tumor described in the present description. In one embodiment, the cancer is a solid tumor, such as the solid tumor described herein, such as 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). lung cancer), pancreatic cancer (eg, pancreatic ductal adenocarcinoma), ovarian cancer, colorectal cancer, and bladder cancer, or any combination thereof. In one embodiment, the disease is pancreatic cancer, eg, metastatic pancreatic ductal adenocarcinoma (PDA), eg, in an individual who has experienced disease progression during at least one previous standard therapy. In one embodiment, the disease is a mesothelioma (eg, malignant pleural mesothelioma), for example, in an individual who has experienced disease progression during at least one previous standard therapy. In one embodiment, the disease is ovarian cancer, eg, malignant serous epithelial ovarian tumor, eg, in an individual who has experienced progression of the disease following at least one previous standard therapy regimen.

In another aspect, the invention relates to a purified or non-natural SLAMF-CAR, e.g., an inhibitory SLAMF-CAR, comprising an extracellular antigen-binding domain, a transmembrane domain, e.g., a transmembrane domain, containing a positively charged group, e.g. a charged side chain, such as the SLAMF transmembrane domain, and a cytoplasmic

- 17 040145 SLAMF domain. In one embodiment, said SLAMF-CAR comprises the cytoplasmic domain of SLAMF, CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, or CD2F-10. In one embodiment, said SLAMF-CAR further comprises a hinge domain located between said transmembrane domain and said extracellular antigen-binding domain.

In another aspect, the invention relates to a nucleic acid, for example, a purified or non-natural nucleic acid, for example, a nucleic acid containing a DNA or RNA sequence, for example mRNA, containing a sequence encoding the SLAMF-CAR described in the present invention.

In another aspect, the invention relates to a cytotoxic cell, for example, a natural or non-natural T cell, NK cell or a line of cytotoxic T cells or NK cells containing the SLAMF-CAR described in the present invention.

In another aspect, the invention relates to a method for producing a cytotoxic cell containing the SLAMF-CAR described in the present invention, comprising introducing into the cytotoxic cell a nucleic acid containing a sequence encoding the SLAMF-CAR described in the present invention. In one of the embodiments, the implementation of the method includes the formation in the cytotoxic cell SLAMF-CAR, described in the present invention.

In another aspect, the invention relates to a method of treating a subject, for example, a method of providing antitumor immunity in a mammal, comprising administering to the mammal an effective amount of a cell containing the SLAMF-CAR described in the present invention.

In another aspect, the invention provides a purified or non-natural FcR-CAR, e.g. a CD16-CAR, e.g. an activating CD16-CAR or a CD64-CAR, for example an activating CD64CAR, comprising an extracellular antigen-binding domain, a transmembrane domain and a CD16 or CD64 cytoplasmic domain. In one embodiment, said FcR-CAR is a CD16-CAR. In one embodiment, said FcR-CAR is a CD64-CAR. In one embodiment, said FcR-CAR may interact with an adapter or intracellular signaling molecule, such as an FcRy or CD3Z domain, such as via a transmembrane domain, such as a transmembrane domain containing a positively charged group, such as an amino acid residue containing a positively charged group , such as a positively charged side chain or, for example, the transmembrane domain of CD16 or CD64. In one embodiment, said FcR-CAR further comprises a hinge domain located between said transmembrane domain and said extracellular antigen-binding domain.

In another aspect, the invention relates to a purified or non-natural nucleic acid, for example, a nucleic acid containing a DNA or RNA sequence, for example mRNA, containing a sequence encoding an FcR-CAR described in the present invention. In one embodiment, the nucleic acid further comprises an adapter molecule or intracellular signaling molecule containing a cytoplasmic activating domain, such as the cytoplasmic domain of FcRy or CD3Z. In one embodiment, said FcR-CAR and said cytoplasmic activating domain are located on separate nucleic acid molecules, eg separate vectors, eg separate viral vectors, eg separate lentiviral vectors.

In another aspect, the invention relates to a cytotoxic cell, for example, a natural or non-natural T cell, NK cell or cytotoxic T cell line or NK cell line containing the FcRCAR described in the present invention. In one embodiment, the cytotoxic cell further comprises a cytoplasmic activating domain, such as the cytoplasmic domain of FcRy or CD3Z.

In another aspect, the invention relates to a method for producing a cell containing an FcR-CAR described in the present invention, comprising introducing into a cytotoxic cell a nucleic acid containing a sequence encoding an FcR-CAR described in the present invention. In one embodiment, the implementation of the method includes the formation in the cytotoxic cell, FcR-CAR, described in the present invention.

In another aspect, the invention relates to a method of treating a subject, for example, a method of providing antitumor immunity in a mammal, comprising administering to the mammal an effective amount of a cell containing an FcR-CAR described in the present invention.

In another aspect, the invention provides a purified or unnatural Ly49-CAR comprising an extracellular antigen-binding domain and a transmembrane domain, eg, a Ly49 transmembrane domain, or a cytoplasmic domain, eg, an ITIM-containing cytoplasmic domain, eg, a Ly49 cytoplasmic domain. In one embodiment, Ly49-CAR contains a transmembrane domain and a Ly49 cytoplasmic domain. In one embodiment, said Ly49-CAR is an activating Ly49-CAR, such as Ly49D or Ly49H. In one

- 18 040145 of the embodiments, said Ly49-CAR contains a positively charged transmembrane domain, for example a positively charged transmembrane domain of Ly49. In one embodiment, said Ly49-CAR may interact with an ITAM-containing cytoplasmic domain, such as DAP 12. In one embodiment, said Ly49-CAR contains a Ly49 transmembrane domain. In one embodiment, said KIR-CAR is an inhibitory Ly49-CAR, such as Ly49A or Ly49C. In one embodiment, said Ly49-CAR contains an ITIM-containing cytoplasmic domain, eg, a Ly49 cytoplasmic domain. In one embodiment, said Ly49-CAR comprises a Ly49 transmembrane domain or a Ly49 cytoplasmic domain independently selected from Ly49A-Ly49W. In one embodiment, said Ly49-CAR further comprises a hinge domain located between said transmembrane domain and said extracellular antigen-binding domain.

In another aspect, the invention relates to a nucleic acid, for example, a purified or non-natural nucleic acid, for example, a nucleic acid containing a DNA or RNA sequence, for example mRNA, containing a sequence encoding the Ly49-CAR described in the present invention. In one embodiment, the nucleic acid further comprises a cytoplasmic activating domain, such as the cytoplasmic domain of DAP12. In one embodiment, said Ly49-CAR and said cytoplasmic activating domain are located on separate nucleic acid molecules, eg separate vectors, eg separate viral vectors, eg separate lentiviral vectors.

In another aspect, the invention relates to a cytotoxic cell, such as a natural or non-natural T cell, NK cell or line of cytotoxic T cells or NK cells containing Ly49CAR, described in the present description. In one embodiment, the cytotoxic cell further comprises a cytoplasmic activating domain, such as the cytoplasmic domain of DAP12.

In another aspect, the invention relates to a method for producing a cell containing the Ly49-CAR described in the present invention, including introducing into a cytotoxic cell, a nucleic acid containing a sequence encoding Ly49-CAR described in the present invention, in the specified cell.

In another aspect, the invention relates to a method for producing a cell containing Ly49-CAR, described in the present invention, including the formation in a cytotoxic cell Ly49-CAR, described in the present description.

In another aspect, the invention relates to a method of treating a subject, for example, a method of providing antitumor immunity in a mammal, comprising administering to the mammal an effective amount of a cell described in the present invention, for example, a Ly49-CAR containing cell described in the present invention.

In another aspect, the invention relates to a cell containing, for example, a cytotoxic cell containing a first non-natural chimeric membrane-immersed receptor containing an antigen-binding domain, and a second non-natural chimeric membrane-immersed receptor containing an antigen-binding domain, where, (i) the antigen-binding domain of one of said first and said second non-natural chimeric membrane-embedded receptor does not contain a light chain variable domain and a heavy chain variable domain, (ii) the antigen-binding domain of one of said first and said second non-natural chimeric membrane-embedded receptor contains scFv and the other is an antigen-binding domain , other than scFv, (iii) when present on the cell surface, the antigen-binding domains of said first and said second non-natural chimeric membrane-embedded receptor interact with each other to a lesser extent than if both were antig. non-binding domains of scFv, (iv) when present on the cell surface, the binding of the antigen-binding domain of said first non-natural chimeric membrane-immersed receptor to its cognate antigen is not substantially reduced by the presence of said second non-natural chimeric membrane-immersed receptor, (v) the antigen-binding domain of one of said first and said second non-natural chimeric membrane-embedded receptor contains a single VH domain, e.g. a single camel, shark or lamprey VH domain, or a single VH domain derived from a human or mouse sequence, or a backbone not belonging to an antibody, e.g. fibronectin, for example, an antibody-like molecule of type III fibronectin, (vi) the antigen-binding domain of one of said first and said second non-natural chimeric membrane-embedded receptor contains scFv, and the other contains a single VH domain, for example, a single camel, shark or lamprey VH domain, or a single VH domain derived from a human or mouse sequence or a non-antibody backbone, e.g., fibronectin, e.g., a type III fibronectin antibody-like molecule, (vii) the antigen-binding domain of one of said first and said second non-natural chimeric membrane-immersed receptor contains scFv and the other contains a nanobody, and (viii) an antigen-binding domain of one of said first and said second

- 19 040145 a non-natural chimeric membrane-embedded receptor contains scFv and the other contains a camelid VHH domain. In one embodiment, said cell is a T cell. In one embodiment, said cell is an NK cell. In one embodiment, said cell is from an NK cell line, such as NK92. In one embodiment, one of said first and said second unnatural chimeric membrane-embedded receptors is a TCAR. In one embodiment, both of said first and said second non-natural chimeric membrane-embedded receptors are CARs. In one embodiment, one of said first and said second unnatural chimeric membrane immersed receptors is NKR-CAR, eg KIR-CAR. In one embodiment, both of said first and said second unnatural chimeric membrane-embedded receptors are NKR-CAR, eg KIR-CAR. In one embodiment, the antigen-binding domain of one of said first and said second non-natural chimeric membrane-immersed receptor does not contain a light chain variable domain and a heavy chain variable domain. In one embodiment, when present on the cell surface, the antigen-binding domains of said first and said second non-natural chimeric membrane-embedded receptor interact with each other to a lesser extent than if both were scFv antigen-binding domains. In one embodiment, when present on the cell surface, the binding of the antigen-binding domain of said first non-natural chimeric membrane-immersed receptor to its cognate antigen is not substantially reduced by said second non-natural chimeric membrane-immersed receptor. In one embodiment, the antigen-binding domain of one of said first and said second non-natural chimeric membrane-embedded receptor comprises a single VH domain, such as a single camel, shark, or lamprey VH domain, or a single VH domain derived from a human or mouse sequence, or a non-antibody backbone, such as fibronectin, such as a type III fibronectin antibody-like molecule. In one embodiment, the antigen-binding domain of one of said first and said second non-natural chimeric membrane-embedded receptor contains scFv and the other contains a single VH domain, such as a single camel, shark, or lamprey VH domain, or a single VH domain derived from a human or mouse, or non-antibody scaffold, for example, fibronectin, for example, an antibody-like molecule of type III fibronectin. In one embodiment, the antigen-binding domain of one of said first and said second non-natural chimeric membrane-immersed receptor contains scFv and the other contains a nanobody. In one embodiment, the antigen-binding domain of one of said first and said second non-natural chimeric membrane-immersed receptor contains scFv and the other contains a camelid VHH domain. In one embodiment, the invention relates to a nucleic acid, for example, a purified or non-natural nucleic acid, containing a sequence encoding the first and second non-natural chimeric membrane-immersed receptor containing the antigen-binding domain described in the present invention. In one of the embodiments, the invention relates to a method for obtaining a cell described in the present invention, including the introduction into the cell of the nucleic acid described in the present invention. In one embodiment, the invention relates to a method for producing a cell described in the present invention, including the formation in a cytotoxic cell of the first and said second non-natural chimeric membrane-immersed receptor described in the present description. In one embodiment, the invention relates to a method of treating a subject, for example, a method of providing antitumor immunity in a mammal, comprising administering to the mammal an effective amount of a cell described in the present invention.

In another aspect, the invention relates to a kit containing a cell or nucleic acid described in the present invention.

In another aspect, the invention relates to an isolated nucleic acid sequence encoding a KIR-CAR (immunoglobulin-like killer cell receptor-chimeric antigen receptor), wherein the isolated nucleic acid sequence comprises an antigen-binding domain nucleic acid sequence and a KIR or a fragment thereof. In one embodiment, the antigen binding domain is selected from the group consisting of a mouse antibody, a humanized antibody, a human antibody, a chimeric antibody, and a fragment thereof. In one embodiment, the fragment is a Fab or scFv. In one embodiment, the antigen-binding domain is the antigen-binding domain described in the present invention, for example, in table. 4. In one embodiment, the KIR is selected from the group consisting of an activating KIR, an inhibitory KIR, and any combination thereof. In one embodiment, at least one hinge region is removed from the activating KIR.

In another aspect, the invention relates to an isolated KIR-CAR (immunoglobulin-like killer cell receptor-chimeric antigen receptor) containing an antigen-binding domain and

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KIR or its fragment. In one embodiment, the antigen binding domain is selected from the group consisting of a mouse antibody, a humanized antibody, a human antibody, a chimeric antibody, and a fragment thereof. In one embodiment, the fragment is a Fab or scFv. In one embodiment, the antigen-binding domain is the antigen-binding domain described in the present invention, for example, in table. 4. In one embodiment, the KIR is selected from the group consisting of an activating KIR, an inhibitory KIR, and any combination thereof. In one embodiment, at least one hinge region is removed from the activating KIR.

In another aspect, the invention relates to a composition comprising at least two KIRCARs, 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 the antigen presented on the tumor, for example, the antigen-binding domain described in the present invention, for example, in table. 4 and the antigen binding domain in the second KIR-CAR is specific for an antigen presented on a normal cell.

In another aspect, the invention relates to a genetically modified T cell containing at least two KIR-CARs, where the first KIR-CAR contains an antigen-binding domain and an activating KIR or fragment thereof, and the second KIR-CAR contains 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 the antigen presented on the tumor, for example, the antigen-binding domain described in the present invention, for example, 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.

In another aspect, the invention relates to a method of providing antitumor immunity in a mammal, where the method includes administering to the mammal an effective amount of a cell containing at least two KIR-CARs, where the first KIR-CAR contains an antigen-binding domain and an activating KIR or fragment thereof, and the second KIR- CAR contains 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 the antigen presented on the tumor, for example, the antigen-binding domain described in the present invention, for example, in table. 4, and the antigen-binding domain in the second KIR-CAR is specific to the antigen presented on the normal cell, thus regulating the side activity of the cell. In one embodiment, the cell is a T cell.

In another aspect, the invention relates to a method of treating an individual suffering from a disease associated with the expression of a tumor antigen, for example, a tumor antigen described in the present description (for example, a proliferative disease, a precancerous condition and a non-malignant tumor associated indication associated with the expression of a tumor antigen) comprising administering to the mammal an effective amount of a cell containing at least two KIR-CARs, where the first KIR-CAR contains an antigen-binding domain and an activating KIR or fragment thereof, and the second KIR-CAR contains an antigen-binding domain and an inhibitory KIR or fragment thereof. In one embodiment, the implementation of the first KIR-CAR contains antigennegative domain described in the present invention, for example, in table. 4.

In one embodiment, the disease associated with the expression of the tumor antigen is a malignant tumor, for example, a malignant tumor described in the present description. In one embodiment, the cancer is a solid tumor, such as the solid tumor described herein, such as 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). lung cancer), pancreatic cancer (eg, pancreatic ductal adenocarcinoma), ovarian cancer, colorectal cancer, and bladder cancer, or any combination thereof. In one embodiment, the disease is pancreatic cancer, eg, metastatic pancreatic ductal adenocarcinoma (PDA), eg, in an individual who has experienced disease progression during at least one previous standard therapy. In one embodiment, the disease is a mesothelioma (eg, malignant pleural mesothelioma), for example, in an individual who has experienced disease progression during at least one previous standard therapy. In one embodiment, the disease is ovarian cancer, eg, malignant serous epithelial ovarian tumor, eg, in an individual who has experienced progression of the disease following at least one previous standard therapy regimen.

Unless otherwise defined, all technical and scientific terms used in the present description have the same meaning as generally understood by a person skilled in the art to which this invention belongs. Although in practical implementation or testing on

- 21 040145 of the present invention, methods and substances similar or equivalent to those described in the present description can be used, suitable methods and substances are described below. All publications, patent applications, patents and other references cited in this specification are incorporated by reference in their entirety. Moreover, the materials, methods, and examples are illustrative only and are not intended to be limiting. Headings, subheadings, or numbered or lettered items, such as (a), (b), (i), etc., are provided for ease of reading only. The use of headings or numbered or lettered elements in this document does not imply that the steps or elements are in alphabetical order, or that the steps or elements must follow one after the other.

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

Brief description of the drawings

The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, the drawings show embodiments which are currently preferred. However, it should be understood that the invention is not limited to the exact locations and technical means of the embodiments shown in the drawings.

Fig. 1 containing FIG. 1A and 1B is a series of schemes demonstrating the structure of naturally occurring inhibitory and activating KIRs (FIG. 1A) and activating scFv-based KIR-CARs (FIG. 1B).

Fig. 2 is a schematic representation of a lentiviral vector used to deliver a KIR-based activating CAR in combination with a DAP12 signaling molecule.

Fig. 3 is an image demonstrating 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/CD28 antibody microbeads and transduced with the indicated CAR or mimicked transduction and expanded ex vivo. Expression was detected using biotinylated polyclonal goat IgG specific for mouse F(ab)2 (Jackson Immunologies) followed by streptavidin-PE staining.

In FIG. 4 demonstrates that T cells expressing actKIR-CAR SS1 had cytotoxic activity against K562 target cells engineered to express mesothelin ligand (KT-meso). Human T cells were stimulated with anti-CD3/CD28 antibody microbeads, transduced with the indicated CAR or mimicked transduction and expanded ex vivo. 105 CFSE-labeled mesothelin-expressing K562 (KT-meso) cells or a wild-type K562 control were incubated with CAR-expressing T cells in various ratios for 16 h at 37° C., 5% CO ₂ . Target K562 cells were then quantified by flow cytometry using CountBright beads and viable cell staining (7AAD). The percentage of lysed K562 cells (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, and then dividing by the number of viable K562. remaining after culturing overnight without effector T cells.

Fig. 5 containing FIG. 5A and 5B is a series of diagrams showing an activating KIR-CAR in which the KIR2DS2 hinge (KIR2S CAR) has been removed. Based on the kinetic model of TCR activation segregation plotted in FIG. 5A, it is believed that the mesothelin-specific KIR-CAR SS1 contains a hinge that is too long to provide adequate segregation. Thus, it is believed that making the KIRCAR mesothelin-specific hinge shorter can improve function. Fig. 5B is a diagram showing that SS1 scFv was fused to a KIR transmembrane domain without two Ig-like domains from KIR2DS2 as a hinge.

Fig. 6 containing FIG. 6A and 6B is a series of images demonstrating that scFv SS1-based KIRS2-CAR has increased cytolytic activity against mesothelin-expressing target cells compared to CARs generated by fusion of scFv SS1 into full-length wild-type KIR2DS2. Primary human T cells were stimulated with CD3/28 microbeads followed by transduction with lentivirus activating KIR-CAR SS1-KIR2DS2, activating KIR CAR SS1-KIRS2, CAR SS1-zeta. Model non-transduced T cells (NTD) were used as controls. T cells were expanded to the end of the logarithmic growth phase. Surface expression of SS1-specific CARs was determined by flow cytometry using a biotinylated goat polyclonal antibody specific for mouse F(ab)2 followed by detection using streptavidin-PE as shown in FIG. 6A. In FIG. 6B demonstrates that K562 target cells with or without mesothelin and stained with CFSE were mixed with the effector T cells characterized in FIG. 6A as indicated using various ratios of effector T cell to target ranging from 10:1 to 1:1. Lysis of K562 target cells was assessed using flow cytometry to determine % viable CFSE+ cells as described for FIG. 4. Data shown is the calculated % lysis of target cells compared to target cells without effector cells.

Fig. 7 containing FIG. 7A and 7B is a series of images showing co-expression of actKIR-CAR CD19 and inhKIR-CAR SS1. Jurkat cells with the NFAT-GFP reporter gene were transduced with the indicated KIR CAR or not transduced (NDT) and mixed 1:1 with target cells with or without CD19 antigens and mesothelin, as. The results show GFP expression 24 hours after mixing Jurkat cells and target cells (FIG. 7A). In FIG. 7B shows surface expression of mesothelin and CD19 idiotypes as determined by staining with mesothelin-Fc fusion protein and anti-CD19 scFv FMC63 idiotype-specific monoclonal antibody.

Fig. 8 containing FIG. 8A, 8B and 8C is a series of images demonstrating the co-expression of wild-type PD-1 with activating KIR-based or TCR-zeta-based CARs directed to mesothelin. Primary human T cells were stimulated with CD3/28 microbeads followed by transduction with a lentivirus activating KIR-CAR SS1-KIRS2 or CAR SS1-zeta. Model non-transduced cells (NTD) were used as a negative control. Cells were expanded for 9 days and surface expression of CAR was determined by mesothelin-Fc staining followed by human Fc-specific goat PE-conjugated antibody (FIG. 8A). K562 cell lines (wild-type [wt], expressing mesothelin [meso], or co-expressing mesothelin and PD-L1 [meso-PDL1]) were stained with a mesothelin-specific CAK1 monoclonal antibody to confirm mesothelin expression on targets (Fig. 8B). Primary human T cells transduced as shown in FIG. 8A, 10 μg of in vitro transcribed RNA encoding wild-type PD1 was electroporated using a BTX ECM830 electroporation device (PD1+) or mimicked transfection (PD1-). Expression on the surface of PD-1 was detected using an anti-PD1 monoclonal antibody conjugated to APC (Fig. 8C).

Fig. 9 is an image demonstrating that the combination of co-expression of wild-type PD-1 with an activating KIR-based CAR and a TCR-zeta-based CAR directed to mesothelin resulted in PD-1 ligand 1 (PDL-1) dependent inhibition of the cytotoxicity of specific to mesothelin activating KIR-CAR. Primary human T cells were stimulated with CD3/28 microbeads followed by transduction with a lentivirus activating KIR-CAR SS1-KIRS2, CAR SS1-zeta, or simulated transduction (NTD). T cells were expanded for 9 days followed by electroporation of 5x106 T cells with 10 μg of in vitro transcribed RNA encoding wild-type PD1 using a BTX ECM830 electroporation device (PD1+) or simulated transfection (PD1-). Surface expression of SS1-specific CAR and PD-1 was determined as shown in FIG. 8. K562 target cells not expressing mesothelin or expressing mesothelin with or without PDL-1 were mixed under various conditions with T cells as indicated using various ratios of effector T cell to target from 30:1 to 1: 1 as shown. Lysis of K562 target cells was assessed by the Calcein AM staining method to quantify the remaining viable cells after 4 hours of incubation. Data shown is the calculated % lysis of target cells compared to target cells without effector cells.

Fig. 10 is an image demonstrating the production of interferon-gamma (IFNγ) and interleukin 2 by donor T cells expressing mesothelin-specific KIR-based activating CAR (SS1-KIRS2) or TCR-zeta-based CAR with a costimulatory domain (SS1-z , SS1-28z or SS1-BBz) or without. Primary human T cells were stimulated followed by transduction with the indicated activating KIR-CAR or TCR-zeta based lentivirus. After expansion, the transduced T cells were mixed with K562 (Kwt) or K562-mesothelin cells (Kmeso) in a ratio of 2:1. Cytokine concentrations were determined in supernatants after 24 hours of stimulation by ELISA for the indicated cytokines from multiple independent donors. Repeated ANOVA showed a significant effect of CAR on IFN-γ (p=0.002) and IL-2 (p=0.0156). SS1KIRS2/DAP12 (SS1KIR) versus sham for IFN-γ (post hoc paired t-test, p=0.0162). SS1-KIR versus SS1-28z for IL-2 (post hoc paired t-test, p=0.0408).

Fig. 11 is a map of the intensity of cytokine concentrations in supernatants as assessed by Luminex-based multiplex immunoassay (Cytokine Human 10-Plex Panel, Life Technologies). A relative concentration intensity map was generated after donor normalization and lowest concentration conditions for each cytokine using the implementation of the heatmap package in the R software for statistical analysis.

In FIG. 12 containing FIG. 12A-12B illustrate the construction of an engineered T cell with a mesothelin-specific KIR-based chimeric antigen receptor (KIR-CAR) with persistent cytotoxic activity. Primary human T cells were stimulated with CD3/28 microbeads followed by transduction with a lentiviral vector expressing GFP and dsRed (control) or DAP12 and dsRed (DAP12). Cells were expanded ex vivo until the end of the logarithmic growth phase. 5x10 ⁶ T cells from each transduced population were electroporated with 10 μg of in vitro transcribed RNA encoding SS1-KIRS2 using a BTX ECM830 (PD1+) electroporation device. Expression of dsRed and SS1-KIRS2 was assessed by flow cytometry, where SS1-KIRS2 was detected using a biotinylated goat polyclonal antibody specific for mouse F(ab)2 followed by streptavidin-PE. On the top panel of Fig. 12A shows a strategy for setting a discrimination window to identify T cells expressing dsRed, which are then analyzed for SS1-KIRS2 expression, as shown in the bottom of the panel. In FIG. 12B demonstrates the ability of the cells characterized in FIG. 12A, mediate cytotoxicity against wild-type K562 cells (K562-wt) or K562 cells that express mesothelin (K562-mesothelin) as assessed using a ⁵¹ Cr release assay for 4 hours.

In FIG. 13 shows that endogenous TCR expression was unaffected by SS1KIRS2 and DAP12 expression. 5×10 ⁶ primary human T cells were electroporated with 10 μg of in vitro transcribed RNA encoding SS1-KIRS2 or mimicked transfection using a BTX ECM830 (PD1+) electroporation device. After overnight incubation, transfected T cells were stained for SS1-KIRS2 expression using a biotinylated goat polyclonal antibody specific for mouse F(ab)2 followed by streptavidin-PE staining. Expression of Ve13.1 was assessed using a PE-conjugated monoclonal antibody specific for this TCR ve chain.

In FIG. 14 illustrates the ability of a mesothelin-specific KIR-based CAR (SS1-KIRS2) to stimulate T cell proliferation that is antigen-dependent but independent of CD28 additional costimulation. Primary human T cells were stimulated with CD3/28 microbeads followed by transduction with lentivirus SS1-KIRS2 and DAP12 or mesothelin-specific CAR TCR-zeta (SS1-zeta). Non-transduced cells under simulated conditions (NTD) were used as a negative control. K562 target cells without mesothelin (K562 wt), or expressing mesothelin (K562-mesothelin), were mixed in various states with T cells as indicated in a ratio of effector T cells to target cells of 2:1. T cells stimulated with K562 mesothelin were further separated depending on the condition with or without a monoclonal anti-CD28 antibody agonist (clone 9.3) at 1 μg/ml. To count the number of population doublings after antigen challenge, the number of viable T cells was determined by flow cytometry using bead-based counts at the indicated time points.

In FIG. 15 containing FIG. 15A, 15B, 15C, 15D, and 15E demonstrate that mesothelin-specific KIR-CAR-modified T cells have enhanced antitumor activity in vivo compared to second-generation TCR-ζ-based CARs carrying CD28 or CD137 (4-1BB) costimulatory domains. In FIG. 15A shows an experiment in which 2×10 ⁶ mesothelin-expressing cells derived from mesothelioma (EM-meso cells) were subcutaneously implanted in NODSCID-y _c ' (NSG) mice. 20 days after tumor implantation, each animal was injected intravenously with 5x10 ⁶ T cells, which were stimulated with anti-CD3/CD28 antibody stimulating beads after lentiviral transduction with a CD3z-based CAR series with a costimulatory domain (SS1-z, SS1-BBz and SS1-28z) or without or mesothelin-specific CAR based on KIR, SS1-KIRS2 with DAP12. Sham transduced T cells (NTD) were used as controls. Tumor volume was measured with a caliper at the indicated time points (n=7 mice per group). In FIG. 15B demonstrates that in vivo KIR-CAR activity is independent of T cell engraftment in the blood, spleen, or tumor. The frequency of human CD45+ T cells was assessed at the end of the experiment by flow cytometry and the data are expressed as percentage of total viable cells in blood, spleen and tumor cleavage product. In FIG. 15C demonstrates that comparable CD3+ TIL frequencies were observed in groups treated with SS1-28z and SS1-KIRS2/DAP12 CAR T cells. The same model was used as shown in Fig. 15A. The frequency of human CD3+ lymphocytes in tumors on day 30 (10 days after CART infusion) was assessed by flow cytometry. In FIG. 15D demonstrates that a mesothelin-specific KIR-based CAR is necessary for tumor eradication with DAP12-modified T cells. The same model was used as shown in Fig. 15A. 4 million T cells expressing DAP12 and dsRed (DAP12), SS1-28z or SS1-KIRS2 and DAP12 (SS1-KIRS2) were injected intravenously on day 20, and long-term tumor volume was assessed by caliper measurement. The arrow indicates the time of TIL isolation used for functional and phenotypic analysis. In FIG. 15E shows the antigen-specific cytotoxic activity of TILs isolated from the mice described in Figure 15D. Antigen-specific cytotoxicity was assessed by co-cultivation with firefly luciferase-expressing EMmeso cells or EMp cells (original EM cells without mesothelin expression) in the indicated ratios.

- 24 040145

E:T within 18 hours.

In FIG. 16 containing FIG. 16A and 16B demonstrate a KIR-based CAR where the specificity of CD19 can induce antigen-specific cytotoxicity against the target cell. After activation with anti-CD3/CD28 antibody beads, T cells were transduced with a bicistronic lentiviral vector expressing DAP12 along with a CD19-specific KIR-based CAR in which an FMC63-derived scFv is fused to a full-length KIR2DS2 (CD19KIR2DS2), or KIR-based CAR. obtained by fusion of scFv FMC63 with the transmembrane and cytoplasmic domain of KIR2DS2 through a short linker [Gly] ₄ -Ser (CD19-KIRS2). The transduced T cells were cultured to the end of the logarithmic growth phase and expression of CD19-specific CARs was assessed based on KIR by flow cytometry using a biotinylated goat anti-mouse F(ab) ₂ polyclonal antibody followed by SA-PE. ⁵¹ Cr-labeled K562 target cells with or without CD19 expression (K562-CD19) (K562-wt) were mixed in various ratios of T cells to target cells (E:T ratio). Cytotoxicity was determined by measuring the fraction of ⁵¹ Cr released into the supernatant for 4 h. Control T cells that were transduced under sham conditions (NTD) or transduced with a CD19-specific (CD19-z) CD3z-based CAR were also included as negative and positive controls. respectively.

In FIG. 17 containing FIG. 17A and 17B demonstrate in vivo CD19-KIRS2 activity. NOD-SCID-yc'/' (NSG) mice were transplanted intravenously by tail vein injection on day 0 with 1 million Nalm-6 CBG tumor cells, a CD19-expressing leukemic cell line. Cells were stimulated with stimulatory beads with anti-CD3/CD28 antibodies followed by lentivirus transduction on day 1 with a series of CD19-specific CD3z-based CARs with or without co-stimulatory domain (CD19z, 19BBz) or CD19-specific CARs based on KIR, CD19KIRS2 with DAP12 (19KIRS2). Non-transduced under simulated T cells (NTD) were used as controls. T cells were expanded to the end of the ex vivo logarithmic growth phase and injected intravenously on day 5 after injection of the leukemic cell line with 2 million CAR T cells per mouse. Tumor burden was assessed by bioluminescent imaging. For each T cell condition, 5 animals were analyzed. In FIG. 17A shows individual bioluminescent photon flux for selected animals on day 5 (baseline before T-cell injection) and day 15 after leukemic cell transplantation. In FIG. 17B shows the average total flow for each processing group over time.

In FIG. 18 demonstrates that NKp46-based NCR-CAR with specificity for mesothelin induces antigen-specific cytotoxicity. After activation with anti-CD3/CD28 antibody beads, T cells were transduced with a bicistronic lentiviral vector expressing DAP12 and SS1-KIRS2 (control), or FcεRγ and mesothelin-specific CAR based on NKp46 (SS1NKp46) or FcεRγ and mesothelin-specific CAR NKp46, in which the natural extracellular domain of NKp46 was truncated (SS1-TNKp46). Expression of mesothelin-specific CARs was assessed by flow cytometry using a biotinylated goat anti-mouse F(ab)2 polyclonal antibody followed by SA-PE as shown in FIG. 18A. T cells were mixed with ⁵¹ Cr-labeled K562 target cells expressing mesothelin in various ratios of effector T cells to K562 target cells (E:T ratio). Cytotoxicity was determined by measuring the ⁵¹ Cr fraction. released into the supernatant within 4 hours compared to spontaneous release as shown in FIG. 18b.

In FIG. 19 is a schematic representation of the receptors used in the experiments shown in FIG. 21-23.

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

In FIG. 21 shows co-expression of SS1-KIRS2 and KIR2DL3 by primary human T cells. Primary human T cells were stimulated with CD3/28 microbeads followed by lentivirus transduction with SS1-KIRS2 and DAP12 (SS1-KIRS2) or sham transduction (NTD) in combination with wild-type KIR2DL3. T cells were expanded to the end of the logarithmic growth phase. Surface expression of mesothelin-specific CAR and KIR2DL3 was determined by mesothelin-Fc staining followed by a PE-conjugated goat anti-human Fc antibody and a monoclonal antibody to the KIR2DL3 ectodomain.

In FIG. 22 demonstrates that KIR2DL3 co-expressed with KIR-CAR can suppress antigen-specific cytotoxicity in the presence of HLA-Cw on target cells. The T cell, which was obtained and characterized as described in FIG. 21 were mixed with ⁵¹ Cr-labeled K562 target cells, which were prepared and characterized as described in FIG. 22. Cytotoxicity was determined by measuring the ⁵¹ Cr fraction released into the supernatant over 4 hours compared to target cells without effector cells.

In FIG. 23 is a schematic representation of the receptors used in the experiments shown in FIG. 24.

In FIG. 24 demonstrates the inability to co-express two scFv-based chimeric receptors on the surface of a T cell while maintaining the appropriate binding specificity of each receptor. Jurkat T cells were transduced using a lentiviral vector encoding SS1-KIR2DL3. These cells were then transduced with a second lentiviral vector encoding CD19KIR2DS2 at various dilutions of the vector. SS1-specific scFv expression was assessed using mesothelin-Fc followed by PE-conjugated goat anti-human Fc. Expression of the CD19-specific scFv was assessed using a PE-conjugated monoclonal antibody specific for the FMC63 idiotype.

Fig. 25 are images demonstrating that expression of the CD19-specific CAR also reduced the expression of mesothelin binding sites on the surface of cells co-expressing the SS1-zeta-mCherry CAR fusion. Primary human T cells were stimulated with CD3/28 microbeads followed by transduction with lentivirus with CAR scFv SS1-zeta carrying an mCherry fusion at the C-terminus (SS1z-mCh) or a CAR specific for CD19 derived from FMC63, 41BB-zeta (19bbz) alone or in combination. Cells transduced in sham mode were used as controls. T cells were expanded to the end of the logarithmic growth phase and expression of dsRed as well as CAR was determined on the surface by flow cytometry after staining with mesothelin-Fc followed by staining with human Fc-specific goat polyclonal antibody conjugated with FITC.

Fig. 26 are images demonstrating that the mutually exclusive expression of scFv SS1 binding sites is not unique to FMC63 scFv. Primary human T cells were stimulated with CD3/28 microbeads followed by transduction with lentivirus with CAR scFv SS1zeta or various CD19-specific CAR 41BB-zeta (19BBz [CARFMC63 scFv, 214d scFv or BL22 scFv with alternating VH and VL orientations [H2 l and L2H ]). NTDs are sham transduced cells used as a staining control. In addition, a separate group of T cells were co-transduced with scFv SS1-zeta CARs and various CD19-specific CARs as described above. T cells were expanded to the end of the logarithmic growth phase and surface CAR expression was determined by staining with biotinylated L protein (recognizing kappa light chain) followed by APC streptavidin staining simultaneously with mesothelin-Fc followed by human Fc-specific goat polyclonal antibody conjugated to PE . For co-transduced cells, it was demonstrated that the mutually exclusive expression observed for FMC63-based CARs is also observed with other scFv-CARs.

In FIG. 27 containing FIG. 27A and 27B illustrate a possible mechanism for the loss of scFv binding when two scFv molecules are co-expressed on the cell surface (FIG. 27A) and the possible avoidance of this interaction when Camelid VHH single domain-based CARs are expressed on the surface of T cells in combination with CARs based on scFv.

In FIG. 28 demonstrates that camelid VHH single domain-based CAR can be expressed on the surface of T cells in combination with scFv-based CAR without noticeable receptor interaction. Jurkat T cells expressing GFP under the control of an NFAT-dependent promoter (NF-GFP) were transduced with mesothelin-specific activating CAR (SS1-CAR), CD19-specific activating (19-CAR), or CAR derived using the camel VHH domain , specific to EGFR (VHH-CAR). Then, after transduction with an activating CAR, cells were transduced with an additional inhibitory CAR recognizing CD19 (19-PD1) to obtain cells expressing both an activating and inhibitory CAR (SS1+19PD1, 19+19PD1, or VHH+19PD1). Transduced Jurkat T cells were cultured for 24 h in association with various cell lines that 1) do not contain any target antigens (K562), 2) express mesothelin (K-meso), CD19 (K-19), or EGFR alone (A431), 3) express a combination of EGFR and mesothelin (A431-mesothelin) or CD19 (A431-CD19) or 4) express a combination of CD19 and mesothelin (K-19/meso). Additional conditions that include no stimulating cells (no stim) or K562 with 1 μg/ml OKT3 (OKT3) were also included as negative and positive controls for NFAT activation, respectively. GFP expression as a marker of NFAT activation was assessed by flow cytometry.

In FIG. 29 shows the KIR2DS2 sequence annotation. The nucleotide sequence provided in FIG. 29 is SEQ ID NO: 342. The amino acid sequence provided in FIG. 29, denote SEQ ID NO: 343.

In FIG. 30 shows the KIR2DL3 sequence annotation. The nucleotide sequence provided in FIG. 30 is SEQ ID NO: 344. The amino acid sequence provided in FIG. 30 are SEQ ID NO: 345.

- 26 040145

In FIG. 31 shows the NKp46 sequence annotation. The nucleotide sequence provided in FIG. 31 is SEQ ID NO: 346. The amino acid sequence provided in FIG. 31 denote SEQ ID NO: 347.

In FIG. 32 shows the SS1-KIRS2 sequence annotation. The nucleotide sequence provided in FIG. 32 is denoted by SEQ ID NO: 348. The amino acid sequence provided in FIG. 32, denote SEQ ID NO: 349.

In FIG. 33 is an annotation for the SS1-KIR2DS2 sequence. The nucleotide sequence provided in FIG. 33 is denoted by SEQ ID NO: 350. The amino acid sequence provided in FIG. 33, denote SEQ ID NO: 351.

In FIG. 34 shows the sequence annotation for SS1-tNKp46. The nucleotide sequence provided in FIG. 34 is denoted by SEQ ID NO: 352. The amino acid sequence provided in FIG. 34, denote SEQ ID NO: 353.

In FIG. 35 shows the SS1-KIRL3 sequence annotation. The nucleotide sequence provided in FIG. 35 is SEQ ID NO: 354. The amino acid sequence provided in FIG. 35, denote SEQ ID NO: 355.

In FIG. 36 demonstrates that CD3Z-based mesothelin-specific CAR and KIR-based CAR have similar in vitro antigen-specific cytotoxicity to mesothelin-derived mesothelin-expressing cells (EM-meso cells). Primary human T cells were stimulated with anti-CD3/CD28 antibody stimulation beads and transduced with a lentiviral vector expressing mesothelin-specific CAR SS1-KIRS2. After expansion, T cells were mixed with ⁵¹ Cr-labeled K562 cells expressing EM-meso at the indicated effector to target ratio (E:T). The % lysis was determined.

In FIG. 37 demonstrates that TIL from mice treated with CART 28ζ lost IFNy secretion when stimulated with mesothelioma-derived mesothelin-expressing cells (EM-meso cells). NOD-SCID-yc'/' (NSG) mice were injected subcutaneously with 2x106 EM-meso cells. 5x106 primary human T cells transduced with the indicated CARs were injected iv on day 16. Eighteen days after the CAR infusion, T cells were isolated with CD45 magnetic beads TIL and mixed with EM-meso at the indicated effector to target ratio (E:T ). Cytokine concentrations were determined in supernatants by ELISA.

In FIG. 38 demonstrates that SS1-KIRS2/DAP12 T cells mediate robust antitumor activity in vivo. NOD-SCID-yc'/' (NSG) mice were injected subcutaneously with 2x106 mesothelin-expressing cells derived from mesothelioma (EM-meso cells). 5x106 primary human T cells transduced with the indicated CAR were injected iv on day 20. Tumor volume was measured with a caliper at the indicated time points.

Fig. 39 containing FIG. 39A, 39B and 39C is a diagram showing the structure of mesothelin-specific CARs. In FIG. 39A shows the mesothelin-specific multi-chain KIR-CAR. In FIG. 39B shows mesothelin-specific single chain KIR-CAR containing DAP12. In FIG. 39C shows a mesothelin-specific CAR containing the CD28 and CD3-zeta signaling domains.

In FIG. 40 shows the in vivo antitumor activity of mesothelin-based CAR constructs, including multi-chain KIRS/DAP12 and single-chain DAP12 constructs.

In FIG. 41A-41F show surface expression of mesothelin-based KIR-CAR on primary human T cells as detected by flow cytometry analysis.

In FIG. 42 containing FIG. 42A and 42B demonstrate the antigen-specific cytotoxic activity of mesothelin-based KIR-CAR against target cells that do not express mesothelin (K562) (FIG. 42A) or target cells engineered to express mesothelin (K562-meso) ( Fig. 42B).

In FIG. 43 containing FIG. 43A and 43B show the production of mesothelin-based KIR-CAR cytokines when cultured in the presence of mesothelin-expressing target cells (K562-meso, black bars) or control target cells that do not express mesothelin (K562, clear bars). In FIG. 43A shows IFN-gamma production; in fig. 43B shows production of the cytokine IL-2.

In FIG. 44 containing FIG. 44A, 44B, 44C, 44D, and 44E depict various single vector configurations, for example, where U6-controlled shRNA is upstream or downstream of EF1-alpha-controlled CAR coding elements. In the exemplary structures shown in FIG. 44A and 44B, transcription is mediated by the U6 and EF1-alpha promoters in the same direction. In the exemplary structures shown in FIG. 51C and 51D, transcription is mediated by the U6 and EF1-alpha promoters in different directions. In FIG. 44E shRNA (and the corresponding U6 promoter) is in the first vector and CAR (and the corresponding EF1-alpha promoter) is in the second vector.

In FIG. 45 illustrates the structures of two exemplary RCAR configurations. Antigen-binding elements include an antigen-binding domain, a transmembrane domain, and a switching domain. Intracellular binding elements include a switch domain, a costimulatory signaling domain, and a primary signaling domain. The two configurations demonstrate that the first and second switch domains described in the present invention may be in different orientations with respect to the antigen binding element and the intracellular binding element. Other RCAR configurations are further described herein.

In FIG. 46 demonstrates that proliferation of CAR-expressing transduced T cells is enhanced by low doses of RAD001 in a cell culture system. CART co-cultured with Nalm-6 cells in the presence of various concentrations of RAD001. The numbers of CAR-positive, CD3-positive T cells (black) and total T cells (grey) were assessed after 4 days of co-culture.

In FIG. 47 illustrates measurements of tumor growth of NALM6-luc cells at daily dosing of RAD001 at 0.3, 1, 3, and 10 mg/kg (mg per kg body weight) or vehicle dosing. Circles indicate carrier; squares represent 10 mg/kg dose of RAD001; triangles represent 3 mg/kg dose of RAD001, inverted triangles represent 1 mg/kg dose of RAD001, and diamonds represent 0.3 mg/kg dose of RAD001.

In FIG. 48 containing FIG. 48A and 48B are pharmacokinetic curves showing the amount of RAD001 in the blood of NSG mice with NALM6 tumors. In FIG. 55A shows PK on day 0 after the first dose of RAD001. In FIG. 48B shows PK on day 14 after the last dose of RAD001. Diamonds represent 10 mg/kg dose of RAD001; squares represent 1 mg/kg dose of RAD001; triangles represent 3 mg/kg dose of RAD001 and x represent 10 mg/kg dose of RAD001.

In FIG. 49 containing FIG. 49A and 49B show in vivo proliferation of humanized CD19 CART cells with and without RAD001 dosing. Low doses of RAD001 (0.003 mg/kg) every day resulted in increased proliferation of CAR T cells above the normal huCAR19 proliferation level. In FIG. 49A shows CD4+CAR T cells; in fig. 49B shows CD8+CAR T cells. Circles indicate PBS; squares mean huCTL019; triangles indicate huCTL019 with 3 mg/kg RAD001; inverted triangles represent huCTL019 with 0.3 mg/kg RAD001; diamonds represent huCTL019 with 0.03 mg/kg RAD001, and circles represent huCTL019 with 0.003 mg/kg RAD001.

Detailed description

In one aspect, the present invention relates to compositions and methods for regulating the specificity and activity of T cells or other cytotoxic cells, such as NK cells. One embodiment relates to a chimeric antigen receptor (CAR), e.g., NK cell receptor (NKR-CAR) based NK cell receptor (NKR), e.g., KIR-CAR, NCR-CAR, SLAMF-CAR, FcR -CAR or Ly49-CAR. In one embodiment, the invention relates to a type of chimeric antigen receptor (CAR), where the CAR is referred to as NKR, for example, KIR-CAR, which is a CAR model containing a receptor component found in natural killer (NK) cells. In one embodiment, the NK receptor includes, but is not limited to, the killer cell immunoglobulin-like receptor (KIR). KIRs can function as an activating KIR or an inhibitory KIR.

One of the advantages of the NKR-CAR, eg KIR-CAR of the invention is that NKRCARs, eg KIR-CAR, provide a way to regulate the specificity of a cytotoxic cell, eg a T cell, to regulate the side activity of the engineered T cell. In some cases, the KIR-CAR of the invention does not require costimulation to proliferate.

NKR-CARs can deliver a signal via an adapter protein, such as an ITAM-containing adapter protein. In one embodiment, the KIR-CARs of the invention comprise an activating KIR that delivers its signal through interaction with an immunoreceptor tyrosine activation motif (ITAM) containing the membrane protein DAP12, which is mediated by residues in the transmembrane domains of such proteins.

In one embodiment, the NKR-CAR may deliver an inhibitory signal via an inhibitory motif. In one embodiment, the KIR-CARs of the invention comprise an inhibitory KIR that delivers a signal through interaction with immunoreceptor tyrosine inhibitory motifs (ITIM). KIRs carrying cytoplasmic domains that contain ITIM abolish the activating signal, resulting in NK inhibition of cytolytic activity and cytokine production activity. However, the invention should not be limited to inhibitory KIRs. Conversely, any inhibitory protein containing a cytoplasmic domain that is associated with an inhibitory signal can be used in the CAR construct of the invention.

Thus, the invention relates to a composition containing NKR-CAR, such as KIR-CAR, vectors containing NKR-CAR, compositions containing NKR-CAR, such as KIR-CAR, vectors packaged in viral particles, and recombinant T cells or other cytotoxic cells containing NKR-CAR, such as KIR-CAR. The invention also includes methods for producing a genetically modified T cell or other cytotoxic cell, such as an NK cell, or a cultured NK cell, such as a NK92 cell expressing an NKR-CAR, such as a KIR-CAR (KIR-28 040145

CART), where the expressed NKR-CAR, for example KIR-CAR, contains the antigen recognition domain of a specific antibody with an intracellular signaling molecule from NKR, for example, KIR. For example, in some embodiments, the intracellular signaling molecule includes, but is not limited to, KIR ITAM, KIR ITIM, and the like.

Thus, the invention relates to compositions and methods for regulating the specificity and activity of T cells or other cytotoxic cells modified to express NKR-CAR, such as KIR-CAR. The present invention also relates to cells containing a set of NKR-CAR types, for example KIR-CAR (for example, activating NKR-CAR, for example KIR-CAR, and inhibitory NKR-CAR, for example KIR-CAR), where the set of NKR-CAR types, for example, KIR-CAR, is involved in signal transduction to regulate T-cell activation. In this aspect, it is preferable to effectively control and regulate NKR-CAR cytotoxic cells, such as KIR-CAR T cells, so that they kill tumor cells while not affecting normal uninvolved cells. Thus, in one embodiment, the present invention also relates to methods for cytolysis of malignant cells while minimizing the depletion of normal non-malignant cells, thereby improving the specificity of NKR-CAR therapy, eg KIR-CAR.

In one embodiment, the NKR-CAR, e.g. KIR-CAR approach involves physically separating the population of CAR types expressed by the cell, where the association of the population of NKR-CAR types, e.g. KIR-CAR, with its target antigen is necessary to activate the cytotoxic NKR- CAR, for example, KIR-CAR T cells. For example, in the KIR-CAR approach, each KIR-CAR in a population of KIR-CARs types contains a different intracellular signaling domain. For example, when a combination of KIR-CAR types is used to induce activation of a KIRCAR T cell, the first KIR-CAR type may contain only the intracellular domain from the activating KIR, and the second CAR type may contain only the intracellular domain from the inhibitory KIR. Thus, a conditional activation of T cells is obtained as a result of the interaction of an activating KIR-CAR (actKIR-CAR) with an antigen on a malignant cell of interest. An inhibitory KIRCAR (inhKIR-CAR), carrying an antigen-binding domain directed against an antigen that is present on a normal but non-cancerous cell, suppresses the activating effects of actKIR-CAR when the T cell encounters normal cells.

In one embodiment, 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, where the first NKR-CAR, e.g. KIR-CAR , is actNKR-CAR, eg actKIR-CAR, and the second NKR-CAR, eg KIR-CAR, is inhNKR-CAR, eg inhKIR-CAR. In one embodiment, the invention provides an inhNKR-CAR, eg inhKIR-CAR, wherein binding of the inhNKR-CAR, eg inhKIR-CAR, to a normal cell results in inhibition of the cytotoxic cell, eg, inhibition of KIR-CAR T cell activity. In one embodiment, binding of an inhNKR-CAR, eg inhKIRCAR, to an antigen associated with a non-malignant cell results in the death of a cytotoxic NKR-CAR cell, eg a KIR-CAR T cell.

In one embodiment, the inhNKR-CAR, eg actKIR-CAR, of the invention can be used in combination with existing CARs to regulate CAR activity. Exemplary CARs are described in PCT/US11/64191, which is incorporated herein by reference in its entirety.

It has been discovered that, in cells containing a constellation of chimeric membrane-embedded receptors containing an antigen-binding domain (CMER), interactions between the CMER antigen-binding domain may be undesirable, for example, because the interaction inhibits the ability of one or more antigen-binding domains to bind to its cognate antigen or may lead to the formation of new binding sites with an unknown cognate antigen. Thus, the present description describes cells containing the first and second non-natural CMER, where the antigen-binding domains minimize such interactions. The present description also describes nucleic acids encoding the first and second non-natural such CMER, as well as methods for obtaining and using such cells and nucleic acids. In one embodiment, the antigen binding domain of one of said first and said second non-natural CMER contains scFv and the other contains a single VH domain, such as a single camel, shark, or lamprey VH domain, or a single VH domain derived from a human or mouse sequence. , or a framework that does not belong to the antibody.

Definitions.

Unless otherwise defined, all technical and scientific terms used in the present description have the same meaning as generally understood by a person skilled in the art to which the invention belongs. While any methods and materials similar or equivalent to those described herein may be used in the practice and/or testing of the present invention, preferred materials and methods are described herein. In describing and disclosing the present invention, the following terminology is used in accordance with how it is defined, where the definition is provided.

It should also be understood that the terminology used in this specification is only intended to describe specific embodiments and is not intended to be limiting.

The singular forms are used herein to refer to one or more than one (eg, at least one) grammatical entity. By way of example, element means one element or more than one element.

Approximately as used herein, references to a measurable quantity such as quantity, duration of time, etc. are intended to include the options ±20% or ±10%, in some cases ±5%, in some cases ±1%, and in some case ±0.1% of the specified value, since such options are suitable for carrying out the described methods.

The term adapter molecule, as used herein, refers to a polypeptide with a sequence that allows interaction with two or more molecules and, in embodiments, promotes the activation or inactivation of a cytotoxic cell. For example, in the case of DAP12, this includes interactions with an activating KIR through charge interactions in the transmembrane domain and interactions with signaling molecules such as ZAP70 or Syk through a phosphorylated ITAM sequence in the cytoplasmic domain.

As used herein, the term antigen or Ag is defined as a molecule that elicits an immune response. Such an immune response may include the formation of antibodies or the activation of specific immunocompetent cells, or both. One skilled in the art will appreciate that any macromolecule, including substantially all proteins or peptides, can serve as an antigen. In addition, antigens can be obtained from recombinant or genomic DNA. One of skill in the art will appreciate that any DNA that contains nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response thus encodes an antigen, as the term is used herein. In addition, one of skill in the art will appreciate that an antigen is not necessarily encoded by the entire nucleotide sequence of a gene alone. It is readily understood that the present invention relates to, but is not limited to, the use of incomplete nucleotide sequences from more than one gene, and that such nucleotide sequences are arranged in various combinations to encode polypeptides that elicit the desired immune response. In addition, one of skill in the art will appreciate that an antigen is not necessarily encoded in its entirety by a gene. It is easy to understand that the antigen can be synthesized or can be isolated from a biological sample. Such a biological sample may include, but is not limited to, a tissue sample, a tumor sample, a cell, or a biological fluid.

As used herein, the term anti-tumor effect refers to a biological effect that can be manifested as a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with a cancerous condition. The antitumor effect can also be manifested in the ability of the peptides, polynucleotides, cells and antibodies of the invention to have a prophylactic effect on the incidence of a tumor in the first place.

As used herein, the term apheresis refers to an art-accepted extracorporeal method in which the blood of a donor or patient is removed from the donor or patient and passed through a device that separates the selected specific component(s) and returns the remainder to the bloodstream of the donor or patient, for example, through retransfusion. Thus, with respect to a sample, apheresis refers to a sample obtained using apheresis.

In accordance with the present invention, the term self-antigen means any self-antigen that is recognized by the immune system as if it were foreign. Self antigens include, but are not limited to, cellular proteins, phosphoproteins, cell surface proteins, cellular lipids, nucleic acids, glycoproteins, including cell surface receptors.

As used herein, the term autoimmune disease is defined as a disorder that results from an autoimmune response. Autoimmune disease is the result of an inadequate and excessive response to self antigen. 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 (type I), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain's syndrome -Barré, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis.

As used herein, the term autologous is intended to refer to any substance that is obtained from a particular individual, where it is then reintroduced to the individual.

As used herein, the term allogeneic refers to any substance derived from another animal of the same species as the individual to which the substance is administered. Two or more individuals are considered to be allogeneic to one another when the genes at one or more loci are not identical. In some aspects, an allogeneic substance from individuals of the same species may be genetically different enough to interact antigenically. In one embodiment, allogeneic refers to a graft obtained from another animal of the same species.

Chimeric antigen receptor: or CAR as the term is used herein, refers to a chimeric polypeptide that shares structural and functional properties with a cell's immune function receptor or adapter molecule, for example, from a T cell or NK cell. CARs include TCAR and NKR-CAR. In embodiments, the CAR contains an antigen-binding domain that binds to a cognate antigen, such as a tumor antigen, as described herein. Once bound to a cognate antigen, a CAR may activate or inactivate the cytotoxic cell in which it resides, or modulate the cell's antitumor activity, or otherwise modulate the cell's immune response.

In some embodiments, the domains in a CAR polypeptide construct are on the same polypeptide chain, eg, contain a chimeric fusion protein. In some embodiments, the domains in a CAR polypeptide construct are not adjacent to each other, eg, are on different polypeptide chains, eg, as provided in RCAR, as described herein.

Natural killer cell immune function receptor-chimeric antigen receptor or NKR-CAR as used herein refers to a CAR that shares functional and structural properties with the natural killer cell immune function receptor (NKR) or adapter molecule from NK- cells. In embodiments, NKRCAR comprises two or all of an antigen-binding domain, a transmembrane domain, eg, an NKR transmembrane domain, and/or a cytoplasmic domain, eg, a NKR cytoplasmic domain.

Fc receptor-chimeric antigen receptor or FcR-CAR as the term is used herein refers to a CAR that shares functional and structural properties with an Fc receptor (FcR). Killer cell immunoglobulin-like receptor-chimeric antigen receptor or KIR-CAR as the term is used herein refers to a CAR that shares functional and structural properties with the killer cell immunoglobulin-like receptor (KIR).

Receptor Ly49 chimeric antigen receptor or Ly49-CAR as the term is used herein refers to a CAR that shares functional and structural properties with the Ly49 receptor (Ly49).

Natural cytotoxicity receptor-chimeric antigen receptor or NCR-CAR, as the term is used herein, refers to a CAR that shares functional and structural properties with the natural cytotoxicity receptor (NCR).

The family of lymphocyte activating signaling molecules-chimeric antigen receptor or SLAM-CAR or SLAMF-CAR as the term is used herein refers to a CAR that shares functional and structural properties with SLAM or SLAMF.

Chimeric T cell antigen receptor or TCAR as the term is used herein refers to CAR. which shares functional and structural properties with an immune function receptor of a cell or an adapter molecule from a T cell. In embodiments, the TCAR comprises an antigenic domain, a primary intracellular signaling domain, and optionally one or more co-stimulatory signaling domains.

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. The antibody may be an intact immunoglobulin derived from natural sources or from recombinant sources, and may be an immunoreactive region of an intact immunoglobulin. The antibodies may be polyclonal or monoclonal, multi or single chain, or intact immunoglobulins and may be obtained from natural sources or from recombinant sources. Antibodies are usually tetramers of immunoglobulin molecules. The antibody molecule described herein may exist in a variety of forms wherein the antigen-binding site of an antibody is expressed as part of a continuous polypeptide chain, including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv), and a humanized or human antibody, for example as described in the present description.

The term antibody fragment refers to at least one region of an intact antibody or recombinant variants thereof and refers to antigen-defining variable regions in

- 31 040145 tact antibody, which is sufficient to ensure recognition and specific binding of the antibody fragment to a target, such as an antigen. Examples of antibody fragments include, but are not limited to, single chain domain antibody (sdAb), Fab, Fab', F(ab')2 and Fv fragments, linear antibodies, scFv antibodies, scFv antibody fragments, linear antibody, single domain antibody such as sdAb (VL or VH), camelid VHH domain, and polyspecific antibodies formed from antibody fragments such as a bivalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region and an isolated CDR or other epitope binding antibody fragments. The antigen binding fragment can also be introduced into single domain antibodies, maxibodies, minibodies, nanobodies, intraantibodies, diabodies, tribodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, Nature Biotechnology, 23:1126-1136 , 2005).

Antigen-binding fragments can also be transplanted into scaffolds based on polypeptides such as type III fibronectin (Fn3) (see US Pat. No. 6,703,199, which describes fibronectin polypeptide mini-antibodies).

The term scFv refers to a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light and heavy chain variable regions are adjacently linked via a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise indicated as used herein, the scFv may contain the VL and VH variable regions in any order, for example, relative to the N-terminus and C-terminus of the polypeptide, the scFv may contain a VL-linker-VH, or may contain a VH-linker- VL.

The term complementarity determining region or CDR, as used herein, refers to amino acid sequences in the variable regions of an antibody that confer antigen specificity and binding affinity. For example, there are generally three CDRs in each heavy chain variable region (eg, HCDR1, HCDR2, and HCDR3) and three CDRs in each light chain variable region (LCDR1, LCDR2, and LCDR3). The precise boundaries of the amino acid sequence of a given CDR can be determined using any of a number of well known schemes, including those described by Kabat et al. (1991), Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (Kabat numbering system), Al-Lazikani et al. (1997) JMB 273,927-948 (Chothia numbering system) or combinations thereof. Under the Kabat numbering system, 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 system, 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 the combined Kabat and Chothia numbering system, in some embodiments, the CDRs correspond to amino acid residues that are part of the Kabat CDR, the Chothia CDR, or both. For example, in some embodiments, the CDRs correspond to amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in a VH, such as a mammalian VH, such as a human VH, and amino acid residues 24-34 (LCDR1) , 50-56 (LCDR2) and 89-97 (LCDR3) in VL, eg mammalian VL, eg human VL.

The portions of the CAR composition of the invention containing an antibody or antibody fragment can be in various forms, where the antigen-binding domain is expressed as part of a continuous polypeptide chain, including, for example, a single-domain antibody (sdAb) fragment, a single-chain antibody (scFv), and a humanized or human antibody. (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, 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 85:58795883; Bird et al., 1988, Science, 242: 423-426). In one aspect, the antigen-binding domain of a CAR composition of the invention comprises an antibody fragment. In an additional aspect, the CAR contains an antibody fragment that contains scFv.

As used herein, the term binding domain or antibody molecule (also referred to herein as a binding domain to a target (e.g., mesothelin)) refers to a protein, e.g., an immunoglobulin chain or fragment thereof, containing at least one immunoglobulin variable domain sequence. . The term binding domain or antibody molecule includes antibodies and antibody fragments. In one embodiment, the antibody molecule is a multispecific antibody molecule, e.g., it comprises a constellation of immunoglobulin variable domain sequences, wherein the first immunoglobulin variable domain sequence of the constellation has binding specificity for a first epitope and the second immunoglobulin variable domain sequence of the population has binding specificity for a second epitope. . In one embodiment, the polyspecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity for no more than two antigens. The bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope.

As used herein, an antibody heavy chain refers to the larger of the two types of polypeptide chains found in all antibody molecules in their natural conformations.

As used herein, an antibody light chain refers to the smaller of the two types of polypeptide chains found in all antibody molecules in their natural conformations. K- and λ-light chains are the two main isotypes of antibody light chains.

As used herein, the term synthetic antibody or recombinant antibody refers to an antibody molecule that is produced using recombinant DNA technology, such as, for example, an antibody molecule expressed by a bacteriophage as described herein. The term should also be interpreted as meaning an antibody molecule that is obtained by synthesizing a DNA molecule encoding an antibody molecule, and where the DNA molecule expresses an antibody protein or an amino acid sequence defining an antibody, where the DNA or amino acid sequence is obtained using DNA synthesis technology or an amino acid sequence that is available and well known in the art.

As used herein, the term cancer is defined as a disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, glioma, etc. The terms tumor and malignant tumor are used interchangeably herein, eg, both terms include solid and fluid, eg, diffuse or circulating tumors. As used herein, the term malignant tumor or tumor includes precancerous as well as malignant tumors and tumors.

The term combination refers to an established combination in a single unit dosage form or combination administration, where a compound of the present invention and a combination partner (e.g., another drug as explained below, also referred to as a therapeutic agent or a co-administration agent) can be administered independently. simultaneously or separately during certain time intervals, especially when such time intervals provide the possibility that the combination partners have a total, for example, synergistic effect. The individual components can be packaged as a set or separately. One or both components (eg powders or liquids) may be reconstituted or diluted to the desired dose prior to administration. The terms co-administration or combined administration, or the like, as used herein, is intended to include the administration of a selected combination partner to one individual in need (e.g., a patient) and is intended to include treatment regimens in which the agents are optionally administered by one and by the same route of administration or simultaneously. As used herein, the term pharmaceutical combination means a product that is the result of mixing or combining more than one active ingredient, and includes stated and unstated combinations of active ingredients. The term fixed combination means that the active ingredients, for example, the compound of the present invention and the combination partner are administered to the patient at the same time in the form of a single unit or dose. The term non-specified combination means that the active ingredients, for example, the compound of the present invention and the combination partner are administered to the patient as separate units simultaneously, simultaneously or sequentially without any particular time limit, where such administration provides therapeutically effective levels of the two compounds in the body. patient. The latter is also used for cocktail therapy, for example, the administration of three or more active ingredients.

As used herein, the term conservative sequence modifications is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody containing an amino acid sequence. Such conservative modifications include substitutions, additions and deletions of amino acids. Modifications can be introduced into an antibody of the invention by standard methods known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced with an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains are defined in the art. These families include amino acids with basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (eg, threonine, valine, isoleucine), and aromatic side chains (eg, tyrosine , phenylalanine, tryptophan, histidine). Thus, for example, one or more amino acid residues in the CDR regions of an antibody of the invention can be replaced with other amino acid residues from the same side chain family, and the modified antibody can be tested for its ability to bind to FRe using the functional assays described herein.

A constitutive promoter is a nucleotide sequence which, when operably linked to a polynucleotide that encodes or defines a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

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

Derived from, as the term is used herein, indicates the relationship between the first and second molecule. Generally refers to a structural similarity between a first molecule and a second molecule and does not impose or include limitation on the method or source for the first molecule that is derived from the second molecule. For example, in the case of an intracellular signaling domain that is derived from a CD3 zeta molecule, the intracellular signaling domain retains the structure of CD3 zeta sufficiently so that it has a necessary function, namely, the ability to generate a signal under suitable conditions. This does not impose or include a limitation on a particular method for obtaining an intracellular signaling domain, for example, it does not mean that in order to provide an intracellular signaling domain, it is necessary to start with a CD3-zeta sequence and delete an undesired sequence or introduce mutations to obtain an intracellular signaling domain.

The term stimulation refers to the primary response induced by the binding of a stimulatory molecule (eg, TCR/CD3 complex) to its cognate ligand, thereby mediating a signaling event such as, but not limited to, signaling through the TCR/CD3 complex. Stimulation may mediate altered expression of certain molecules, such as decreased expression of TGF-β, and/or recognition of cytoskeletal structures, and the like.

The term stimulatory molecule refers to a molecule expressed by a T cell that provides primary cytoplasmic signaling sequence(s) that regulates the primary activation of the TCR complex via the stimulatory pathway, at least some aspect of T cell signaling pathway. In one aspect, the primary signal initiates, for example, the binding of a TCR/CD3 complex to a peptide-loaded MHC molecule, and which results in the mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. The primary cytoplasmic signaling sequence (also referred to as the primary signaling domain) that acts in a stimulatory manner may contain a signaling motif, which is known as an immunoreceptor tyrosine activating motif or ITAM. Examples of ITAMs containing a primary cytoplasmic signal sequence that finds particular use in the invention include, but are not limited to, ITAMs derived from TCR-zeta, FcR-gamma, FcR-beta, CD3-gamma, CD3-delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as ICOS), FcεRI, CD66d, DAP10 and DAP12. In a particular CAR of the invention, the intracellular signaling domain in any one or more CARs of the invention contains an intracellular signal sequence, for example, the primary signal sequence of CD3-zeta. In a particular CAR of the invention, the primary CD3-zeta signal sequence is the sequence provided as SEQ ID NO: 9, or equivalent residues from non-human species, e.g., mouse, rodent, monkey, primate, and the like. In a particular CAR of the invention, the primary CD3-zeta signal sequence is the sequence as provided in SEQ ID NO: 10, or equivalent residues from non-human species, e.g., mouse, rodent, monkey, primate, and the like.

The term antigen presenting cell or APC refers to a cell of the immune system, such as a helper cell (e.g., B cell, dendritic cell, etc.), which presents a foreign antigen complexed with major histocompatibility complex (MHC) molecules on its surface. . T cells can recognize these complexes using their T cell receptors (TCRs). APCs process antigens and present them to T cells.

Intracellular signaling domain, as the term is used in the present description, refers to the intracellular part of the molecule. An intracellular signaling domain can generate a signal that provides immune function to an effector containing a CAR cell, such as a CART cell or a CAR expressing NK cell. Examples of effector immune function, e.g. in CART

- 34 040145 cell or expressing CAR NK cell include cytolytic activity and activity of helpers, including the secretion of cytokines. In embodiments, an intracellular signaling domain transduces an effector function signal and directs the cell to perform a specialized function. While a complete intracellular signaling domain can be used, in many cases the use of a complete chain is not necessary. Where such a truncated portion of the intracellular signaling domain is used, such a truncated portion may be used in place of the intact strand, provided that it converts the effector function signal. Thus, the term intracellular signaling domain is intended to include any truncated portion of an intracellular signaling domain sufficient to signal an effector function.

In one embodiment, the intracellular signaling domain may comprise a primary intracellular signaling domain. Illustrative primary intracellular signaling domains include primary intracellular signaling domains derived from molecules that provide primary stimulation or antigen-dependent stimulation. In one embodiment, the intracellular signaling domain may comprise a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include costimulatory intracellular signaling domains derived from molecules providing costimulatory signals or antigen-dependent stimulation. For example, in the case of a CAR-expressing effector cell of the immune system, such as a CAR-expressing T cell or a CAR-expressing NK cell, the primary intracellular signaling domain may contain a cytoplasmic T-cell receptor sequence, and the costimulatory intracellular signaling domain may contain a cytoplasmic sequence from a co-receptor or co-stimulatory molecules.

The primary intracellular signaling domain may contain a signaling motif, which is known as an immunoreceptor tyrosine activating motif or ITAM. Examples of ITAMs containing primary cytoplasmic signal sequences include, but are not limited to, ITAMs derived from CD3-zeta, FcR-gamma, FcR-beta, CD3-gamma, CD3-delta, CD3-epsilon, CD5, CD22, CD79a, CD79b , CD278 (ICOS), FceRI, CD66d, DAP10 and DAP12.

The term zeta or alternatively zeta chain, CD3 zeta or TCR zeta is defined as a protein provided as a protein with GenBan accession number BAG36664.1 or equivalent residues from a non-human species, e.g. mouse, rodent, monkey, primate, etc. and the zeta stimulatory domain or alternatively the CD3 zeta stimulatory domain or the TCR zeta stimulatory domain is defined as amino acid residues from the cytoplasmic domain of the zeta chain that are sufficient to functionally transmit the initial signal required for T cell activation. In one aspect, the cytoplasmic zeta domain contains residues 52 to 164 of GenBank accession number BAG36664.1, or equivalent residues from non-human species, eg, mouse, rodent, monkey, primate, etc., that are their functional orthologues. In one aspect, the zeta stimulatory domain or CD3 zeta stimulatory domain is the sequence provided 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.

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 essential for an effective immune response. Costimulatory molecules include, but are not limited to, MHC class I molecule, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, lymphocyte-activating signaling molecules (SLAM proteins), NK cell-activating receptors, BTLA, Toll-like receptor, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a/CD18), 41BB (CD137), B7-H3, 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, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, L AT, GADS, SLP-76, PAG/Cbp, CD19a, and a ligand that specifically binds to CD83.

A costimulatory intracellular signaling domain refers to the intracellular region of a costimulatory molecule.

An intracellular signaling domain may comprise a complete intracellular region or a complete native intracellular signaling domain of the molecule from which it is derived, or a functional fragment thereof.

The term 4-1BB refers to a member of the TNFR superfamily with the amino acid sequence

- 35 040145 value provided as GenBank accession number AAA62478.2, or equivalent remnants from non-human species, eg mouse, rodent, monkey, primate, etc.; and co-stimulatory domain 4-1BB is defined as amino acid residues 214-255 of GenBank accession number AAA62478.2 or equivalent residues from non-human species, eg, mouse, rodent, monkey, primate, and the like. In one aspect, the 4-1BB co-stimulatory domain is the sequence provided as SEQ ID NO: 7, or equivalent residues from a non-human species, eg, mouse, rodent, monkey, primate, and the like.

An effector cell of the immune system, as the term is used herein, refers to a cell that is involved in an immune response, for example, in activating an immune effector response. Examples of immune system effector cells include T cells, such as alpha/beta T cells and gamma/delta T cells, B cells, natural killer (NK) cells, natural killer T(NKT) cells, mast cells, and myeloid phagocytes.

Immune effector function or immune effector response, as the term is used herein, refers to a function or response, for example, of an effector cell of the immune system that enhances or activates an immune attack on a target cell. For example, immune effector function or response refers to a property of a T or NK cell that promotes cytolysis or inhibition of growth or proliferation of a target cell. In the case of a T cell, examples of immune effector function or response are primary stimulation and costimulation.

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

The term coding refers to the natural property of particular nucleotide sequences in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as templates for the synthesis of other polymers and macromolecules in biological processes with a particular nucleotide sequence (e.g., rRNA, tRNA, and mRNA) or a particular amino acid sequence. and the biological properties associated with them. Thus, a gene encodes a protein if the transcription and translation of the mRNA corresponding to such a gene results in the production of a protein in a cell or other biological system. A coding strand, a nucleotide sequence that is identical to an mRNA sequence and generally provided in sequence listings, and a non-coding strand used as a template for transcription of a gene or cDNA may be referred to as encoding a protein or other product of that gene or cDNA.

Unless otherwise indicated, a nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate variants of one another and that encode the same amino acid sequence. Nucleotide sequences that code for proteins and RNA may contain introns.

The term effective amount or therapeutically effective amount is used interchangeably herein and refers to an amount of a compound, compound, substance or composition as described herein effective to achieve a particular biological result. Such results may include, but are not limited to, inhibition of viral infection, as determined by any means suitable in the art.

As used herein, endogenous refers to any substance from an organism, cell, tissue, or system, or a substance produced in an organism, cell, tissue, or system.

As used herein, the term exogenous refers to any substance introduced into an organism, cell, tissue, or system, or produced outside the organism, cell, tissue, or system.

The term expression, as used herein, is defined as the transcription and/or translation of a specific nucleotide sequence driven by its promoter.

The term transfer vector refers to a composition of matter which contains an isolated nucleic acid and which can be used to deliver the isolated nucleic acid into a cell. Various vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides linked to ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term transfer vector includes an autonomously replicating plasmid or virus. The term is also to be construed to further include non-plasmid and viral compounds that facilitate the transfer of nucleic acid into cells, such as, for example, a polylysine compound, a liposome, and the like. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.

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

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.

As used herein, the term homologous or identity refers to the sequence identity of a subunit of two polymeric molecules, for example two nucleic acid molecules, such as two DNA molecules or two RNA molecules, or two polypeptide molecules. When the position of a subunit in both of the two molecules is occupied by the same monomeric subunit; for example, if a position in each of two DNA molecules is occupied by adenine, then they are homologous at that position. The homology of two sequences depends directly on the number of matching or homologous positions; for example, if half (eg, five positions in a polymer ten subunits long) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the sequences (eg, 9 out of 10) match or are homologous, the two sequences are 90% homologous.

Humanized forms of non-human antibodies (e.g., mice) are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antibody antigen-binding subsequences) that contain the minimum sequence obtained from non-human immunoglobulin. Advantageously, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the complementarity determining region (CDR) of the recipient are replaced with residues from a CDR belonging to a non-human species (donor antibody) such as mice, rats or rabbits, with the desired specificity, affinity and capacity. In some instances, framework region (FR) residues of the human Fv immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies may contain residues that are not found in either the recipient antibody or the imported CDR or framework sequences. Such modifications are conducive to further improve and optimize antibody performance. Typically, a humanized antibody contains substantially all of at least one, and typically two, variable domains wherein all or substantially all of the CDRs correspond to CDRs of a non-human immunoglobulin and all or substantially all of the FRs are FR sequences of human immunoglobulin. The humanized antibody optimally also comprises at least an immunoglobulin constant region (Fc) region, typically a human immunoglobulin constant region region. For more 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.

Wholly human refers to an immunoglobulin, such as an antibody or antibody fragment, where the entire molecule is human or consists of an amino acid sequence identical to the human form of the antibody or immunoglobulin.

As used herein, educational material includes a publication, record, chart, or any other medium of expression that can be used to communicate the usefulness of the compositions and methods of the invention. The training material of the kit according to the invention can, for example, be attached to a container that contains the nucleic acid, peptide and/or composition of the invention, or supplied together with a container that contains the nucleic acid, peptide and/or composition. Alternatively, the training material may be supplied separately from the container, with the intent that the training material and connection are shared by the recipient.

The term isolated means altered or removed from the natural state. For example, a nucleic acid or peptide naturally present in a living animal is not isolated, but the same nucleic acid or peptide, partially or completely separated from the accompanying substances from its natural state, is isolated. An isolated nucleic acid or protein may exist in a substantially purified form, or may exist in a non-natural environment such as, for example, a host cell.

In the context of the present invention, the following abbreviations are used for commonly occurring nucleic acid bases. A refers to adenosine, C refers to cytosine, G refers to guanosine, T refers to thymidine, and U refers to uridine.

Unless otherwise indicated, a nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate variants of each other and that encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA may also include introns in cases where such nucleotide sequence encoding a protein may, in a certain embodiment, contain intron(s).

- 37 040145

As used herein, lentivirus belongs to a genus in the family Retroviridae. Lentiviruses are unique among retroviruses in that they are able to infect non-dividing cells; they can deliver a significant amount of genetic information to the DNA of the host cell, thus they represent one of the most efficient vector methods for delivering genes. HIV, SIV and FIV are examples of lentiviruses. Vectors derived from lentiviruses provide a means of obtaining significant levels of gene transfer in vivo.

The term lentiviral vector refers to a vector derived from at least a portion of the lentivirus genome, in particular including a self-inactivating lentiviral vector as provided by Milon e et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentiviral vectors that can be used in the clinic include, but are not limited to, for example, LENTIVECTOR® gene delivery technology from Oxford BioMedica, the LENTIMAX™ vector system from Lentigen, and the like. Types of lentiviral vectors for non-clinical use are also available and known to those skilled in the art.

The term flexible polypeptide linker or linker, as used in relation to scFv, refers to a peptide linker that consists of amino acids, such as glycine and/or serine residues, used alone or in combination to link heavy chain variable regions and light chain variable regions to each other. friend. In one embodiment, the flexible polypeptide linker is a Gly/Ser linker and contains the amino acid sequence (Gly-Gly-Gly-Ser)n (SEQ ID NO: 38) where n is a positive integer equal to or greater than 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 one embodiment, flexible polypeptide linkers comprise, but are not limited to, (Gly4 Ser)4 (SEQ ID NO: 27) or (Gly4 Ser)3 (SEQ ID NO: 28). In another embodiment, the linkers contain multiple repeats of (Gly2Ser), (GlySer) or (Gly3Ser) (SEQ ID NO: 29). Also within the scope of the invention are the linkers described in WO 2012/138475, incorporated herein by reference.

An NK cell immune function receptor, or NKR as the term is used herein, refers to an endogenous natural transmembrane protein expressed in NK cells that can interact with a ligand on an antigen presenting cell and modulate the NK cell's immune response, for example, it can modulate the cytolytic cytokine activity or secretion of NK cells. NKR can promote activation (activating NKR or actNKR) or inhibition (inhibitory NKR or inhNKR). Typically, NKR contains an extracellular ligand-binding domain (ECD), a transmembrane domain (TM), and an intracellular cytoplasmic domain (ICD). NKR includes the killer cell immunoglobulin-like receptor (KIR) receptor family such as KIR2DS2, the NK cell receptor (NCR) family such as NKp46 (NCR1), the signaling lymphocyte-activating receptor (SLAM) receptor (SLAMF) family such as 2B4, and Fc binding receptors such as the IgG binding receptor, CD16 (FcyRIII). Examples of NK cell immune responses modulated by NKR include target cell killing (often referred to as cytotoxicity or cytolysis), cytokine secretion and/or proliferation. Typically, the NKR suitable for use in the methods and compositions described herein is human NKR (or hNKR). In one embodiment, the Mus musculus Ly49 receptor family is also included, which has evolved convergently to provide the same function as KIR in murine NK and T cells.

The term operably linked refers to a functional relationship between a regulatory sequence and a heterologous nucleic acid sequence resulting from the expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in an operative 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. In general, operably linked DNA sequences are contiguous and necessarily join two protein coding regions in the same reading frame.

Parenteral administration of an immunogenic composition includes, for example, subcutaneous (SC), intravenous (IV), intramuscular (IM), or intrasternal injection or infusion techniques.

The term nucleic acid, nucleic acid molecule, or polynucleotide, as used interchangeably herein, is defined as a chain of nucleotides. In addition, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has general knowledge that nucleic acids are polynucleotides that can be hydrolyzed to monomeric nucleotides. Monomeric nucleotides can be hydrolyzed to nucleotides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences that any

- 38 040145 by all methods available in this field, including without limitation, recombinant methods, i.e. cloning nucleic acid sequences from a recombinant library or cell genome using conventional cloning technology and PCR™, and the like, and synthetic methods. In embodiments, nucleic acids as used herein are deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or a combination of DNA or RNA, and polymers thereof in single or double stranded form. The term nucleic acid includes a gene, cDNA or mRNA. In one embodiment, the nucleic acid molecule is synthetic (eg, chemically synthesized) or recombinant. Unless specifically limited, the term includes nucleic acids containing analogues or derivatives of natural nucleotides that have similar binding properties as a reference nucleic acid and are metabolized in a similar manner to natural nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes its conservatively modified variants (eg, degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly specified. In particular, degenerate codon substitutions can be generated by generating sequences in which the third position of one or more selected (or all) codons is replaced 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)).

As used herein, the terms peptide, polypeptide, and protein are used interchangeably and refer to a compound 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 restriction on the maximum number of amino acids that a protein or peptide sequence may contain. Polypeptides include any peptide or protein containing two or more amino acids connected to each other by peptide bonds. As used herein, the term refers to both shorter chains, which are also commonly referred to in the art, such as peptides, oligopeptides, and oligomers, and longer chains, which are generally referred to in the art as proteins, for which there are many types. Polypeptides include, for example, among other biologically active fragments, essentially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

As used herein, the term promoter is defined as a DNA sequence that is recognized by a cell's synthesis machinery or an input synthesis machinery necessary to initiate a specific transcription of a polynucleotide sequence.

As used herein, the term promoter/regulatory sequence means a nucleic acid sequence that is necessary for the expression of a gene product operably linked to a promoter/regulatory sequence. In some cases, such a sequence may be a core promoter sequence, and in other cases, such a sequence may also contain an enhancer sequence and other regulatory elements that are necessary for the expression of the gene product. The promoter/regulatory sequence may, for example, be a sequence that expresses the gene product in a tissue-specific manner.

An inducible promoter is a nucleotide sequence which, when operably linked to a polynucleotide that encodes or establishes a gene product, causes the gene product to be formed in a cell essentially only when the promoter is present in the cell.

A tissue-specific promoter is a nucleotide sequence which, when operably linked to a polynucleotide that encodes or establishes a gene product, causes the gene product to be generated in a cell substantially only if the cell is a tissue type cell.

The term signal transduction pathway refers to the biochemical relationship between different signal transduction molecules that play a role in transmitting a signal from one part of a cell to another part of the cell. The phrase cell surface receptor includes molecules and complexes of molecules capable of receiving a signal and transmitting a signal across the cell membrane.

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

As used herein, a substantially purified cell refers to a cell that is substantially free of other cell types. A substantially purified cell also refers to a cell that has been separated from other cell types with which it is normally associated in its natural state. In some instances, a population of substantially purified cells refers to a homogeneous population of cells. In other cases, the term simply refers to a cell that has been separated from the cells with which it is naturally associated in its natural state. In some variants

- 39 040145 implementation cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

As used herein, a 5' cap (also called an RNA cap, 7-methylguanosine RNA cap, or m ⁷ G-cap RNA) is a modified guanine nucleotide that is added to the front or 5' end of the eukaryotic messenger RNA immediately after the start of transcription. The 5' cap consists of an end group that is linked to the first nucleotide to be transcribed. Its presence is critical for ribosome recognition and protection against RNases. The addition of a cap is transcriptionally coupled and occurs co-transcriptionally, such that one influences the other. Immediately after the start of transcription, the 5' end of the synthesized mRNA is bound by the synthesizing cap complex associated with RNA polymerase. This enzymatic complex catalyzes the chemical reactions that are necessary for mRNA capping. Synthesis takes place in the form of multi-stage biochemical reactions. The capping group can be modified to modulate the functionality of the mRNA, such as its stability or translation efficiency.

As used herein, in vitro transcribed RNA refers to RNA, preferably mRNA, that has been synthesized in vitro. Typically, in vitro transcribed RNA is derived from an in vitro transcription vector. An in vitro transcription vector contains a template that is used to generate in vitro transcribed RNA.

As used herein, poly(L) is a series of adenosines attached via polyadenylation to an mRNA. In a preferred embodiment, the construct for transient expression of polyA is between 50 and 5000 (SEQ ID NO: 30), preferably greater than 64, more preferably greater than 100, most preferably greater than 300 or 400. Poly(L) sequences may be chemically or enzymatically modified to modulate functionality of the mRNA, such as localization, stability, or translation efficiency.

As used herein, polyadenylation refers to the covalent attachment of a polyadenyl group, or a modified variant thereof, to a messenger RNA molecule. In eukaryotic organisms, most messenger RNA (mRNA) molecules are polyadenylated at the 3' end. The 3'-poly(A) tail is a long sequence of adenine nucleotides (usually several hundred) added to the pre-mRNA by the action of the enzyme polyadenylate polymerase. In higher eukaryotes, a poly(L) tail is added to a transcript that contains a specific sequence, a polyadenylation signal. The poly(L) tail and its protein bond help protect mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, mRNA export from the nucleus, and translation.

Polyadenylation occurs in the nucleus immediately after the transcription of DNA into RNA, but additionally can also occur later in the cytoplasm. After transcription is terminated, the mRNA chain is cleaved by the action of the endonuclease complex associated with RNA polymerase. The cleavage site is typically characterized by the presence of the LLLLL base sequence near the cleavage site. After mRNA cleavage, adenosine residues are added to the free 3'-end at the cleavage site.

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

As used herein, the term therapeutic means treatment and/or prophylaxis. The therapeutic effect is obtained by suppression, remission or eradication of the disease state.

As used herein, the term transfected or transformed or transduced refers to a method by which an exogenous nucleic acid is transferred or introduced into a host cell. A transfected or transformed or transduced cell is a cell that has been transfected, transformed or transduced with an exogenous nucleic acid. A cell includes an individual's primary cell and its progeny.

As used herein, the terms treat, treatment, and treating refer to reducing or ameliorating the progression, severity, and/or duration of a proliferative disorder, or ameliorating one or more symptoms (preferably one or more prominent symptoms) of a proliferative disorder that results from one or more therapies (eg, one or more therapeutic agents such as the CLR of the invention). In specific embodiments, the terms treat, treatment, and treating refer to an improvement in at least one measurable physical parameter of a proliferative disorder, such as tumor growth, not necessarily in a patient. In other embodiments, the terms treat, treatment, and treating refer to inhibiting the progression of a proliferative disorder physically, eg, by stabilizing a significant symptom, physiologically, eg, by stabilizing a physical parameter, or both. In others

- 40 040145 embodiments, the terms treat, treatment, and treating refer to reducing or stabilizing the size of a tumor or the number of malignant cells.

As used herein, the term prophylaxis means the prevention or protective treatment of a disease or disease condition.

As used herein, the term tumor antigen refers to a molecule (usually a protein, carbohydrate, or lipid) that is expressed on the surface of a cancer cell or tumor cell, in whole or in fragment form (e.g., MHC/peptide), and which is suitable for preferentially targeting the pharmacological agent to the malignant cell or tumor cell. In some embodiments, the tumor antigen is a marker expressed by normal cells and malignant cells, such as a differentiation marker, such as CD19 or CD123 on B cells. In some embodiments, the tumor antigen is a cell surface molecule that is overexpressed in the malignant cell compared to the normal cell, e.g., 1-fold overexpression, 2-fold overexpression, 3-fold overexpression or more compared to a normal cell. In some embodiments, the tumor antigen is a cell surface molecule that is inappropriately synthesized in the cancer cell, for example, a molecule that contains deletions, additions, or mutations compared to a molecule expressed by a normal cell. In some embodiments, the tumor antigen is expressed only on the cell surface of the malignant cell in whole or as a fragment (eg, MHC/peptide) and is not synthesized or expressed on the surface of a normal cell.

In some embodiments, the CARs of the present invention refer to CARs containing an antigen-binding domain (eg, an antibody or antibody fragment) that binds to an MHC-provided peptide. Typically, peptides derived from endogenous proteins fill pockets of the major histocompatibility complex (MHC) class I molecule and are recognized by T cell receptors (TCRs) on CD8+ T lymphocytes. Class I MHC 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 antigens or tumor antigens against human leukocyte antigen (HLA)-A1 or HLA-A2 have been described (see, for example, Sastry et al., J Virol., 2011 85(5):1935-1942; Sergeeva et al., Blood, 2011 117(16):4262-4272; Verma et al., J. Immunol., 2010 184(4):2156-2165; Willemsen et al. ., Gene Ther., 2001 8(21):1601-1608; Dao et al., Sci. Transl. Med., 2013 5(176):176ra33; Tassev et al., Cancer Gene Ther., 2012 19(2 ):84-100). For example, a TCR-like antibody can be identified based on a screening of a library, such as a human scFv phage display library.

As used herein, the phrase under transcriptional control or operably linked means that the promoter is in the correct position and orientation with respect to the polynucleotide to control the initiation of transcription by the RNA polymerase and the expression of the polynucleotide.

A vector is a composition of matter which contains an isolated nucleic acid and which can be used to deliver the isolated nucleic acid into a cell. Various vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides linked to ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term transfer vector includes an autonomously replicating plasmid or virus. The term is also to be construed to further include non-plasmid and viral compounds that facilitate the transfer of nucleic acid into cells, such as, for example, a polylysine compound, a liposome, and the like. Examples of viral transfer vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.

As used herein, the term specifically binds refers to an antibody or ligand that recognizes and binds to a cognate binding partner protein contained in a sample, but where the antibody or ligand does not substantially recognize or bind to other molecules in the sample.

By the term stimulation is meant the primary response induced by the binding of a stimulatory molecule to its cognate ligand, thus mediating a signaling event, such as, but not limited to, signaling through the corresponding NK receptor.

Xenogeneic refers to a transplant obtained from an animal of a different species.

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

- 41 040145

As used herein, a regulated chimeric antigen receptor (RCAR) refers to a group of polypeptides, typically two in the simplest embodiments, which, when located in an effector cell of the immune system, provide the cell with specificity for a target cell, typically a malignant cell. and regulated intracellular signal generation. In some embodiments, an RCAR comprises at least an extracellular antigen-binding domain, a transmembrane and cytoplasmic signaling domain (also referred to herein as an intracellular signaling domain) containing a functional signaling domain derived from a stimulatory molecule and/or a co-stimulatory molecule as defined herein. with respect to the CAR molecule. In some embodiments, the groups of polypeptides in an RCAR are not adjacent to each other, eg, are on different polypeptide chains. In some embodiments, RCARs comprise a dimerization switch that, in the presence of a dimerization-inducing molecule, can cause the polypeptides to pair with one another, for example, can lead to the pairing of an antigen-binding domain with an intracellular signaling domain. In some embodiments, an RCAR is expressed in a cell (eg, an effector cell of the immune system) as described herein, eg, an RCAR expressing cell (also referred to herein as an RCARX cell). In one embodiment, the RCARX cell is a T cell and is referred to as a RCART cell. In one embodiment, the RCARX cell is a NK cell and is referred to as an RCARN cell. An RCAR can provide an RCAR-expressing cell with specificity for a target cell, typically a malignant cell, and with regulated intracellular signal generation or proliferation that can optimize the immune effector properties of the RCAR-expressing cell. In embodiments, an RCAR cell is based at least in part on an antigen-binding domain, which provides specificity for a target cell that contains an antigen bound by the antigen-binding domain.

Membrane anchor or membrane anchor domain, as the term is used herein, refers to a polypeptide or group, such as a myristoyl group, sufficient to anchor an extracellular or intracellular domain in the plasma membrane.

A switch domain, as the term is used herein, for example when referring to RCAR, refers to a structural unit, typically based on a polypeptide, which, in the presence of a dimerization-inducing molecule, is associated with another switch domain. The association results in operable linking of a first entity associated, for example, fused to the first switching domain, and a second entity associated, for example, fused to the second switching domain. The first and second switch domains are collectively referred to as the dimerization switch. In embodiments, the first and second switch domains are similar, for example, they may be polypeptides containing the same primary amino acid sequence and are collectively referred to as a homodimerization switch. In embodiments, the first and second switch domains are different from each other, for example, they are polypeptides containing different primary amino acid sequences and are collectively referred to as a heterodimerization switch. In embodiments, the switch is intracellular. In embodiments, the switch is extracellular. In embodiments, the switch domain is a polypeptide-based entity, eg, FKBP or FRB-based, and the dimerization-inducing molecule is a small molecule, eg, rapalogue. In embodiments, the switch domain is a polypeptide-based entity, such as scFv, that binds to the myc peptide, and the dimerization-inducing molecule is a polypeptide, a fragment thereof, or a polypeptide multimer, such as a myc ligand or myc ligand multimers that bind to one or more scFv myc. In embodiments, the switch domain is a polypeptide-based entity, eg, the myc receptor, and the dimerization-inducing molecule is an antibody or fragments thereof, eg, a myc antibody.

Dimerization-inducing molecule, as the term is used herein, for example, in relation to RCAR, refers to a molecule that facilitates the formation of a link between the first switch domain and the second switch domain. In embodiments, the dimerization-inducing molecule does not naturally occur in the individual or does not occur at concentrations that result in significant dimerization. In embodiments, the dimerization-inducing molecule is a small molecule, such as rapamycin or rapalogue, such as RAD001.

The term bioequivalent refers to the amount of an agent other than a reference compound (eg, RAD001) required to produce an effect equivalent to that of a reference dose or reference amount of a reference compound (eg, RAD001). In one embodiment, the effect is the level of mTOR inhibition, e.g., as measured by P70S6 kinase inhibition, e.g., as assessed in an in vivo or in vitro assay, e.g.

- 42 040145 as measured by the assay described in the present invention, for example, the Boulay assay, or by measuring levels of phosphorylated S6 by Western blotting. In one embodiment, the effect is a change in the ratio of PD-1 positive/PD-1 negative effector cells of the immune system, eg T cells or NK cells, as measured by cell sorting. In one embodiment, a bioequivalent amount or dose of an mTOR inhibitor is an amount or dose that provides the same level of P70S6 kinase inhibition as provided by a reference dose or a reference amount of a reference compound. In one embodiment, a bioequivalent amount or dose of an mTOR inhibitor is an amount or dose that provides the same level of change in the ratio of PD-1 positive/PD-1 negative immune system effector cells, e.g., T cells or NK cells, as provided by a reference dose or a reference amount of a reference compound.

The term low immune-enhancing dose when used in combination with an mTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g., RAD001 or rapamycin, or a catalytic mTOR inhibitor, refers to a dose of an mTOR inhibitor that partially, but not completely, inhibits mTOR activity, e.g., as measured by inhibition of P70S6 kinase activity. Methods for assessing mTOR activity, such as P70S6 kinase inhibition, are described herein. The dose is insufficient to result in complete immune suppression, but is sufficient to enhance the immune response. In one embodiment, a low immune-boosting dose of an mTOR inhibitor results in a decrease in PD-1 positive immune system effector cells, e.g., T cells or NK cells, and/or an increase in PD1-negative immune system effector cells, e.g., T cells or NK cells, or an increase in the ratio of PD-1 negative T cells/PD-1 positive effector cells of the immune system, such as T cells or NK cells.

In one embodiment, a low immune-boosting dose of an mTOR inhibitor results in an increase in the number of intact effector cells of the immune system, such as T cells or NK cells. In one embodiment, a low immune-boosting dose of an mTOR inhibitor results in one or more of the following:

increased expression of one or more of the following markers: CD62L ^high , CD127 ^hlgh , CD27+ and BCL2, eg on memory T cells, eg memory T cell progenitors;

decreased expression of KLRG1, e.g., on memory T cells, e.g., memory T cell progenitors, and increased memory T cell progenitors, e.g., cells with any one or combination of the following: increased CD62L ^high , increased CD127 ^high , increased CD27+, decreased KLRG1 and increased BCL2;

where any of the changes described above occurs, for example, at least transiently, for example, compared with an untreated individual.

As used herein, refractory refers to a disease, such as cancer, that does not respond to treatment. In embodiments, a refractory cancer may be resistant to treatment before or during treatment. In other embodiments, a refractory cancer may become resistant during treatment. Refractory cancer is also called resistant cancer.

As used herein, relapsing or relapse refers to the return or recurrence of a disease (e.g., cancer) or signs and symptoms of a disease, such as cancer, after a period of improvement or response, e.g., after prior treatment with a therapy, e.g., therapy malignant tumor. The initial response period may include a decrease in the level of malignant cells below a certain threshold value, for example, below 20, 1, 10, 5, 4, 3, 2, or 1%. Reappearance may include an increase in the level of malignant cells above a certain threshold value, for example, above 20, 1, 10, 5, 4, 3, 2, or 1%. For example, with respect to B-ALL, reappearance may include, for example, reappearance of blasts in the blood, bone marrow (>5%) or any extramedullary site after a complete response. A complete response in this context may include <5% BM blasts. More generally, in one embodiment, a response (eg, complete response or partial response) may include no detectable MRD (minimal residual disease). In one embodiment, the initial response period lasts at least 1, 2, 3, 4, 5, or 6 days, at least 1, 2, 3, or 4 weeks, at least 1, 2, 3, 4, 6 , 8, 10, or 12 months, or at least 1, 2, 3, 4, or 5 years.

In some embodiments, a therapy that involves the administration of a CD19 inhibitor, such as CD19 CAR therapy, may result in relapse or treatment-refractory disease. Relapse or resistance may be caused by loss of CD19 (eg, loss of antigen mutation) or other change in CD19 that reduces the level of CD19 (eg, caused by clonal selection of CD19-negative clones). A malignant tumor characterized

- 43 040145 such CD19 loss or change is referred to herein as CD19 negative cancer or CD19 negative recurrent cancer. It should be understood that a CD19-negative cancer does not necessarily have a 100% loss of CD19, but a sufficient decrease to reduce the effectiveness of CD19 therapy such that such a cancer recurs or becomes refractory. In some embodiments, the CD19-negative cancer results from CD19 CAR therapy.

Ranges: Throughout this description, various aspects of the invention may be provided in range format. It should be understood that the description in range format is for convenience and brevity only and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to specifically describe all possible subranges as well as individual numerical values within that range. For example, a description of a range such as 1 to 6 should be considered to specifically describe subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 up to 6, and so on, as well as individual numbers in that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

NKR-CAR.

Disclosed herein are compositions and methods for regulating the specificity and activity of cytotoxic cells, eg, T cells or NK cells, eg, with a non-natural chimeric antigen receptor (CAR). In one embodiment, the CAR is NKR-CAR. NKR-CAR is a CAR that shares functional and structural properties with the NK cell immune function receptor (or NKR). NKR and NKR-CAR are described in the present description, for example, in the section below. As discussed below, the NKR series can serve as the basis for the NKR-CAR.

NK cell immune function receptors (NKR) and NK cells.

As discussed herein, an NK cell immune function receptor (or NKR) refers to an endogenous natural transmembrane protein expressed by NK cells that can interact with a ligand on an antigen presenting cell and modulate the NK cell's immune response, e.g., it can modulate cytolytic activity or secretion of cytokines by NK cells.

NK cells are mononuclear cells that develop in the bone marrow from lymphoid progenitors, and morphological features and biological properties typically include expression of the cluster determinant (CD) of CD16, CD56 and/or CD57; absence of TCR alpha/beta or gamma/delta complex on the cell surface; the ability to bind to and cause death of target cells that are unable to express their major histocompatibility complex (MHC)/human leukocyte antigen (HLA) proteins; and the ability to induce the death of 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 have a regulatory effect on the immune system; and can undergo several stages of cell division and produce daughter cells with biological properties similar to the parent cell. Upon activation by interferons and/or cytokines, NK cells mediate the lysis of tumor cells and cells infected with intracellular pathogens through mechanisms that require direct physical contact between the NK cell and the target cell. Target cell lysis involves the release of cytotoxic granules by the NK cell onto the surface of the bound target and effector proteins such as perforin and granzyme B, which penetrate the target's plasma membrane and induce apoptosis or programmed cell death. Normally, healthy cells are protected from lysis by NK cells.

NK cell activity is regulated by a complex mechanism that includes stimulatory and inhibitory signals.

Briefly, the lytic activity of NK cells is regulated by various cell surface receptors that translate positive or negative intracellular signals upon interaction with ligands on the target cell. The balance between positive and negative signals transmitted through such receptors determines whether the NK cell lyses or does not lyse (cause death) of the target cell. NK cell stimulatory signals can be 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, as well as certain inhibitory KIRs that recognize major histocompatibility complex (MHC) class I molecules (Karre et al., Nature, 1986; 319:6758; Ohlen et al., Science, 1989;246:666-8). These inhibitory receptors bind to polymorphic

- 44 040145 determinants of MHC class I molecules (including HLA class I) present on other cells and inhibit NK cell mediated lysis.

KIR-CAR.

Disclosed herein is a chimeric antigen receptor (CAR) molecule comprising an antigen-binding domain and a killer cell immunoglobulin-like receptor domain (KIR-CAR). In one embodiment, the implementation of the KIR-CAR according to the invention is expressed on the surface of the effector cells of the immune system, for example, T cells or NK cells.

NKCAR based on KIR-CAR.

KIRs, referred to as killer cell immunoglobulin-like receptors, have been characterized in humans and non-human primates, and are type 1 polymorphic transmembrane molecules present on certain subgroups 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, various KIRs are stimulatory or inhibitory for NK cells.

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

KIRs are a family of cell surface proteins found on NK cells. They regulate the cytolytic function of such cells by interacting with class I MHC molecules, which are expressed by all cell types. This interaction allows them to detect virus-infected cells or tumor cells. Most KIRs are inhibitory, which means that their recognition of MHC suppresses the cytotoxic activity of the NK cell that expresses them. Only a limited number of KIRs have the ability to activate cells.

The KIR gene family contains at least 15 gene loci (KIR2DL1, KIR2DL2/L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1/S1, KIR3DL2, KIR3DL3 and two pseudogenes KIR2DP1 and KIR3DP1) encoded in 100-200 kb region leukocyte receptor complex (LRC) complex located on chromosome 19 (19q13.4). LRC is a large 1 mbp. and a dense cluster of rapidly evolving immune response genes that contain genes encoding other cell surface molecules with distinct Ig-like extracellular domains. In addition, the extended LRC contains genes encoding the transmembrane adapter molecules DAP10 and DAP12.

The KIR genes vary in length from 4 to 16 kb. (complete genomic sequence) and may contain from four to nine exons. KIR genes are classified as belonging to one of three groups according to their structural features: (1) type I KIR2D genes, which encode two extracellular domain proteins with D1 and D2 conformations; (2) structurally divergent type II KIR2D genes that encode two extracellular domain proteins with D0 and D2 conformations; and finally (3) the KIR3D genes encoding proteins with three extracellular Ig-like domains (D0, D1 and D2).

The type I KIR2D genes, which encode the KIR2DP1 pseudogene, as well as the KIR2DL1-3 and KIR2DS1-5 genes, contain eight exons, as well as the sequence of pseudoexon 3. This pseudoexon is inactivated in type I KIR2D. In some cases, this is due to a nucleotide substitution located at the intron 2-exon 3 splicing site, where its nucleotide sequence has a high degree of identity with KIR3D exon 3 sequences and is characterized by a characteristic three-base-pair deletion. In other cases, a premature stop codon initiates exon 3 differential splicing. In the type I KIR2D gene group, KIR2DL1 and KIR2DL2 share a deletion in exon 7, distinguishing them from all other KIRs in that exon, resulting in a shorter exon 7 sequence. Similarly, in KIR2D type I, KIR2DL1-3 differs from KIR2DS1-5 only in the length of the region encoding the cytoplasmic tail in exon 9. The structure of the KIR2DP1 pseudogene differs from that of KIR2DL1-3 in that the former has a shorter exon 4 sequence due to the deletion of one base pair.

Type II KIR2D genes include KIR2DL4 and KIR2DL5. In contrast to KIR3D and KIR2D type I, KIR2D type II characteristically deletes the region corresponding to exon 4 in all other KIRs. In addition, type II KIR2D genes differ from type I KIR2D genes in that the former contain a translated exon 3, while type I KIR2D genes contain an untranslated pseudoexon 3 sequence in its place. In type I IKIR2D genes, KIR2DL4 further differs from KIR2DL5 (and from other KIR genes) by the length of its exon 1 sequence. Exon 1 in KIR2DL4 was found to be six nucleotides longer and contain a start codon different from the start codon contained in other KIR genes. This start codon matches the Kozak consensus transcription start sequence better than the second possible start codon in KIR2DL4, which matches the start codon found in other KIR genes.

The KIR3D genes contain nine exons and include the structurally related genes KIR3DL1, KIR3DS1, KIR3DL2, and KIR3DL3. The nucleotide sequences of KIR3DL2 are the longest of all KIR genes and span 16256 bp. in genome-wide sequences and 1368 b.p. V

- 45 040145 cDNA. In the KIR3D group, 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 length (in some KIR3DS1 alleles) to 108 amino acid residues in length (in KIR2DL4 proteins). Furthermore, KIR3DS1 differs from KIR3DL1 or KIR3DL2 in that the former contains a shorter exon 8 sequence. KIR3DL3 differs from other KIR sequences in that it does not contain exon 6 at all. This gene fragment contains absolutely no exon 69, and in rare cases only exon 2. The remaining portions of the gene that are contained (exon 1, 3, 4 and 5) have a high level of sequence identity with other KIR3D sequences, in particular with KIR3DL3 sequences.

The KIR proteins contain characteristic Ig-like domains in their extracellular regions, which in some KIR proteins are involved in HLA class I ligand binding. They also contain transmembrane and cytoplasmic regions that are functionally relevant as they determine the type of signal that is transduced in the NK cell. KIR proteins may contain two or three Ig-like domains (thus KIR2D or KIR3D) as well as short or long cytoplasmic tails (provided as KIR2DS or KIR2DL). The two KIR domain proteins are divided into two groups depending on the origin of the membrane-distal Ig-like domains contained. Type I KIR2D proteins (KIR2DL1, KIR2DL2, KIR2DL3, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, and KIR2DS5) contain a membrane-distal Ig-like domain, similar in origin to the Ig-like D1 domain of KIR3D, but do not contain the D0 domain. This Ig-like D1 domain is encoded mainly by the fourth exon of the corresponding KIR genes. Type I IKIR2D proteins, KIR2DL4 and KIR2DL5, contain a membrane-distal Ig-like domain of a sequence similar to the D0 domain found in KIR3D proteins, however, type I IKIR2D does not contain a D1 domain. Long cytoplasmic tails typically contain two immunoreceptor tyrosine inhibitory motifs (ITIMs) that transduce inhibitory signals in the NK cell. The short cytoplasmic tails contain a positively charged amino acid residue in their transmembrane region, which allows them to bind to the DAP12 signaling molecule capable of generating an activating signal.

The exception to this is KIR2DL4, which contains only one ITIM at the N-terminus. In addition, KIR2DL4 also contains a charged residue (arginine) in its transmembrane domain, a property that allows this receptor to elicit both inhibitory and activating signals. KIR regulates the response of human NK cells by delivering inhibitory or activating signals upon recognition of class I MHC ligands on the surface of possible target cells.

The length of KIR proteins varies from 306 to 456 amino acid residues. Although the differences in protein length are mainly due to the number of Ig-like domains contained, the difference in the length of the cytoplasmic regions is also a factor influencing. The length of the leader peptide of most KIR proteins is 21 amino acid residues. However, the presence of a different start codon leads to the formation of a correspondingly longer leader peptide in the KIR2DL4 proteins.

The length of the Ig-like D0 domain contained in IKIR2D type I proteins and KIR3D proteins is approximately 96 amino acid residues. The length of the D1 domain of the type I KIR2D and KIR3D proteins is 102 amino acid residues, while the length of the D2 domain of all KIR proteins is 98 amino acid residues. The length of the stem region varies from the 24 amino acid residues found in most KIR proteins to just seven amino acid residues in the divergent KIR3DL3 protein. The transmembrane region is 20 amino acid residues long for most KIR proteins, but one residue is shorter in KIR2DL1 and KIR2DL2 proteins as a result of three base pair deletions in exon 7. In conclusion, the cytoplasmic region of KIR proteins is characterized by large ranges of length change, ranging from 23 amino acid residues in some KIR3DS1 alleles, up to 96 amino acid residues contained in the KIR3DL2 proteins.

Amino acid sequences of human (Homo sapiens) KIR polypeptides are available from the NCBI e database, see e.g. .1 (GI:338968852) NP_006728.2 (GI:134268642) 6912472) .1 (GI: 124107604), NP_036445.1 (GI: 6912474).

The nomenclature for KIR is based on the number of extracellular domains (where KIR2D and KIR3D contain two and three extracellular Ig domains, respectively) and whether the cytoplasmic tail is long (KIR2DL or KIR3DL) or short (KIR2DS or KIR3DS). The presence or absence of a given KIR varies from one NK cell to another in the NK population present in one individual. Among humans, there is also a relatively high level of KIR gene polymorphism, where

- 46 040145 certain KIR genes are present in some but not all individuals. The expression of KIR alleles in NK cells is stochastically regulated, which means that in a given individual, a given lymphocyte may express one, two, or more different KIRs, depending on the individual's genotype.

NK cells from a single individual typically express different combinations of KIRs, providing a repertoire of NK cells with different specificities for class I MHC molecules.

Certain products of the KIR genes cause stimulation of lymphocyte activity when bound to the appropriate ligand. All activating KIRs contain a short cytoplasmic tail with a charged transmembrane residue that binds to an adapter molecule containing immunoreceptor tyrosine activating motifs (ITAMs) that transduce stimulatory signals into the NK cell. In contrast, inhibitory KIRs contain a long cytoplasmic tail containing an immunoreceptor tyrosine inhibitory motif (ITIM) that transduces inhibitory signals in the NK cell upon interaction with their MHC class I ligands. Known inhibitory KIRs include members of the KIR2DL and KIR3DL subfamilies. Inhibitory KIRs containing two Ig domains (KIR2DL) recognize HLA-C allotypes: KIR2DL2 (formerly referred to as p58.2) and the closely related allelic KIR2DL3 gene product recognize group 1 HLA-C allotypes (including HLA-Cw1, -3, -7 and -8), while KIR2DL1 (p58.1) recognizes group 2 HLA-C allotypes (such as HLA-Cw2, -4, -5 and -6). Recognition of KIR2DL1 is due to the presence of a Lys residue at position 80 of the HLA-C allele. Recognition of KIR2DL2 and KIR2DL3 is due to the presence of an Asn residue at position 80 in HLA-C. It should be noted that the vast majority of HLA-C alleles contain an Asn or Lys residue at position 80. Thus, KIR2DL1, -2 and -3 collectively recognize essentially all HLA-C allotypes found in humans. One KIR with three Ig domains, KIR3DL1 (p70), recognizes an epitope common to HLA-Bw4 alleles. Finally, KIR3DL2 (p140), a homodimer of molecules with three Ig domains, recognizes HLA-A3 and -A11.

However, the invention should not be limited to inhibitory KIRs containing a cytoplasmic tail containing ITIM. Conversely, any inhibitory protein containing a cytoplasmic domain that is expressed with an inhibitory signal can be used in the construction of a CAR of the invention. Non-limiting examples of an inhibitory protein include, but are not limited to, CTLA-4, PD-1, and the like. These proteins are known to inhibit T cell activation.

Thus, the invention relates to a KIR-CAR containing an extracellular domain that contains a target-specific binding element, otherwise referred to as an antigen-binding domain, fused to a KIR or a fragment thereof. In one embodiment, the KIR is an activating KIR that contains a short cytoplasmic tail that binds to an adapter molecule containing immunoreceptor tyrosine activation motifs (ITAMs) that transduce stimulatory signals into the NK cell (referred to elsewhere herein as actKIR -CAR). In one embodiment, the KIR is an inhibitory KIR that contains a long cytoplasmic tail containing an immunoreceptor tyrosine inhibitory motif (ITIM) that transduces inhibitory signals (referred to elsewhere herein as inhKIR-CAR). In some cases, when constructing an actKIR-CAR, it is desirable to remove the hinge region for activating KIRs. This is because the invention is based in part on the discovery that an activating KIR-CAR in which the KIR2DS2 hinge was removed to produce CAR KIR2S, such CAR KIRS2 had increased cytolytic activity compared to actKIR-CAR containing full-length wild-type KIR2DS2.

Nucleic acid sequences encoding the desired molecules of the invention can be obtained by recombinant methods known in the art, such as, for example, screening libraries from cells expressing the gene, delivering the gene from a vector known to contain the gene, or isolating directly from cells and tissues containing the gene by standard methods. Alternatively, the gene of interest may be synthesized rather than cloned.

The present invention relates to retroviral and lentiviral vector constructs expressing KIR-CAR that can be directly transduced into a cell. The present invention also relates to an RNA construct that can be directly transduced into a cell. The method for obtaining mRNA for use in transfection includes in vitro transcription (IVT) of a template with specially designed primers, followed by the addition of poly(A) to obtain a construct containing a 3'- and 5'-untranslated sequence (UTR), a 5'-cap and/ or an internal ribosome binding site (IRES), a gene to be expressed, and a poly(A) tail, typically 50-2000 bases long. The RNA obtained in this way can be efficiently transfected into various types of cells. In one embodiment, the template includes KIR-CAR sequences.

In one embodiment, the KIR-CAR contains an antigen-binding domain and a transmembrane domain of the KIR. In one embodiment, the KIR-CAR comprises an antigen-binding domain and a KIR intracellular domain, such as the inhKIR intracellular domain.

- 47 040145

Domain D KIR, as this term is used in the present description, refers to the domain D0, D1 or

D2KIR.

A KIR D domain, as the term is used herein, refers to a polypeptide domain having the structural and functional properties of a KIR D domain.

The D0 KIR domain, as the term is used herein, refers to the D0 KIR domain. In one embodiment, the D0 domain of the KIR KIR-CAR. has at least 70%, 80%, 85%, 90%, 95% or 99% homology with a reference sequence, for example, a natural KIR D0 domain or a KIR D0 domain described in the present invention. In embodiments, the KIRCAR D0 domain of KIRCAR differs by no more than 15%, 10%, 5%, 2, or 1% of its residues from a reference sequence, such as a natural KIR D0 domain or a KIR D0 domain described herein. In embodiments, the KIR-CAR D0 KIR domain differs by no more than 5, 4, 3, 2, or 1 residue from a reference sequence, such as a natural KIR D0 domain or a KIR D0 domain described herein. In embodiments, the KIR D0 KIR domain of the KIR-CAR does not differ or has 100% homology to a reference sequence, such as a natural KIR D0 domain or a KIR D0 domain described in the present invention.

A KIR D1 domain, as the term is used herein, refers to a polypeptide domain having the structural and functional properties of a KIR D1 domain. In one embodiment, the KIR D1 KIR domain of a KIR-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology to a reference sequence, e.g., a natural KIR D1 domain or a KIR D1 domain described in the present invention. In embodiments, the KIR-CAR D1 KIR domain differs by no more than 15%, 10%, 5%, 2%, or 1% of its residues from a reference sequence, e.g., a natural KIR D1 domain or a KIR D1 domain described herein. In embodiments, the KIR D1 KIR domain of a KIR-CAR differs by no more than 5, 4, 3, 2, or 1 residue from a reference sequence, such as a natural KIR D0 domain or a KIR D1 domain described herein. In embodiments, the KIR D1 domain of a KIR-CAR does not differ or has 100% homology to a reference sequence, such as a natural KIR D1 domain or a KIR D1 domain described in the present invention.

A KIR D2 domain, as the term is used herein, refers to a polypeptide domain having the structural and functional properties of a KIR D2 domain. In one embodiment, the KIR D2 domain of a KIR-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology to a reference sequence, e.g., a natural KIR D2 domain or a KIR D2 domain described in the present invention. In embodiments, the KIR-CAR D2 KIR domain differs by no more than 15%, 10%, 5%, 2%, or 1% of its residues from a reference sequence, such as a natural KIR D2 domain or a KIR D2 domain described herein. In embodiments, the KIR-CAR D2 domain of KIR differs by no more than 5, 4, 3, 2, or 1 residue from a reference sequence, such as a natural KIR D2 domain or a KIR D2 domain described herein. In embodiments, the KIR D2 domain of a KIR-CAR does not differ or has 100% homology to a reference sequence, such as a natural KIR D2 domain or a KIR D2 domain described in the present invention.

A KIR hinge or stem domain, as the term is used herein, refers to a polypeptide domain having the structural and functional properties of a KIR hinge or stem domain. In one embodiment, the KIR hinge or stem domain of KIR-CAR has at least 70%, 80, 85, 90, 95, or 99% homology with a reference sequence, e.g., a native KIR hinge or stem domain or a KIR hinge or stem domain described by in the present invention. In embodiments, the KIR hinge or stem KIR domain differs by no more than 15%, 10%, 5%, 2%, or 1% of its residues from a reference sequence, e.g., a naturally occurring KIR hinge or stem domain or a KIR hinge or stem domain as described herein. description. In embodiments, the KIR hinge or stem domain of KIR-CAR differs by no more than 5, 4, 3, 2, or 1 residue from a reference sequence, e.g., a naturally occurring KIR hinge or stem domain or a KIR hinge or stem domain described herein. In embodiments, the KIR hinge or stem domain of KIR-CAR does not differ or has 100% homology with a reference sequence, for example, a natural KIR hinge or stem domain or a KIR hinge or stem domain described in the present invention.

A KIR transmembrane domain, as the term is used herein, refers to a polypeptide domain having the structural and functional properties of a KIR transmembrane domain. In one embodiment, the KIR transmembrane domain of KIR-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology to a reference sequence, e.g., a natural KIR transmembrane domain or a KIR transmembrane domain described in the present invention. In embodiments, the KIR transmembrane domain of KIR-CAR differs by no more than 15%, 10%, 5%, 2%, or 1% of its residues from a reference sequence, e.g., a natural KIR transmembrane domain or a KIR transmembrane domain as described in the present invention. In embodiments, the KIR transmembrane domain of a KIR-CAR differs by no more than 5, 4, 3, 2, or 1 residue from a reference sequence, e.g., a natural KIR transmembrane domain or a KIR transmembrane domain described in the present invention. In embodiments, the KIR transmembrane domain of KIR-CAR does not differ or has 100% homology to a reference sequence, eg, a natural KIR transmembrane domain or a KIR transmembrane domain described in the present invention.

An intracellular KIR domain, as the term is used herein, refers to a polypeptide domain having the structural and functional properties of an intracellular KIR domain. The intracellular KIR domains comprise inhibitory KIR intracellular domains (herein referred to as inhKIR intracellular domains) and activating KIR intracellular domains (herein referred to as actKIR intracellular domains). In one embodiment, the inhKIR intracellular domain contains the ITIM sequence. In one embodiment, the intracellular KIR domain of KIR-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology with a reference sequence, e.g., a natural KIR intracellular domain or a KIR intracellular domain described in the present invention. In embodiments, the intracellular KIR domain of KIR-CAR differs by no more than 15%, 10%, 5%, 2%, or 1% of its residues from a reference sequence, e.g., a native KIR intracellular domain or a KIR intracellular domain described herein. In embodiments, the intracellular KIR domain of KIR-CAR differs by no more than 5, 4, 3, 2, or 1 residue from a reference sequence, for example, a naturally occurring KIR intracellular domain or a KIR intracellular domain described herein. In embodiments, the intracellular KIR domain of KIR-CAR does not differ or has 100% homology with a reference sequence, for example, a natural intracellular KIR domain or an intracellular KIR domain described herein.

NCR.

The NKR-CAR described in the present invention includes NCR-CARs that share functional and structural properties with NCRs.

Natural killer (NK) cells are cytotoxic lymphoid cells specialized in the destruction of tumors 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 a large number of cell surface receptors to surface markers on the target cell. NK cell surface receptors can be distinguished depending on whether they activate or inhibit NK cell mediated cytotoxicity. Different interactions between different receptors seem to lead to the formation of synapses between NK cells and target cells. The integration of activating and inhibitory signals at the synapse determines whether or not NK cells exhibit their cytolytic function on the target cell. Among the activating receptors, a family of Ig-like molecules are referred to as natural cytotoxicity receptors (NCRs). Such natural cytotoxicity receptors include the NKp30, NKp44 and NKp46 molecules. NCRs are key NK cell activating receptors in tumor cell recognition. All three NCRs are involved in the clearance of tumor and virus-infected cells. In the latter, antiviral activity is initiated by the interaction of NKp44 with hemagglutinin of the influenza virus or the Sendai virus. NKp46 targets virus-infected cells by binding to influenza virus hemagglutinin or Sendai virus hemagglutinin-neuraminidase. In contrast, NK cell-mediated cytotoxicity has been shown to be inhibited by binding of NKp30 to the human cytomegalovirus pp65 protein (see, for example, Arnon et al., Nat. Immunol., (2005) 6:515-523).

Amino acid sequences of human (Homo sapiens) NCR polypeptides are available from the NCBI database, see e.g. 47605775), NP_001138939.1 (GI:224586865) and/or NP_001138938.1 (GI:224586860).

In one embodiment, the NCR-CAR comprises an antigen-binding domain and an NCR transmembrane domain. In one embodiment, the KIR-CAR contains an antigen binding domain and an NCR intracellular domain.

An NCR extracellular domain, as the term is used herein, refers to a polypeptide domain having the structural and functional properties of an NCR extracellular domain. In one embodiment, the NCR extracellular domain of an NCR-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology to a reference sequence, e.g., a natural NCR extracellular domain or an NCR extracellular domain described in the present invention. In embodiments, the NCR-CAR extracellular domain of NCR differs by no more than 15%, 10%, 5%, 2%, or 1% of its residues from a reference sequence, e.g., a natural NCR extracellular domain or an NCR extracellular domain described herein. In embodiments, the NCR extracellular domain of an NCR-CAR differs by no more than 5, 4, 3, 2, or 1 residue from a reference sequence, e.g., a natural NCR extracellular domain or an NCR extracellular domain described herein. In embodiments, the extracellular NCR domain of an NCR-CAR does not differ or has 100% homology with a reference sequence, eg, a natural NCR extracellular domain or an NCR extracellular domain described in the present invention.

An NCR hinge or stem domain, as used herein, refers to a polypeptide domain having the structural and functional properties of an NCR hinge or stem domain. In one embodiment, the NCR hinge or stem domain of an NCR-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology to a reference sequence, e.g., a natural NCR hinge or stem domain or an NCR hinge or stem domain described by in the present invention. In embodiments, the NCR-CAR hinge or stem NCR domain differs by no more than 15%, 10%, 5%, 2%, or 1% of its residues from a reference sequence, e.g., a native NCR hinge or stem domain or an NCR hinge or stem domain as described herein. description. In embodiments, the NCR-CAR NCR hinge or stem domain differs by no more than 5, 4, 3, 2, or 1 residue from a reference sequence, e.g., a natural NCR hinge or stem domain or an NCR hinge or stem domain described herein. In embodiments, the NCR hinge or stem domain of an NCR-CAR does not differ or has 100% homology to a reference sequence, for example, a natural NCR hinge or stem domain or an NCR hinge or stem domain described in the present invention.

An NCR transmembrane domain, as the term is used herein, refers to a polypeptide domain having the structural and functional properties of an NCR transmembrane domain. In one embodiment, the NCR transmembrane domain of an NCR-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology to a reference sequence, e.g., a natural NCR transmembrane domain or an NCR transmembrane domain described in the present invention. In embodiments, the NCR transmembrane domain of an NCR-CAR differs by no more than 15%, 10%, 5%, 2%, or 1% of its residues from a reference sequence, e.g., a natural NCR transmembrane domain or an NCR transmembrane domain described herein. In embodiments, the NCR-CAR NCR transmembrane domain differs by no more than 5, 4, 3, 2, or 1 residue from a reference sequence, e.g., a natural NCR transmembrane domain or an NCR transmembrane domain described herein. In embodiments, the NCR-CAR NCR transmembrane domain does not differ or has 100% homology to a reference sequence, eg, a natural NCR transmembrane domain or an NCR transmembrane domain described in the present invention.

An NCR intracellular domain, as the term is used herein, refers to a polypeptide domain having the structural and functional properties of an NCR intracellular domain. In one embodiment, the intracellular NCR domain of an NCR-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology with a reference sequence, e.g., a natural NCR intracellular domain or an intracellular NCR domain described in the present invention. In embodiments, the intracellular NCR domain of NCR-CAR differs by no more than 15%, 10%, 5%, 2%, or 1% of its residues from a reference sequence, e.g., a natural NCR intracellular domain or an NCR intracellular domain described herein. In embodiments, the intracellular NCR domain of an NCR-CAR differs by no more than 5, 4, 3, 2, or 1 residue from a reference sequence, e.g., a natural NCR intracellular domain or an NCR intracellular domain described herein. In embodiments, the intracellular NCR domain of an NCR-CAR does not differ or has 100% homology with a reference sequence, eg, a native NCR intracellular domain or an intracellular NCR domain described in the present invention.

SLAM receptors.

The NKR-CARs described in the present invention include SLAMF-CARs that share functional and structural properties with SLAMF.

The lymphocyte-activating signaling molecule (SLAM) family of immune cell receptors is closely related to the CD2 family of the immunoglobulin (Ig) superfamily of molecules. The SLAM family (SLAMF) currently includes nine members, called SLAM, CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME and CD2F-10. In general, SLAM molecules contain two to four extracellular Ig domains, a transmembrane segment, and an intracellular tyrosine-rich region. The molecules are differentially expressed by different cell types of the immune system. Several of these are self-liganding, and SLAM has been identified as the human measles virus receptor. Several small SH2-containing adapter proteins are known to bind to the intracellular domains of members of the SLAM family and modulate 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 SAP adapter and then the Fyn Src kinase. The next cascade of signal transduction influences the result of interactions between T-cell-antigen-presenting cell and NK-cell-target cell.

The amino acid sequences of the human (Homo sapiens) SLAM polypeptide receptor are available from the NCBI database, see e.g. NP_001171808.1 (GI: 296434285) (GI: 55925578).

In one embodiment, the SLAMF-CAR comprises an antigen-binding domain and a SLAMF transmembrane domain. In one embodiment, the SLAMF-CAR comprises an antigen-binding domain and an intracellular SLAMF domain.

The SLAMF extracellular domain, as the term is used herein, refers to a polypeptide domain having the structural and functional properties of the SLAMF extracellular domain. In one embodiment, the SLAMF SLAMF-CAR extracellular domain has at least 70%, 80%, 85%, 90%, 95%, or 99% homology with a reference sequence, e.g., the natural SLAMF extracellular domain or the SLAMF extracellular domain described in the present invention. In embodiments, the SLAMF SLAMF-CAR extracellular domain differs by no more than 15%, 10%, 5%, 2%, or 1% of its residues from a reference sequence, e.g., the natural SLAMF extracellular domain or the SLAMF extracellular domain described herein. In embodiments, the SLAMF SLAMF-CAR extracellular domain differs by no more than 5, 4, 3, 2, or 1 residue from a reference sequence, e.g., the natural SLAMF extracellular domain or the SLAMF extracellular domain described herein. In embodiments, the SLAMF SLAMF-CAR extracellular domain does not differ or has 100% homology to a reference sequence, eg, the natural SLAMF extracellular domain or the SLAMF extracellular domain described in the present invention.

SLAMF hinge or stem domain, as the term is used herein, refers to a polypeptide domain having the structural and functional properties of a SLAMF hinge or stem domain. In one embodiment, the SLAMF SLAMF-CAR hinge or stem domain has at least 70%, 80%, 85%, 90%, 95%, or 99% homology to a reference sequence, e.g., a natural SLAMF hinge or stem domain or a SLAMF hinge or stem domain described by in the present invention. In embodiments, the SLAMF SLAMF-CAR hinge or stem domain differs by no more than 15%, 10%, 5%, 2%, or 1% of its residues from a reference sequence, e.g., a native SLAMF hinge or stem domain or a SLAMF hinge or stem domain as described herein. description. In embodiments, the SLAMF SLAMF-CAR hinge or stem domain differs by no more than 5, 4, 3, 2, or 1 residue from a reference sequence, e.g., a natural SLAMF hinge or stem domain or a SLAMF hinge or stem domain described herein. In embodiments, the SLAMF SLAMF-CAR hinge or stem domain is the same or has 100% homology to a reference sequence, e.g., a natural SLAMF hinge or stem domain or a SLAMF hinge or stem domain described in the present invention.

A SLAMF transmembrane domain, as the term is used herein, refers to a polypeptide domain having the structural and functional properties of a SLAMF transmembrane domain. In one embodiment, the SLAMF SLAMF-CAR transmembrane domain has at least 70%, 80%, 85%, 90%, 95%, or 99% homology to a reference sequence, e.g., a natural SLAMF transmembrane domain or a SLAMF transmembrane domain described in the present invention. In embodiments, the SLAMF SLAMF-CAR transmembrane domain differs by no more than 15%, 10%, 5%, 2%, or 1% of its residues from a reference sequence, e.g., a natural SLAMF transmembrane domain or a SLAMF transmembrane domain described herein. In embodiments, the SLAMF SLAMF-CAR transmembrane domain differs by no more than 5, 4, 3, 2, or 1 residue from a reference sequence, such as a natural SLAMF transmembrane domain or a SLAMF transmembrane domain described herein. In embodiments, the SLAMF SLAMFCAR transmembrane domain does not differ or has 100% homology to a reference sequence, for example, a natural SLAMF transmembrane domain or a SLAMF transmembrane domain described in the present invention.

An intracellular SLAMF domain, as the term is used herein, refers to a polypeptide domain having the structural and functional properties of an intracellular SLAMF domain. In one embodiment, the SLAMF intracellular domain of the SLAMF-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology to a reference sequence, e.g., the natural SLAMF intracellular domain or the SLAMF intracellular domain described in the present invention. In embodiments, the SLAMF SLAMFCAR intracellular domain differs by no more than 15%, 10%, 5%, 2%, or 1% of its residues from the reference sequence,

- 51 040145 for example, natural intracellular domain SLAMF or intracellular domain SLAMF described in the present description. In embodiments, the SLAMF SLAMFCAR intracellular domain differs by no more than 5, 4, 3, 2, or 1 residue from a reference sequence, such as a natural SLAMF intracellular domain or a SLAMF intracellular domain described herein. In embodiments, the intracellular SLAMF domain of the SLAMF-CAR does not differ or has 100% homology with a reference sequence, eg, the natural SLAMF intracellular domain or the SLAMF intracellular domain described in the present invention.

Fc-binding receptors.

NKR-CARs as described herein include Fc receptor-based CARs, FcRCARs, eg CD16-CARs and CD64-CARs, which share functional and structural properties with CD16 and CD64.

When activated, NK cells produce large amounts of cytokines and chemokines and simultaneously exhibit high cytolytic activity. Activation of NK cells can occur through direct binding of NK cell receptors to ligands on the target cell, as can be seen in direct tumor cell cytolysis, or through Fc receptor (CD16; FcyRIII) cross-linking through binding of the Fc region of antibodies associated with antigen-bearing cell. This involvement of CD16 (CD16 crosslinking) initiates NK cell responses via intracellular signals that are received through one or both of the CD16-associated adapter chains, FcRy or CD3Z. Engagement of CD16 leads to phosphorylation of the y- or ζ-chain, which in turn recruits tyrosine kinases, syk and ZAP-70, initiating a signal transduction cascade leading to rapid and efficient effector functions. The most well known effector function is the release of cytoplasmic granules carrying toxic proteins to kill nearby target cells through a process of antibody-dependent cellular cytotoxicity. Cross-linking with CD16 also results in the production of cytokines and chemokines, which in turn activate and regulate a series of immune responses.

However, unlike T and B lymphocytes, NK cells are believed to have only limited target recognition capability 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 Fc activating receptor CD16, which recognizes IgG-coated target cells, thus enhancing target recognition (Ravetch and Bolland, Annu. Rev. Immunol., 19:275-290 (2001); Lanier, Nat. Immunol. , 9(5):495-502 (2008); Bryceson and Long, Curr Opin Immunol., 20(3):344-352 (2008)). For expression and signaling activity of several NK cell activating receptors, physically coupled adapters are essential that transduce signals via immunoreceptor tyrosine activating motifs (ITAMs). Among these adapters, FcRy and CD3Z chains can bind to CD16 and natural cytotoxicity receptors (NCRs) as disulfide-like homodimers or heterodimers, and such chains were thought to be expressed by all mature NK cells.

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

In one embodiment, the FcR-CAR comprises an FcR antigen-binding domain and a transmembrane domain. In one embodiment, the FcR-CAR comprises an antigen-binding domain and an intracellular FcR domain.

The CD16 extracellular domain, as the term is used herein, refers to a polypeptide domain having the structural and functional properties of the CD16 extracellular domain. In one embodiment, the CD16 extracellular domain of a CD16-CAR shares at least 70%, 80%, 85%, 90%, 95%, or 99% homology with a reference sequence, e.g., native CD16 extracellular domain or CD16 extracellular domain described in the present invention. In embodiments, the CD16 extracellular domain of a CD16-CAR differs by no more than 15%, 10%, 5%, 2%, or 1% of its residues from a reference sequence, e.g., a native CD16 extracellular domain or a CD16 extracellular domain described herein. In embodiments, the CD16 extracellular domain of a CD16-CAR differs by no more than 5, 4, 3, 2, or 1 residue from a reference sequence, e.g., native CD16 extracellular domain or CD16 extracellular domain described herein. In embodiments, the CD16 extracellular domain of a CD16-CAR does not differ or has 100% homology to a reference sequence, eg, a natural CD16 extracellular domain or a CD16 extracellular domain described in the present invention.

A CD16 hinge or stem domain, as used herein, refers to a polypeptide domain having the structural and functional properties of a CD16 hinge or stem domain. In one embodiment, the CD16 hinge or stem domain of a CD16-CAR shares at least 70%, 80%, 85%, 90%, 95%, or 99% homology with a reference sequence.

- 52 040145 values, for example, the natural hinge or stem domain of CD16 or the hinge or stem domain of CD16, are described in the present description. In embodiments, the CD16 hinge or stem domain of a CD16-CAR differs by no more than 15%, 10%, 5%, 2%, or 1% of its residues from a reference sequence, e.g., a naturally occurring CD16 hinge or stem domain or a CD16 hinge or stem domain described herein. description. In embodiments, the CD16 hinge or stem domain of a CD16-CAR differs by no more than 5, 4, 3, 2, or 1 residue from a reference sequence, e.g., a native CD16 hinge or stem domain or a CD16 hinge or stem domain described herein. In embodiments, the CD16 hinge or stem domain of a CD16-CAR does not differ or has 100% homology to a reference sequence, eg, a native CD16 hinge or stem domain or a CD16 hinge or stem domain described in the present invention.

A CD16 transmembrane domain, as the term is used herein, refers to a polypeptide domain having the structural and functional properties of a CD16 transmembrane domain. In one embodiment, the CD16 transmembrane domain of a CD16-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology to a reference sequence, e.g., a natural CD16 transmembrane domain or a CD16 transmembrane domain described in the present invention. In embodiments, the CD16 transmembrane domain of a CD16-CAR differs by no more than 15%, 10%, 5%, 2%, or 1% of its residues from a reference sequence, e.g., a natural CD16 transmembrane domain or a CD16 transmembrane domain described herein. In embodiments, the CD16 transmembrane domain of a CD16-CAR differs by no more than 5, 4, 3, 2, or 1 residue from a reference sequence, such as a natural CD16 transmembrane domain or a CD16 transmembrane domain described herein. In embodiments, the CD16 transmembrane domain of a CD16-CAR does not differ or has 100% homology to a reference sequence, eg, a natural CD16 transmembrane domain or a CD16 transmembrane domain described in the present invention.

The CD16 intracellular domain, as the term is used herein, refers to a polypeptide domain having the structural and functional properties of the CD16 intracellular domain. In one embodiment, the CD16 intracellular domain of a CD16-CAR shares at least 70%, 80%, 85%, 90%, 95%, or 99% homology with a reference sequence, e.g., a natural CD16 intracellular domain or a CD16 intracellular domain described in the present invention. In embodiments, the CD16 intracellular domain of a CD16-CAR differs by no more than 15%, 10%, 5%, 2%, or 1% of its residues from a reference sequence, e.g., a natural CD16 intracellular domain or a CD16 intracellular domain described herein. In embodiments, the CD16 intracellular domain of a CD16-CAR differs by no more than 5, 4, 3, 2, or 1 residue from a reference sequence, e.g., a natural CD16 intracellular domain or a CD16 intracellular domain described herein. In embodiments, the CD16 intracellular domain of a CD16-CAR does not differ or has 100% homology with a reference sequence, eg, a natural CD16 intracellular domain or a CD16 intracellular domain described in the present invention.

The CD64 extracellular domain, as the term is used herein, refers to a polypeptide domain having the structural and functional properties of the CD64 extracellular domain. In one embodiment, the CD64 extracellular domain of a CD64-CAR shares at least 70%, 80%, 85%, 90%, 95%, or 99% homology with a reference sequence, e.g., native CD64 extracellular domain or CD64 extracellular domain described in the present invention. In embodiments, the CD64 extracellular domain of a CD64-CAR differs by no more than 15%, 10%, 5%, 2%, or 1% of its residues from a reference sequence, e.g., native CD64 extracellular domain or CD64 extracellular domain described herein. In embodiments, the CD64 extracellular domain of a CD64-CAR differs by no more than 5, 4, 3, 2, or 1 residue from a reference sequence, e.g., native CD64 extracellular domain or CD64 extracellular domain described herein. In embodiments, the CD64 extracellular domain of a CD64-CAR does not differ or has 100% homology to a reference sequence, eg, a native CD64 extracellular domain or a CD64 extracellular domain described herein.

A CD64 hinge or stem domain, as the term is used herein, refers to a polypeptide domain having the structural and functional properties of a CD64 hinge or stem domain. In one embodiment, the CD64 hinge or stem domain of a CD64-CAR has at least 70%, 80, 85, 90, 95, or 99% homology to a reference sequence, e.g., a natural CD64 hinge or stem domain or a CD64 hinge or stem domain described by in the present invention. In embodiments, the CD64 hinge or stem domain of a CD64-CAR differs by no more than 15%, 10%, 5%, 2%, or 1% of its residues from a reference sequence, e.g., a naturally occurring CD64 hinge or stem domain or a CD64 hinge or stem domain described herein. description. In options

- 53 040145 the CD64 hinge or stem domain of a CD64-CAR differs by no more than 5, 4, 3, 2, or 1 residue from a reference sequence, such as a native CD64 hinge or stem domain or a CD64 hinge or stem domain described herein . In embodiments, the CD64 hinge or stem domain of a CD64-CAR does not differ or has 100% homology to a reference sequence, eg, a native CD64 hinge or stem domain or a CD64 hinge or stem domain described in the present invention.

A CD64 transmembrane domain, as the term is used herein, refers to a polypeptide domain having the structural and functional properties of a CD64 transmembrane domain. In one embodiment, the CD64 transmembrane domain of a CD64-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology to a reference sequence, e.g., a natural CD64 transmembrane domain or a CD64 transmembrane domain described in the present invention. In embodiments, the CD64 transmembrane domain of a CD64-CAR differs by no more than 15%, 10%, 5%, 2%, or 1% of its residues from a reference sequence, e.g., a natural CD64 transmembrane domain or a CD64 transmembrane domain described herein. In embodiments, the CD64 transmembrane domain of a CD64-CAR differs by no more than 5, 4, 3, 2, or 1 residue from a reference sequence, such as a natural CD64 transmembrane domain or a CD64 transmembrane domain described herein. In embodiments, the CD64 transmembrane domain of a CD64-CAR does not differ or has 100% homology to a reference sequence, eg, a natural CD64 transmembrane domain or a CD64 transmembrane domain described in the present invention.

The CD64 intracellular domain, as the term is used herein, refers to a polypeptide domain having the structural and functional properties of the CD64 intracellular domain. In one embodiment, the CD64 intracellular domain of a CD64-CAR shares at least 70%, 80%, 85%, 90%, 95%, or 99% homology with a reference sequence, e.g., a natural CD64 intracellular domain or a CD64 intracellular domain as described herein. In embodiments, the CD64 intracellular domain of a CD64-CAR differs by no more than 15%, 10%, 5%, 2%, or 1% of its residues from a reference sequence, e.g., a natural CD64 intracellular domain or a CD64 intracellular domain described herein. In embodiments, the CD64 intracellular domain of a CD64-CAR differs by no more than 5, 4, 3, 2, or 1 residue from a reference sequence, e.g., a natural CD64 intracellular domain or a CD64 intracellular domain described herein. In embodiments, the intracellular CD64 domain of a CD64-CAR does not differ or has 100% homology with a reference sequence, eg, a natural CD64 intracellular domain or a CD64 intracellular domain described in the present invention.

Ly49 and related lectin-like killer cell receptors.

NKR-CARs described herein include Ly49-CARs that share functional and structural properties with Ly49.

Ly49 receptors are derived from at least 23 identified genes (Ly49A-W) in the mouse. These receptors play many of the same roles in murine NK cells and T cells that KIR played in humans, despite their distinct structure (type II integral membrane proteins of the C-type lectin superfamily), and they are also characterized by a significant degree of genetic variability. like the kir of man. The marked functional similarity between the Ly49 and KIR receptors suggests that these groups of receptors evolved independently, even similarly, to perform the same physiological functions in NK cells and T cells.

Similarly to KIR in humans, different Ly49 receptors recognize different MHC class I alleles and are differentially expressed by subpopulations of NK cells. The original classical receptors Ly49, Ly49A and Ly49C contain a cytoplasmic domain bearing two immunoreceptor tyrosine inhibitory motifs (ITIMs) analogous to inhibitory KIRs such as KIR2DL3.

Such domains have been identified to recruit phosphatase, SHP-1 and, like inhibitory KIRs, serve to limit the activation of NK cells and T cells. In addition to inhibitory Ly49 molecules, several members of the family, such as Ly49D and Ly49H, have lost their ITIM-containing domains and instead gained the ability to interact with a signal adapter molecule, DAP12, similar to activating KIRs such as KIR2DS2 in humans.

The amino acid sequences of members of the Ly49 family are available from the NCBI database, see e.g. 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).

In one embodiment, Ly49-CAR contains an antigen-binding domain and a Ly49 transmembrane domain. In one embodiment, Ly49-CAR contains an antigen-binding domain and an intracellular Ly49 domain.

The LY49 extracellular domain, as the term is used herein, refers to a polypeptide domain having the structural and functional properties of an extracellular domain.

- 54 040145

LY49. In one embodiment, the LY49 extracellular domain of LY49-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology to a reference sequence, e.g., native LY49 extracellular domain or a LY49 extracellular domain as described in the present invention. In embodiments, the LY49 extracellular domain of LY49-CAR differs by no more than 15%, 10%, 5%, 2%, or 1% of its residues from a reference sequence, e.g., the natural LY49 extracellular domain or the LY49 extracellular domain described herein. In embodiments, the LY49 LY49 extracellular domain of LY49-CAR differs by no more than 5, 4, 3, 2, or 1 residue from a reference sequence, e.g., native LY49 extracellular domain or LY49 extracellular domain described herein. In embodiments, the LY49 extracellular domain of LY49-CAR does not differ or has 100% homology with a reference sequence, for example, the natural LY49 extracellular domain or the LY49 extracellular domain described in the present invention.

A LY49 hinge or stem domain, as the term is used herein, refers to a polypeptide domain having the structural and functional properties of a LY49 hinge or stem domain. In one embodiment, the LY49 hinge or stem domain of LY49-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology to a reference sequence, e.g., a natural LY49 hinge or stem domain or a LY49 hinge or stem domain described by in the present invention. In embodiments, the LY49 hinge or stem domain of LY49-CAR differs by no more than 15%, 10%, 5%, 2%, or 1% of its residues from a reference sequence, e.g., a native LY49 hinge or stem domain or a LY49 hinge or stem domain described herein. description. In embodiments, the LY49 LY49 hinge or stem domain of LY49-CAR differs by no more than 5, 4, 3, 2, or 1 residue from a reference sequence, e.g., a natural LY49 hinge or stem domain or a LY49 hinge or stem domain described herein. In embodiments, the LY49 LY49 hinge or stem domain of LY49-CAR does not differ or has 100% homology to a reference sequence, for example, a natural LY49 hinge or stem domain or a LY49 hinge or stem domain described in the present invention.

A LY49 transmembrane domain, as the term is used herein, refers to a polypeptide domain having the structural and functional properties of a LY49 transmembrane domain. In one embodiment, the LY49 transmembrane domain of LY49-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology to a reference sequence, e.g., a natural LY49 transmembrane domain or a LY49 transmembrane domain described in the present invention. In embodiments, the LY49 transmembrane domain of LY49-CAR differs by no more than 15%, 10%, 5%, 2%, or 1% of its residues from a reference sequence, e.g., a natural LY49 transmembrane domain or a LY49 transmembrane domain described herein. In embodiments, the LY49 transmembrane domain of LY49-CAR differs by no more than 5, 4, 3, 2, or 1 residue from a reference sequence, e.g., a natural LY49 transmembrane domain or a LY49 transmembrane domain described herein. In embodiments, the LY49 transmembrane domain of LY49-CAR does not differ or has 100% homology to a reference sequence, for example, a natural LY49 transmembrane domain or a LY49 transmembrane domain described in the present invention.

The LY49 intracellular domain, as the term is used herein, refers to a polypeptide domain having the structural and functional properties of the LY49 intracellular domain. In one embodiment, the LY49 intracellular domain of LY49-CAR shares at least 70%, 80%, 85%, 90%, 95%, or 99% homology with a reference sequence, e.g., a natural LY49 intracellular domain or a LY49 intracellular domain described in the present invention. In embodiments, the LY49 intracellular domain of LY49-CAR differs by no more than 15%, 10%, 5%, 2%, or 1% of its residues from a reference sequence, e.g., a native LY49 intracellular domain or a LY49 intracellular domain described herein. In embodiments, the LY49 intracellular domain of LY49-CAR differs by no more than 5, 4, 3, 2, or 1 residue from a reference sequence, e.g., a natural LY49 intracellular domain or a LY49 intracellular domain described herein. In embodiments, the LY49 intracellular domain of LY49-CAR does not differ or has 100% homology with a reference sequence, for example, the natural LY49 intracellular domain or the LY49 intracellular domain described in the present invention.

Intracellular signaling domains or adapter molecules such as DAP12.

Some NKR-CARs interact with other molecules, such as those containing an intracellular signaling domain, such as ITAM. In one embodiment, the intracellular signaling domain is DAP12.

DAP12 is so named because of its structural features and putative function. Certain cell surface receptors do not contain a characteristic functional group that could hypothetically interact with another partner protein, presumably the 12 kDa protein.

- 55 040145

The signaling mechanism may include the ITAM signal.

DAP12 was identified from sequence databases based on a hypothetical association with CD3 (see Olcese et al. (1997) J. Immunol., 158:5083-5086), presence of the ITAM sequence (see Thomas, (1995) J. Exp. Med ., 181:1953-1956), prediction of a certain size (see Olcese and Takase et al. (1997) J. Immunol., 159:741-747, and other features. In particular, it was assumed that the transmembrane domain contains a charged residue , which provides a salt bridge to the appropriate transmembrane segments of its putative receptor partners, the CD94 KIR protein and likely other similar proteins See Daeron et al (1995) Immunity, 3:635-646.

In fact, many of the known receptor molecules KIR, MIR, ILT, and CD94/NKG2 may actually function with an accessory protein that is part of a functional receptor complex. See Olces et al. (1997) J. Immunol., 158:5083-5086; and Takase et al. (1997) J. Immunol., 159:741-747.

A DAP 12 domain, as the term is used herein, refers to a polypeptide domain having the structural and functional properties of a DAP 12 cytoplasmic domain and typically contains an ITAM domain. In one embodiment, the KIR-CAR DAP 12 domain shares at least 70%, 80%, 85%, 90%, 95%, or 99% homology with a reference sequence, eg, native DAP 12 or DAP 12 as described herein. In embodiments, the KIR-CAR DAP 12 domain differs by no more than 15%, 10%, 5%, 2%, or 1% of its residues from a reference sequence, eg, natural DAP12 or DAP12 as described herein. In embodiments, the KIR-CAR DAP 12 domain differs by no more than 5, 4, 3, 2, or 1 residue from a reference sequence, eg, natural DAP12 or DAP12 as described herein. In embodiments, the KIR-CAR DAP 12 domain does not differ or has 100% homology to a reference sequence, eg, native DAP12 or DAP12 as described herein.

DAP10 was identified in part by its homology to DAP12 and durim traits. In particular, unlike DAP12, which contains an ITAM activating motif, DAP10 contains an ITIM inhibitory motif. MDL-1 was identified by its functional relationship with DAP12.

A functional interaction between, for example, DAP12 or DAP10 and its accessory receptor may allow the use of a structural combination in receptors that is not normally found in the truncated form of the receptor. Thus, the signaling mechanism through accessory proteins such as DAP12 and DAP10 may allow interesting engineering of other KIR-like receptor complexes, for example with the KIR, MIR, ILT and CD94 NKG2 receptor types. It is possible to construct truncated forms of intact receptors that interact with DAP12 or DAP10 to form a functional signaling complex.

The primate DAP12 nucleotide sequence corresponds to nucleotides 1 to 339 of SEQ ID NO: 332; the amino acid sequence corresponds to amino acids 1 to 113 of SEQ ID NO: 333. The signal sequence appears to run from met(-26) to gln(-1) or ala1; the mature protein should run from about ala1 (or gln2), the extracellular domain from about ala1 to pro14; the extracellular domain contains two cysteines at 7 and 9, which likely provide disulfide bonds to additional homotypic or heterotypic accessory proteins; the transmembrane region runs from about gly15 or val16 to about gly39; and ITAM motif tyr65 to leu79 (YxxL-6/8x-YxxL) (SEQ ID NO: 341). EST LVA03A was identified and used to extract other overlapping sequences. Also see Human Genbank EST, which are part of human DAP12; some, but not all, including Genbank access numbers AA481924, H39980, W60940, N41026, R49793, W60864, W92376, H12338, T52100, AA480109, H12392, W74783, and T55959.

Inhibitory NKR-CAR.

The present invention relates to compositions and methods for limiting the depletion of non-malignant cells by a type of therapy based on CAR T cells. As described herein, a type of CAR T cell therapy involves the use of NK receptors including, but not limited to, NK cell activating and inhibitory receptors known as the killer cell immunoglobulin-like receptor (KIR). Thus, the invention relates to compositions and methods of using NKR-CAR, for example KIR-CAR, including, but not limited to, activating NKR-CAR (actNKR-CAR), for example, activating KIR-CAR (actKIR-CAR) and inhibitory NKR -CAR (inhNKR-CAR), e.g. inhibitory KIR-CAR (inhKIR-CAR).

In some embodiments, the inhKIR-CAR KIR is an inhibitory KIR that contains a long cytoplasmic tail containing an immunoreceptor tyrosine inhibitory motif (ITIM) that transduces inhibitory signals (referred to elsewhere herein as inhKIR-CAR).

In some embodiments, the inhKIR-CAR contains the cytoplasmic domain of an inhibitory molecule other than KIR. In some embodiments, such inhibitory molecules may reduce the ability of a cell to elicit an immune effector response. Cytoplasmic domains of inhibitory molecules can be connected, for example, by fusion with the transmembrane domains of KIR. Illustrative inhibitory molecules are shown in table. 1.

Table 1

inhibitory molecules

PD1 TIGIT KIR PD-L1 LAIR1 A2aR PD-L2 CD160 MNS class I CTLA4 TIM3 SEACAM (e.g. SEACAM-1, SEACAM-3 and/or SEACAM-5) LAG3 2B4 CD80 CD86 B7-NC (CD276) MNS class II GAL9 adenosine TGFR (eg. TGFR beta) VISTA B7-H4 (VTCN1) BTLA HVEM (TNFRSF14 or CD270)

In some embodiments, the inhKIR-CAR contains a PD1 cytoplasmic domain. A PD1 cytoplasmic domain, as the term is used herein, refers to a polypeptide domain having the structural and functional properties of a PD1 cytoplasmic domain. In one embodiment, the PD1 cytoplasmic domain of KIR-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology with a reference sequence, e.g., a natural PD1 cytoplasmic domain or a PD1 cytoplasmic domain described in the present invention (SEQ ID NO: 338). In embodiments, the PD1 cytoplasmic domain of KIR-CAR differs by no more than 15%, 10%, 5%, 2%, or 1% of its residues from a reference sequence, e.g., a natural PD1 cytoplasmic domain or a PD1 cytoplasmic domain described herein. In embodiments, the KIRCAR PD1 cytoplasmic domain differs by no more than 5, 4, 3, 2, or 1 residue from a reference sequence, such as a native PD1 cytoplasmic domain or a PD1 cytoplasmic domain described herein. In embodiments, the PD1 cytoplasmic domain of KIR-CAR does not differ or has 100% homology with a reference sequence, eg, a natural PD1 cytoplasmic domain or a PD1 cytoplasmic domain described in the invention.

In some embodiments, the inhKIR-CAR contains the cytoplasmic domain of CTLA-4. A CTLA-4 cytoplasmic domain, as the term is used herein, refers to a polypeptide domain having the structural and functional properties of a CTLA-4 cytoplasmic domain. In one embodiment, the CTLA-4 cytoplasmic domain of KIR-CAR has at least 70%, 80%, 85%, 90%, 95%, or 99% homology with a reference sequence, e.g., a natural CTLA-4 cytoplasmic domain or a CTLA-4 cytoplasmic domain described in the present invention (SEQ ID NO: 339). In embodiments, the CTLA-4 cytoplasmic domain of KIR-CAR differs by no more than 15%, 10%, 5%, 2%, or 1% of its residues from a reference sequence, e.g., the natural CTLA-4 cytoplasmic domain or the CTLA-4 cytoplasmic domain described herein. description. In embodiments, the CTLA-4 cytoplasmic domain of KIR-CAR differs by no more than 5, 4, 3, 2, or 1 residue from a reference sequence, e.g., a natural CTLA-4 cytoplasmic domain or a CTLA-4 cytoplasmic domain described herein. In embodiments, the CTLA-4 cytoplasmic domain of KIR-CAR does not differ or has 100% homology with a reference sequence, for example, a natural CTLA-4 cytoplasmic domain or a CTLA-4 cytoplasmic domain described in the present invention.

In one embodiment, the inhNKR-CAR, eg inhKIR-CAR, when bound to an antigen on a non-target or non-participating cell, inactivates the cytotoxic cell containing the inhNKR-CAR. While most of the description below is with inhKIR-CARs, the invention includes similar uses for other inhNKR-CARs.

In one embodiment, T cells expressing actKIR-CAR have an antitumor property when bound to their target, while T cells expressing inhKIR-CAR result in inhibition of cell activity when inhKIR-CAR binds to its target.

Regardless of the type of KIR-CAR, KIR-CARs are designed to contain an extracellular domain containing an antigen-binding domain fused to a cytoplasmic domain. In one embodiment, KIR-CAR, when expressed in a T cell, is able to redirect antigen recognition in a manner dependent on antigen specificity. An exemplary antigen is CD19 as this antigen is expressed in B-cell lymphoma. However, CD19 is also expressed by normal B cells, and thus CARs containing an anti-CD19 domain can deplete normal B cells. Depletion of normal B cells can make the individual receiving treatment susceptible to infection, since B cells usually help T cells control infection. The present invention relates to compositions and methods for limiting normal tissue depletion during KIR-CAR T cell therapy. In one embodiment, the present invention relates to methods for treating cancer and other disorders using KIR-CAR T cell therapy while limiting the depletion of healthy non-participating cells.

In one embodiment, the invention provides for the control or regulation of T cell activity with KIR-CAR. In one embodiment, the invention relates to compositions and methods relating to the genetic modification of T cells to express a set of KIR-CAR types, wherein activation of a T cell with KIR-CAR is dependent on binding of a set of KIR-CAR types to their target receptor. Dependence on the binding of a set of KIR-CAR types improves the specificity of the T cell's lytic activity with KIR-CAR, thus reducing the likelihood of depletion of normal healthy tissue.

In another embodiment, the invention comprises compositions and methods relating to the genetic modification of a T cell with an inhibitory KIR-CAR. In one embodiment, the inhibitory KIR-CAR comprises an extracellular antigen-binding domain that recognizes an antigen associated with a normal non-malignant cell and an inhibitory cytoplasmic domain.

In one embodiment, the invention relates to a dual KIR-CAR, wherein the T cell is genetically modified to express inhKIR-CAR and actKIR-CAR. In one embodiment, binding of the inhKIR-CAR to a normal non-cancerous cell results in inhibition of the dual KIR-CAR T cell. For example, in one embodiment, binding of an inhKIR-CAR to a normal non-malignant cell results in the death of the double-KIR-CAR T cell. In another embodiment, binding of inhKIR-CAR to a normal non-cancerous cell results in inhibition of actKIR-CAR signaling. In yet another embodiment, binding of inhKIR-CAR to a normal non-cancerous cell results in the induction of signal transduction that prevents the actKIR-CAR T cell from exhibiting antitumor activity. Thus, a dual KIR-CAR comprising at least one inhKIR-CAR and at least one actKIR-CAR according to the invention refers to a dual KIR-CAR T cell activity regulation mechanism.

In one embodiment, the present invention relates to methods for treating cancer and other disorders using T-cell based therapies with KIRCAR while minimizing depletion of normal healthy tissue. The malignant tumor may be a hemoblastosis, a solid tumor, a primary tumor, or a metastatic tumor. Other diseases that are monotreatable using the compositions and methods of the invention include viral, bacterial and parasitic infections, as well as autoimmune diseases.

extracellular hinge domain.

The extracellular hinge domain, as the term is used herein, refers to the NKR-CAR polypeptide sequence located between the transmembrane domain and the antigen-binding domain. In one embodiment, the extracellular hinge domain provides sufficient distance from 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 one embodiment, the extracellular hinge domain is sufficiently short or flexible that it does not interfere with the interaction of a cell that contains the NKR-CAR with an antigen-bearing cell, eg, a target cell. In one embodiment, the extracellular hinge domain is 2 to 20, 5 to 15, 7 to 12, or 8 to 10 amino acids in length. In one embodiment, the hinge domain contains at least 50, 20, or 10 residues. In embodiments, the hinge length is 10 to 300, 10 to 250, or 10 to 200 residues. In one embodiment, the distance that the hinge extends from the cell is short enough that the hinge does not interfere with interaction with the surface of the target cell. In one embodiment, the hinge is rubbed less than 20, 15, or 10 nanometers from the surface of the cytotoxic cell. Thus, the suitability of a hinge can be affected by the linear length, the number of amino acid residues, and the flexibility of the hinge. The length of the IgG4 hinge can be 200 amino acids, but the distance it extends from the surface of the cytotoxic cell is shorter due to the folding of the Ig domain. The CD8 alpha domain, which consists of ~43 amino acids, is fairly linear at ~8 nm in length. In contrast, the length of the IgG4 C2 and C3 hinge is ~200 amino acids, but its distance from the cytotoxic cell surface is comparable to that of the CD8 alpha hinge. Without wishing to be bound by theory, the similarity in elongation is affected by flexibility.

In some cases, the extracellular hinge domain is, for example, a hinge of

- 58 040145 human protein, fragment or short oligo- or polypeptide linker.

In some embodiments, the implementation of the hinge is an artificial sequence. In one embodiment, the hinge is a short oligopeptide linker containing a glycine-serine doublet.

In some embodiments, the implementation of the hinge is a natural sequence. In some embodiments, the hinge may be a human Ig (immunoglobulin) hinge or a fragment thereof. In one embodiment, for example, the hinge comprises (eg, consists of) an IgG4 hinge amino acid sequence (SEQ ID NO: 3). In one embodiment, for example, the hinge comprises (eg, consists of) an IgD hinge amino acid sequence (SEQ ID NO: 4). In some embodiments, the hinge may be a human CD8 hinge or a fragment thereof. In one embodiment, for example, the hinge contains (eg, consists of) the CD8 hinge amino acid sequence (SEQ ID NO: 2). Additional sequences of exemplary hinge domains are provided in Table. 5.

TCAR.

In some embodiments, the CAR cell-based therapy of the present invention comprises NKR-CAR in combination with TCAR. In embodiments, the CAR cell-based therapy of the present invention comprises an NKR-CAR expressing cell as described herein, further comprising, for example, expressing TCAR. In alternative embodiments, the CAR cell-based therapy of the present invention comprises a first cell expressing an NKR-CAR of the present invention and a second cell expressing a TCAR.

In one embodiment, the TCAR contains an antigen-binding domain fused to an intracellular domain, such as a cytoplasmic domain. In embodiments, the cytoplasmic domain contains an intracellular signaling domain and produces an intracellular signal, where an extracellular domain, such as an antigen-binding domain to which it is fused, binds to a counterligand. Intracellular signaling domains may include primary intracellular signaling domains and costimulatory signaling domains. In one embodiment, a TCAR molecule can be engineered for expression in an effector cell of the immune system, such as a T cell or NK cell, such that the TCAR molecule contains a domain, such as a primary intracellular signaling domain, a co-stimulatory signaling domain, inhibitory domains, and etc., which are derived from a polypeptide, which are usually associated with an immune cell. For example, a TCAR for expression in an effector cell of the immune system, such as a T cell or NK cell, may contain a 4-1BB domain and a CD3 zeta domain. In this case, both the 4-1BB and CD3 zeta domains will be derived from polypeptides associated with an effector cell of the immune system, such as a T cell or NK cell. In another embodiment, the TCAR molecule can be designed to be expressed in an effector cell of the immune system, such as a T cell or NK cell, such that the TCAR molecule contains a domain that is derived from a polypeptide that is not normally associated with the effector cell. immune system. Alternatively, a TCAR for expression in a NK cell may contain a 4-1BB domain and a CD3 zeta domain derived from a T cell (see, for example, WO 2013/033626, incorporated herein by reference).

The following sections relate to the construction and expression of NKR-CAR and TCAR described in the present description, and how to use them.

antigen binding domain.

The CARs described herein, for example, the KIR-CARs and TCARs described herein, contain an antigen-binding domain in the extracellular region.

Antigen binding domain, as the term is used herein, refers to a molecule that has affinity for a target antigen, typically an antigen on a target cell, eg, a cancer cell. An exemplary antigen-binding domain comprises a polypeptide, such as an antibody molecule (which includes the antibody and its antigen-binding fragments, such as immunoglobulin, single-domain antibody (sdAb), and scFv), or a non-antibody backbone, such as fibronectin, and the like. In embodiments, the antigen binding domain is a single polypeptide. In embodiments, the antigen binding domain comprises one, two or more polypeptides.

The choice of antigen binding domain may depend on the type and number of ligands or receptors that define the surface of the target cell. For example, an antigen binding domain can be selected to recognize a ligand or receptor that acts as a cell surface marker on target cells associated with a particular disease state. Examples of cell surface markers that can act as ligands or receptors include a cell surface marker associated with a particular disease state, such as cell surface markers for viral diseases, bacterial diseases, parasitic infections, autoimmune diseases, and disorders associated with unwanted cell proliferation. , for example, a malignant tumor, for example, a malignant tumor described in the present invention.

In relation to the present invention, a tumor antigen or an antigen of a proliferative disorder or an antigen associated with a proliferative disorder refers to antigens that are common to specific proliferative disorders. In certain aspects, the proliferative disorder antigens of the present invention are derived from cancers, including, but not limited to, primary or metastatic melanoma, thioma, lymphoma, sarcoma, lung cancer (e.g., NSCLC or SCLC), liver cancer, non-Hodgkin's lymphoma, lymphoma Hodgkin's, leukemias, multiple myeloma, glioblastoma, neuroblastoma, uterine cancer, cervical cancer, kidney cancer, thyroid cancer, bladder cancer, kidney cancer and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer glands, colon cancer, etc. In some embodiments, the cancer is B-cell acute lymphoid leukemia (BALL), T-cell acute lymphoid leukemia (TALL), acute lymphoid leukemia (ALL), acute myelogenous leukemia (AML); one or more chronic leukemias including, but not limited to, chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL); additional hematologic malignancies or hematologic conditions including, but not limited to, B-cell prolymphocytic leukemia, blast plasmacytoid dendritic cell neoplasia, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large follicular lymphoma , malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone cell lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasia, Waldenström's macroglobulinemia.

In one embodiment, the tumor antigen comprises one or more cancer antigenic epitopes immunologically recognized by tumor infiltrating lymphocytes (TILs) derived from a mammalian cancer.

Tumor antigens are proteins produced by tumor cells that elicit an immune response, in particular T-cell mediated immune responses. The choice of antigen-binding domain of the invention depends on the particular type of cancer to be treated. Tumor antigens are well known in the art and include, for example, the tumor antigen described in International Application PCT/US2015/020606. In embodiments, the tumor antigen is selected from glioma-associated antigen, cancer embryonic antigen (CEA), EGFRvIII, interleukin 11 receptor alpha (IL-11Ra), interleukin 13 receptor alpha-2 subunit (IL-13Ra or CD213A2), epidermal growth factor receptor ( EGFR), B7H3 (CD276), Kit (CD117), carbonic anhydrase (CA-IX), CS-1 (also referred to as CD2 subset 1), cell surface-associated mucin 1 (MUC1), BCMA, an oncogenic fusion protein consisting from the punctate break cluster region (BCR) and bcr-abl homologue 1 of the Abelson murine leukemia viral oncogene (Abl), tyrosine protein kinase receptor ERBB2 (HER2/neu), human β-chorionic gonadotropin, alpha-fetoprotein (AFP), anaplastic lymphoma kinase (ALK), CD19, CD123, cyclin B1, lectin-reactive AFP, adrenergic receptor beta 3 associated with Fos antigen 1, (ADRB3), thyroglobulin, tyrosinase; ephrin type A receptor 2 (EphA2), glycation end products receptor (RAGE-1), renal ubiquitin antigen 1 (RU1), renal ubiquitin antigen 2 (RU2), synovial sarcoma antigen, punctate gap X 2 (SSX2), anchor protein 4 A-kinases (AKAP-4), lymphocyte-specific protein tyrosine kinase (LCK), proacrosin-binding protein sp32 (OY-TES1), domain pair protein Pax-5 (PAX5), squamous cell carcinoma antigen recognized by T cells 3 (SART3) , lectin-like molecule 1 type C (CLL-1 or CLECL1), fucosyl GM1, hexasaccharide moiety of globo-H glycoceramide (GloboH), MN-CA IX, epithelial cell adhesion molecule (EPCAM), EVT6-AML, transglutaminase 5 (TGS5), human telomerase reverse transcriptase (hTERT), polysialic acid, placental antigen 1 (PLAC1), gut carboxylesterase, Lewis Y antigen, sialylated Lewis adhesion molecule (sLe), lymphocyte antigen complex 6, locus K 9 (LY6K), mutant heat shock protein 70 -2 (mut hsp70-2), M-CSF, a v-myc homologue derived from the oncogenic neuroblastoma avian myelocytomatosis virus (MYCN), member of the C homologues of the Ras family (RhoC), tyrosinase-related protein 2 (TRP-2), cytochrome P450 1B1 (CYP1B1), similar to binding CCCTC factor (zinc finger protein) antigen (BORIS or Brother of the Regulator of Imprinted Sites), prostate, prostate specific antigen (PSA), paired domain Pax-3 protein (PAX3), prostate acid phosphatase ( PAP), cancer/testes antigen 1 (NYESO-1), cancer/testes antigen 2 (LAGE-1a), LMP2, neutral cell adhesion molecule (NCAM), p53 tumor protein (p53), p53 mutant, rat sarcoma mutant (Ras), glycoprotein 100 (gp100), protein, OR51E2, pannexin 3 (PANX3), prostate specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), melanoma-associated high molecular weight a antigen (HMWMAA), cell receptor 1 virus

- 60 040145 hepatitis A (HAVCR1), vascular endothelial growth factor receptor 2 (VEGFR2), platelet growth factor receptor beta (PDGFR-beta), legumain, human papillomavirus E6 (HPV E6), human papillomavirus E7 (HPV E7), survivin, telomerase, sperm protein 17 (SPA17), fetal stage-specific antigen 4 (SSEA-4), tyrosinase, TCR gamma alternative reading frame protein (TARP), Wilms tumor protein (WT1), prostate carcinoma tumor antigen 1 ( PCTA-1), melanoma inhibitor of apoptosis (ML-IAP), MAGE, melanoma-associated antigen 1 (MAGE-A1), testicular cancer-testicular antigen 1 in melanoma (MAD-CT-1), testicular cancer-testicular antigen 2 in melanoma melanoma (MAD-CT-2), melanoma T cell-recognized antigen 1 (MelanA/MART1), antigen family X, member 1A (XAGE1), mutant elongation factor 2 (ELF2M), ERG (TMPRSS2 ETS fusion gene), N - acetylglucosaminyltransferase V (NA17), neutrophil elastase , translocation breakpoints in sarcoma, breast differentiation antigen (NY-BR-1), eFrin-B2, CD20, CD22, CD24, Cd30, CD33, CD38, CD44v6, CD97, CD171, CD179a, androgen receptor, insulin growth factor ( IGF)-I, IGF-II, IGF-I receptor, GD2 ganglioside (GD2), o-acetyl-GD2-ganglioside (OAcGD2), GD3 ganglioside (aNeu5Ac(28)aNeu5Ac(2-3)bDGalp(1-4) bDGlcp(1-1)Cer), Gm3 ganglioside (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(11)Cer), representative of D group 5 class C G-protein coupled receptor (GPRC5D), receptor 20 coupled with G protein (GPR20), X chromosome open reading frame 61 (CXORF61), folate receptor (FRa), folate receptor beta, orphan receptor tyrosine kinase 1 (ROR1), Fms-like tyrosine kinase 3 (Flt3), tumor-associated glycoprotein 72 (TAG72) ), Tn antigen (TN Ag or (GalNAca-Ser/Thr)), angiopoietin-binding cell surface receptor 2 (Tie 2), endothelial tumor marker 1 (TEM1 or CD248), endothelial-related antigen tumor marker 7 (TEM7R), claudin 6 (CLDN6), thyroid stimulating hormone receptor (TSHR), uroplakin 2 (UPK2), mesothelin, serine protease 21 (testisin or PRSS21), epidermal growth factor receptor (EGFR), fibroblast activation protein alpha (FAP), olfactory receptor 51E2 (OR51E2), gene 6 of the ETS translocation variant localized on chromosome 12p (ETV6-AML), CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); an IgA receptor Fc fragment (FCAR or CD89); a member of the 2 subfamily A leukocyte immunoglobulin-like receptor (LILRA2); CD300 molecular-like member of the f family (CD300LF); member A family 12 lectin domain type C (CLEC12A); bone marrow stromal cell antigen 2 (BST2); containing an EGF-like module mucin-like hormone receptor 2 (EMR2); lymphocyte antigen 75 (LY75); glypican 3 (GPC3); Fc receptor-like 5 (FCRL5) and lamb-like polypeptide immunoglobulin 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 of them. combinations.

In one embodiment, the tumor antigen comprises one or more cancer antigenic epitopes associated with the cancer.

Cancers express a number of proteins that can act as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase and GP 100 in melanoma and prostate acid phosphatase (PAP), and prostate specific antigen (PSA) in prostate cancer. Other target antigens include transformation-associated molecules such as the HER2/Neu/ErbB-2 oncogene. Yet another group of target antigens are oncofetal antigens such as cancer embryonic antigen (CEA). In B-cell lymphoma, the tumor-specific idiotypic immunoglobulin is a true tumor-specific immunoglobulin antigen that is unique to the individual tumor. B cell differentiation antigens such as CD19, CD20 and CD37 are other candidate antigen targets in B cell lymphoma.

Non-limiting examples of tumor antigens include the following: differentiation antigens such as MART-1/MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multilinear antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutant tumor suppressor genes such as p53, Ras, HER-2/neu; unique tumor antigens resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4RET, IGH-IGK, MYL-RAR; and viral antigens such as Epstein-Barr EBVA antigens and human papillomavirus (HPV) E6 and E7 antigens. 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, p15, p16, 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, MG7Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2\cyclophilin C-associated protein, TAAL6, TAG72, TLP and TPS.

Depending on the desired antigen that is chosen as a target, the CAR of the invention

- 61 040145 can be designed to contain an appropriate antigen-binding domain that is specific for the desired target antigen.

Antigen binding domains derived from an antibody molecule.

The antigen binding domain can be derived from an antibody molecule, e.g., one or more monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, single domain antibodies, e.g., heavy chain variable domain (VH), light chain variable domain (VL), and variable domain (VHH), for example, from a human or a camel. In some cases, it is beneficial when the antigen-binding domain is derived from the same species for which the CAR is ultimately used, e.g., for use in humans, it may be beneficial that the antigen-binding domain of a CAR, e.g., KIR-CAR, e.g. , contained a human-owned or humanized antigen-binding domain. Antibodies can be obtained by methods known in the art.

In certain aspects, the scFv is contiguous and in the same reading frame as the leader sequence. In one aspect, the leader sequence is the amino acid sequence provided as SEQ ID NO: 1.

In one aspect, the antigen binding domain is a fragment, such as a single chain variable fragment (scFv). In one aspect, the antigen binding domain is a Fv, Fab, (Fab')2, or a bifunctional (eg, bispecific) hybrid antibody (eg, Lanzavecchia et al., Eur. J. Immunol., 17, 105 (1987)). In one aspect, the antibodies and fragments thereof of the invention bind to a tumor antigenic protein or fragment thereof with wild-type specificity or increased affinity.

In some cases, scFvs can be prepared in a manner known in the art (see, for example, 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 made by linking the VH and VL chains to each other using flexible polypeptide linkers. In some embodiments, the scFv molecules comprise a flexible polypeptide linker with an optimized length and/or amino acid composition. The length of the flexible polypeptide linker can greatly influence how scFv variable regions fold and interact. In fact, if a short polypeptide linker is used (eg, in the range of 5-10 amino acids), intra-chain folding is prevented. Interchain folding is also necessary to bring two variable regions closer together to form a functional epitope binding site. For examples of linker orientation and size, see, for example, Hollinger et al., 1993 Proc. Natl. Acad. sci. U.S.A., 90:64446448, US Patent Application Publication Nos. 2005/0100543, 2005/0175606, 2007/0014794, and PCT Publication Nos. WO 2006/020258 and WO 2007/024715, which are incorporated herein by reference.

scFv may contain a linker of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 , 35, 40, 45, 50 or more amino acid residues between its VL and VH regions. The linker sequence may contain any naturally occurring amino acid. In one embodiment, the scFv peptide linker consists of amino acids, such as glycine and/or serine residues, used alone or in combination to link the heavy chain variable region and the light chain variable region to each other. In one embodiment, the flexible polypeptide linker is a Gly/Ser linker and, for example, contains the amino acid sequence (Gly-Gly-Gly-Ser)n, where n is a positive integer equal to or greater than 1 (SEQ ID NO: 40). 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 one embodiment, flexible polypeptide linkers include, but are not limited to, (Gly4 Ser)4 (SEQ ID NO: 27) or (Gly4 Ser) 3 (SEQ ID NO: 28). In another embodiment, the linkers contain multiple repeats of (Gly2Ser), (GlySer) or (Gly3Ser) (SeQ ID NO: 29).

In some embodiments, the antigen-binding domain is single-domain antigen-binding (SDAB) molecules. The SDAB molecule includes molecules whose complementarity determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain variable domains binding molecules not naturally containing light chains, single domains derived from conventional 4 chain antibodies, engineered domains, and single domain scaffolds other than those derived from antibodies (e.g., those described more details below). The SDAB molecules may be any of the existing single domain molecules in the art or any future single domain molecules. SDAB molecules can be obtained from any species, including, but not limited to, mouse, human, camel, llama, fish, shark, goat, rabbit, and cattle. The term also includes natural single domain antibody molecules from species other than Camelidae and sharks.

In one aspect, the SDAB molecule can be derived from an immunoglobulin variable region found in fish, such as, for example, a variable region derived from an immunoglobulin isotype known as a novel antigen receptor (NAR) found in shark sera. Methods for making single domain molecules derived from the NAR variable region (IgNAR) are described in WO 03/014161 and Streltsov (2005), Protein Sci., 14:2901-2909.

In another aspect, the SDAB molecule is a naturally occurring single domain antigen binding molecule known as a heavy chain without light chains. Such single domain molecules are described, for example, in WO 9404678 and Hamers-Casterman C. et al. (1993) Nature, 363:446-448. For clarity, such a variable domain derived from a heavy chain molecule naturally lacking a light chain is known herein as a VHH or nanobody to differentiate its conventional VH of four-chain immunoglobulins. Such a VHH molecule can be obtained from Camelidae species such as camel, llama, dromedary, alpaca and guanaco. Other species along with the Camelidae can produce heavy chain molecules that do not naturally contain a light chain; such VHHs are within the scope of the invention.

In certain embodiments, the SDAB molecule is a single chain fusion polypeptide containing one or more single domain molecules (e.g., nanobodies) that do not contain a complementary variable domain or constant region, e.g., Fc, of an immunoglobulin that binds to one or more target antigens.

SDAB molecules can be recombinant, CDR-grafted, humanized, camelized, deimmunized, and/or produced in vitro (eg, selectable by phage display).

In one embodiment, the antigen-binding domain portion comprises a human antibody or fragment thereof.

In some embodiments, the non-human antibody is humanized, where specific sequences or regions of the antibody are modified to increase similarity to an antibody naturally produced in humans. In one embodiment, the antigen binding domain is humanized.

Non-human antibodies can be humanized in a variety of ways known in the art, such as CDR grafting (see, for example, EP 239,400; WO 91/09967; and US 5225539, 5530101 and 5585089, each incorporated herein by reference in its entirety), veneering or surface modification (see, for example, EP 592.106 and EP 519.596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnicka et al ., 1994, Protein Engineering, 7(6):805-814; and Roguska et al., 1994, PNaS, 91:969-973, each of which is incorporated herein in its entirety by reference), strand permutation (see, for example, US Pat. No. 5,565,332, which is incorporated herein by reference in its entirety), and by the methods described, for example, in US Patent Application Publication No. US2005/0042664, US Patent Application Publication No. US2005/0048617, US Patent No. 6407213, US Pat. No. 5766886, International Publication No. WO 9317105, Tan et al., 2002, J. Immunol., 169:1119-25; Caldas et al., 2000, Protein Eng., 13(5):353-60; Morea et al., 2000, Methods, 20:267-79; Baca et al., 1997, J. Biol. Chem. 272:10678-84; Roguska et al., 1996, Protein Eng., 9(10):895-904; Couto et al., 1995, Cancer Res., 55:5973s-5977; Couto et al., 1995, Cancer Res., 55(8):1717-22; Sandhu, 1994, Gene, 150(2):40910 and Pedersen et al., 1994, J. Mol. Biol., 235(3):959-73, each of which is incorporated herein by reference in its entirety. Typically, framework residues in framework regions are replaced with the corresponding residue from the CDR of the donor antibody to alter, eg, improve, antigen binding. Such framework substitutions are identified by methods well known in the art, for example, modeling interactions of CDRs and framework residues to identify framework residues important for antigen binding, and sequence comparison to identify unusual framework residues at specific positions. (See, for example, Queen et al., US Pat. No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323, which are incorporated herein by reference in their entirety.) In preferred embodiments, the humanized antibody molecule contains the sequence described in the present description, for example, the variable region of the light chain and/or the variable region of the heavy chain described in the present description, for example, the humanized variable region of the light chain and/or the variable region of the heavy chain described in table. 4.

A humanized antibody contains one or more amino acid residues introduced into it from a non-human source. Such non-human amino acid residues are generally referred to as import residues, which are typically removed from the imported variable domain. Thus, humanized antibodies contain one or more CDRs from non-human immunoglobulin molecules and framework regions from a human. Humanization of antibodies is well known in the art and can essentially be carried out in the following manner by Winter et al. (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323327 (1988) ; Verhoeyen et al., Science, 239:1534-1536 (1988)), by replacing the rodent CDR or CDR sequences with the corresponding human antibody sequences, i.e. CDR grafting (EP 239400; PCT Publication No. WO 91/09967 and US Pat. Nos. 4,816,567; 6,331,415; 5,225,539; In such humanized chimeric antibodies, there is essentially less than an intact human variable domain.

- 63 040145 ka was replaced with the corresponding sequence from non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some framework (FR) residues are replaced with residues from similar regions in rodent antibodies. Antibody humanization can also be obtained by vining or resurfacing (EP 592106; EP 519596; Padlan, 1991, Molecular Immunology, 28(4/5): 489-498; Studnicka et al., Protein Engineering, 7(6):805-814 (1994) and Roguska et al., PNAS, 91:969-973 (1994)) or by strand permutation (US Pat. No. 5,565,332), the contents of which are incorporated herein by reference in their entirety.

In some embodiments, an antibody of the invention is further prepared using an antibody comprising one or more of the VH and/or VL sequences described herein, which can be used as a starting material for constructing a modified antibody, where the modified antibody may have altered properties from with the original antibody. In various embodiments, an antibody is constructed by modifying one or more amino acids in one or both variable regions (i.e., VH and/or VL), for example, in one or more CDR regions and/or in one or more framework regions.

In another aspect, the antigen binding domain is a T cell receptor (TCR) or a fragment thereof, eg, single chain TCR (scTCR). Methods for producing such TCRs are known in the art. See, for example, Willemsen R.A. et al., Gene Therapy, 7: 1369-1377 (2000); Zhang T. et al., Cancer Gene Ther., 11: 487-496 (2004); Aggen et al., Gene Ther., 19(4):365-74 (2012) (references incorporated herein by its entirety). For example, scTCRs can be constructed that contain the Va and Ve genes from a T cell clone linked by a linker (eg, a flexible peptide). This approach is very suitable for a cancer-associated target, which itself is intracellular, however, a fragment of such an antigen (peptide) is presented on the surface of MHC cancer cells.

In embodiments, the NKR-CAR or TCAR molecule described herein contains an antigen-binding domain containing an scFv that specifically binds to the tumor antigen described herein. Amino acid sequences of scFvs that specifically bind to the tumor antigens described herein are provided in Table 1. 4. In some embodiments, the implementation of the CDR sequences of scFv provided in table. 4 is underlined. The exact boundaries of the amino acid sequence of a given CDR can be determined using any of a number of well known numbering systems, including the numbering systems described by Kabat or Chothia, or using a combination of the Kabat and Chothia numbering systems.

The scFv sequences provided in Table. 4 contain a linker sequence that connects the variable regions of the scFv heavy and light chains. The linker sequence can be any of the linker sequences described in the present description, for example, the linker sequence provided in table. 5.

It should also be noted that some of the scFv sequences provided in Table. 4, additionally contain a leader sequence, for example, the amino acid sequence of SEQ ID NO: 1, with some of the scFv sequences provided in table. 4 do not contain a leader sequence, such as the amino acid sequence of SEQ ID NO: 1.

The scFv sequences provided in Table. 4, with or without a leader sequence, such as SEQ ID NO: 1, are also included in the invention. A person skilled in the art can easily use the sequences provided in table. 4 to generate scFv or CAR antigen-binding domains with or without a leader sequence, such as the amino acid sequence of SEQ ID NO: 1.

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Exemplary Antigen Binding Domains

Table 4

Antigen target Name Amino acid sequence SEQ ID NO: CD19 huscFvl EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLI YHTSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPY TFGQGTKLEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTC TVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYSSSLKSRVTIS KDN S KNQVS LKL SSVTAADTAVYYCAKHYYYGGS YAMDYWGQGT LVTV SS 161 CD19 huscFv2 Eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlli yhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpy tfgqgtkleikggggsggggsggggsqvqlqesgpglvkpsetlsltc tvsgvslpdygvswirqppgkglewigviwgsettyyqsslksrvtis kdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtv ss 162 CD19 huscFv3 Qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewi gviwgsettyyssslksrvtiskdnsknqvslklssvtaadtavyyca khyyyggsyamdywgqgtlvtvssggggsggggsggggseivmtqspa tlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsg iparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkle ik 163 CD19 huscFv4 Qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewi gviwgsettyyqsslksrvtiskdnsknqvslklssvtaadtavyyca khyyyggsyamdywgqgtlvtvssggggsggggsggggseivmtqspa tlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsg iparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkle ik 164 CD19 huscFv5 Eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlli yhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpy tfgqgtkleikggggsggggsggggsggggsqvqlqesgpglvkpset Isltctvsgvslpdygvswirqppgkglewigviwgsettyyssslks rvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqg tlvtvss 165 CD19 huscFv6 Eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlli yhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpy tfgqgtkleikggggsggggsggggsggggsqvqlqesgpglvkpset Isltctvsgvslpdygvswirqppgkglewigviwgsettyyqsslks rvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqg 166

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tlvtvss CD19 huscFv7 Qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewi gviwgsettyyssslksrvtiskdnsknqvslklssvtaadtavyyca khyyyggsyamdywgqgtlvtvssggggsggggsggggsggggseivm. tqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhts rlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgq gtkleik 167 CD19 huscFv8 Qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglew igviwgsettyyqsslksrvtiskdnsknqvslklssvtaadtavyy cakhyyyggsyamdywgqgtlvtvssggggsggggsggggsggggse ivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlli yhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlp ytfgqgtkleik 168 CD19 huscFv9 Eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlli yhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpy tfgqgtkleikggggsggggsggggsggggsqvqlqesgpglvkpset Isltctvsgvslpdygvswirqppgkglewigviwgsettyynsslks rvtiskdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqg tlvtvss 169 CD19 Hu scFvlO Qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewi gviwgsettyynsslksrvtiskdnsknqvslklssvtaadtavyyca khyyyggsyamdywgqgtlvtvssggggsggggsggggsggggseivm tqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhts rlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgq gtkleik 170 CD19 Hu scFvll Eivmtqspatlslspgeratlscrasqdiskylnwyqqkpgqaprlli yhtsrlhsgiparfsgsgsgtdytltisslqpedfavyfcqqgntlpy tfgqgtkleikggggsggggsggggsqvqlqesgpglvkpsetlsltc tvsgvslpdygvswirqppgkglewigviwgsettyynsslksrvtis kdnsknqvslklssvtaadtavyycakhyyyggsyamdywgqgtlvtv ss 171 CD19 Hu scFvl2 Qvqlqesgpglvkpsetlsltctvsgvslpdygvswirqppgkglewi gviwgsettyynsslksrvtiskdnsknqvslklssvtaadtavyyca khyyyggsyamdywgqgtlvtvssggggsggggsggggseivmtqspa tlslspgeratlscrasqdiskylnwyqqkpgqaprlliyhtsrlhsg iparfsgsgsgtdytltisslqpedfavyfcqqgntlpytfgqgtkle ik 172 CD19 muCTL019 Diqmtqttsslsaslgdrvtiscrasqdiskylnwyqqkpdgtvklli yhtsrlhsgvpsrfsgsgsgtdysltisnleqediatyfcqqgntlpy tfgggtkleitggggsggggsggggsevklqesgpglvapsqslsvtc tvsgvslpdygvswirqpprkglewlgviwgsettyynsalksrltii kdnsksqvfIkmnslqtddtaiyycakhyyyggsyamdywgqgtsvtv 173

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ss CD123 Mull72 DIVLTQ S PAS LAVS LGQRATIS CRASESVDNYGNTFMHWYOOKP GQ P P К L LIYRASNLESGIPARFS GS GS RT D FT LΤIΝ PVEADDVATYYCQQSN EDPPTFGAGTKLELKGGGGSGGGGSSGGGSOIOLVOSGPELKKPGETV KIS CKASGYIFTNYGMNWVKQAPGKSFKWMGWINTYTGESTYSADFKG RFAFSLETSASTAYLHINDLKNEDTATYFCARSGGYDPMDYWGQGTSV TVSS 174 CD123 Mull76 DVOITOSPSYLAASPGETITINCRASKSISKDLAWYOEKPGKTNKLLI YSGSTLQSGIPSRFSGSGSGTDFTLTISSLEPEDFAMYYCQQHNKYPY TFGGGTKLEIKGGGGSGGGGSSGGGSQVQLQQPGAELVRPGASVKLSC KAS GYT FTSYWMNWVKQRPDQGLEWIGRIDPYDSETHYNQKFKDKAIL TVDKSSSTAYMOLSSLTSEDSAVYYCARGNWDDYWGOGTTLTVSS 175 CD123 huscFvl Divltqspdslavslgeratincrasesvdnygntfmhwyqqkpgqpp klliyrasnlesgvpdrfsgsgsrtdftltisslqaedvavyycqqsn edpptfgqgtkleikggggsggggsggggsggggsqiqlvqsgselkk pgasvkvsckasgyiftnygmnwvrqapgqglewmgwintytgestys adfkgrfvfsldtsvstaylqinalkaedtavyycarsggydpmdywg qgttvtvss 176 CD123 huscFv2 Divltqspdslavslgeratincrasesvdnygntfmhwyqqkpgqpp klliyrasnlesgvpdrfsgsgsrtdftltisslqaedvavyycqqsn edpptfgqgtkleikggggsggggsggggsggggsqiqlvqsgaevkk pgasvkvsckasgyiftnygmnwvrqapgqrlewmgwintytgestys adfkgrvtitldtsastaymelsslrsedtavyycarsggydpmdywg qgttvtvss 177 CD123 huscFv3 Eivltqspatlslspgeratlscrasesvdnygntfmhwyqqkpgqap rlliyrasnlesgiparfsgsgsrtdftltisslepedvavyycqqsn edpptfgqgtkleikggggsggggsggggsggggsqiqlvqsgselkk pgasvkvsckasgyiftnygmnwvrqapgqglewmgwintytgestys adfkgrfvfsldtsvstaylqinalkaedtavyycarsggydpmdywg qgttvtvss 178 CD123 huscFv4 Eivltqspatlslspgeratlscrasesvdnygntfmhwyqqkpgqap rlliyrasnlesgiparfsgsgsrtdftltisslepedvavyycqqsn edpptfgqgtkleikggggsggggsggggsggggsqiqlvqsgaevkk pgasvkvsckasgyiftnygmnwvrqapgqrlewmgwintytgestys adfkgrvtitldtsastaymelsslrsedtavyycarsggydpmdywg qgttvtvss 179 CD123 huscFv5 Qiqlvqsgselkkpgasvkvsckasgyiftnygmnwvrqapgqglewm gwintytgestysadfkgrfvfsldtsvstaylqinalkaedtavyyc arsggydpmdywgqgttvtvssggggsggggsggggsggggsdivltq spdslavslgeratincrasesvdnygntfmhwyqqkpgqppklliyr asnlesgvpdrfsgsgsrtdftltisslqaedvavyycqqsnedpptf gqgtkleik 180

- 67 040145

CD123 huscFv6 Qiqlvqsgselkkpgasvkvsckasgyiftnygmnwvrqapgqglewm gwintytgestysadfkgrfvfsIdtsvstaylqinalkaedtavyyс arsggydpmdywgqgttvtvssggggsggggsggggsggggseivltq spatlslspgeratls erasesvdnygntfmhwyqqkpgqaprlliyr asnlesgiparfsgsgsrtdftltisslepedvavyycqqsnedpptf gqgtkleik 181 CD123 huscFv7 Qiqlvqsgaevkkpgasvkvsckasgyiftnygmnwvrqapgqrlewm gwintytgestysadfkgrvtitldtsastaymelsslrsedtavyyc arsggydpmdywgqgttvtvssggggsggggsggggsggggsdivltq spdslavslgeratincrasesvdnygntfmhwyqqkpgqppklliyr asnlesgvpdrfsgsgsrtdftltisslqaedvavyycqqsnedpptf gqgtkleik 182 CD123 huscFv8 Qiqlvqsgaevkkpgasvkvsckasgyiftnygmnwvrqapgqrlewm gwintytgestysadfkgrvtitldtsastaymelsslrsedtavyyc arsggydpmdywgqgttvtvssggggsggggsggggsggggseivltq spatlslspgeratls erasesvdnygntfmhwyqqkpgqaprlliyr asnlesgiparfsgsgsrtdftltisslepedvavyycqqsnedpptf gqgtkleik 183 CD123 123-1 human malpvtalllplalllhaarpqvqlvqsgaevkkpgasvkvsckasgy tftgyymhwv rqapgqgle wmg winpnsggtnyagkfg g rvtmtrdts i s t a ymeIsrlrsddtavyyca rdmnilatvpfdiwgqgtmvtvs s g g qgsggggsggggsdiqmtqspsslsasvgdrvtitcrasqsistylnw yqqkpgkapnlliyaafs_lc[s_gvpsrf sgsgsgtdf tltinslqpedf atууcgggdsvpltfgggtk1eik 184 CD123 123-2 human malpvtalllplalllhaarpqvqlvqsgaevkkpgasvkvsckasgy tftqyymhwvrqapgqglewmgwinpnsggtnyagkfggrv111 rdt s i s t vymeIsrlrsddtavyyca rdmnilatvpfdiwqqqtmvtvss g g ggsggggsggggsdigmtgspsslsasvgdrvtitcrasgsissylnw yggkpgkapklliyaassigsgypsrfsgsgsgtdftltvnslqpedf atууcgggdsvpltfgggtr1eik 185 CD123 123-3 human malpvtalllplalllhaarpqvqlvqsgaevkkpgasvkvsckasgy iftgyyihwvrqapgqgle wmgwinpnsggtnyagkfggrvtmtrdts istaymelsglrsddpavvycardmnilatvpfdiwgggtlvtvssgg ggsggggsggggsdigltgspsslsasvgdrvtitcrasgsissylnw yggkpgkapklliyaassigsgypsrfsgsgsgtdftltvnslgpedf atyycgggdsvpltfgggtkveik 186 CD123 123-4 human malpvtalllplalllhaarpgvglggsgaevkksgasvkvsckasgy tftdyymhwlrqapgqglewmgwinpnsgdtnyagkfggrvtltrdts i s tvymels rlrsddtavvycardmnilatvpfdiwgqgtmvtvss a s qgggsggrasggggsdigmtgspsslsasvgdrvtitcrasgsissyl nwyggkpgkapklliyaassigsgypsrfsgsgsgtdftltisslqpe dfatyycgggdsvpltfgggtkveik 187

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CD123 hzCAR-1 MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGY TFTSYWMNWVROAPGQGLEWMGRIDPYDSETHYNQKFKDRVTMTVDК S TSTAYMELSSLRSEDTAVYYCARGNWDDYWGOGTTVTVSSGGGGSGGG GSGGGGSGGGGSDVQLTQSPSFLSASVGDRVTITCRASKSISKDLAWY OQKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFA TYYCQQHNKYPYTFGGGTKVEIK 188 CD123 hzCAR-2 MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGY TFTSYWMNWVROAPGQGLEWMGRIDPYDSETHYNQKFKDRVTMTVDК S TSTAYMELSSLRSEDTAVYYCARGNWDDYWGOGTTVTVSSGGGGSGGG GSGGGGSGGGGSEWLTQSPATLSLSPGERATLSCRASKSISKDLAWY QQKPGQAPRLLIYSGSTLQSGIPARFSGSGSGTDFTLTISSLEPEDFA VYYCQQHNKYPYTFGGGTKVEIK 189 CD123 hzCAR-3 MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGY TFTSYWMNWVROAPGOGLEWMGRIDPYDSETHYNQKFKDRVTMTVDК S TSTAYMELSSLRSEDTAVYYCARGNWDDYWGOGTTVTVSSGGGGSGGG GSGGGGSGGGGSDWMTQSPAFLSVTPGEKVTITCRASKSISKDLAWY OOKPDOAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTFTISSLEAEDAA TYYCQQHNKYPYTFGGGTKVEIK 190 CD123 hzCAR-4 MALPVTALLLPLALLLHAARPQVQLVQSGAEVKKPGASVKVSCKASGY TFTSYWMNWVROAPGOGLEWMGRIDPYDSETHYNQKFKDRVTMTVDК S TSTAYMELSSLRSEDTAVYYCARGNWDDYWGOGTTVTVSSGGGGSGGG GSGGGGSGGGGSDWMTQSPDSLAVSLGERATINCRASKSISKDLAWY OOKPGOPPKLLIYSGSTLQSGVPDRFSGSGSGTDFTLTISSLOAEDVA VYYCQQHNKYPYTFGGGTKVEIK 191 CD123 hzCAR-5 MALPVTALLLPLALLLHAARPDVQLTQSPSFLSASVGDRVTITCRASK SISKDLAWYOOKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTEFTLTI SSLQPEDFATYYCQQHNKYPYTFGGGTKVEIKGGGGSGGGGSGGGGSG GGGS QVQLVQ S GAEVKK P GASVKVS C KAS GYT FT SYWMNWVROAPGQ G L EWMGRIDPYDSETHYNQKFKDRVTMTVDКS T S TAYMELSSLRSEDTA VYYCARGNWDDYWGOGTTVTVSS 192 CD123 hzCAR-6 MAL PVT AL L L P LAL L L HAAR P EWL T Q S PAT L S L S P G E RAT L S C RASK SISKDLAWYOOKPGQAPRLLIYSGSTLQSGIPARFS GSGSGTDFTLTI SSLEPEDFAVYYCQQHNKYPYTFGGGTKVEIKGGGGSGGGGSGGGGSG GGGS QVQLVQ S GAEVKK P GASVKVS C KAS GYT FT SYWMNWVROAPGQ G L EWMGRIDPYDSETHYNQKFKDRVTMTVDКS T S TAYMELSSLRSEDTA VYYCARGNWDDYWGOGTTVTVSS 193 CD123 hzCAR-7 MALPVTALLLPLALLLHAARPDWMTQSPAFLSVTPGEKVTITCRASK SISKDLAWYOOKPDQAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTFTI SSLEAEDAATYYCQQHNKYPYTFGGGTKVEIKGGGGSGGGGSGGGGSG GGGS QVQLVQ S GAEVKK P GASVKVS C KAS GYT FT SYWMNWVROAPGQ G L EWMGRIDPYDSETHYNQKFKDRVTMTVDКS T S TAYMELSSLRSEDTA VYYCARGNWDDYWGOGTTVTVSS 194

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CD123 hzCAR-8 MAL P VT AL L L P L AL L L H AAR P D WMT Q S P D S L AVS L G E RAT IN C RASK SISKDLAWYQQКPGQPPKLLIYSGSTLQSGVPDRFSGSGSGTDFTLTI SSLOAEDVAVYYCOQHNKYPYTFGGGTKVEIKGGGGSGGGGSGGGGSG GGGS QVQLVQ S GAEVKK P GASVKVS C KAS GYT FT SYWMNWVROAPGQ G L EWMGRIDPYDSETHYNQKFKDRVTMTVDКS T S TAYMELSSLRSEDTA VYYCARGNWDDYWGOGTTVTVSS 195 CD123 hzCAR-9 MALPVTALLLPLALLLHAARPQVQLVQSGSELKKPGASVKVSCKASGY TFTSYWMNWVROAPGQGLEWMGRIDPYDSETHYNQKFKDRFVFSVDК S VSTAYLOISSLKAEDTAVYYCARGNWDDYWGOGTTVTVSSGGGGSGGG GSGGGGSGGGGSDVQLTQSPSFLSASVGDRVTITCRASKSISKDLAWY QQKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFA TYYCQQHNKYPYTFGGGTKVEIK 196 CD123 hzCAR-10 MALPVTALLLPLALLLHAARPQVQLVQSGSELKKPGASVKVSCKASGY TFTSYWMNWVROAPGOGLEWMGRIDPYDSETHYNQKFKDRFVFSVDК S VSTAYLOISSLKAEDTAVYYCARGNWDDYWGOGTTVTVSSGGGGSGGG GSGGGGSGGGGSEWLTQSPATLSLSPGERATLSCRASKSISKDLAWY OOKPGOAPRLLIYSGSTLQSGIPARFSGSGSGTDFTLTISSLEPEDFA VYYCQQHNKYPYTFGGGTKVEIK 197 CD123 hzCAR-11 MALPVTALLLPLALLLHAARPQVQLVQSGSELKKPGASVKVSCKASGY TFTSYWMNWVROAPGOGLEWMGRIDPYDSETHYNQKFKDRFVFSVDК S VSTAYLOISSLKAEDTAVYYCARGNWDDYWGOGTTVTVSSGGGGSGGG GSGGGGSGGGGSDWMTQSPAFLSVTPGEKVTITCRASKSISKDLAWY OOKPDOAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTFTISSLEAEDAA TYYCQQHNKYPYTFGGGTKVEIK 198 CD123 hzCAR-12 MALPVTALLLPLALLLHAARPQVQLVQSGSELKKPGASVKVSCKASGY TFTSYWMNWVROAPGOGLEWMGRIDPYDSETHYNQKFKDRFVFSVDК S VSTAYLOISSLKAEDTAVYYCARGNWDDYWGQGTTVTVSSGGGGSGGG GSGGGGSGGGGSDWMTOSPDSLAVSLGERATINCRASKSISKDLAWY OOKPGOPPKLLIYSGSTLQSGVPDRFSGSGSGTDFTLTISSLOAEDVA VYYCQQHNKYPYTFGGGTKVEIK 199 CD123 hzCAR-13 MALPVTALLLPLALLLHAARPDVOLTOSPSFLSASVGDRVTITCRASK SISKDLAWYOOKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTEFTLTI SSLQPEDFATYYCQQHNKYPYTFGGGTKVEIKGGGGSGGGGSGGGGSG GGGSOVOLVOSGSELKKPGASVKVSCKASGYTFTSYWMNWVROAPGOG LEWMGRIDPYDSETHYNQKFKDRFVFSVDKSVSTAYLOISSLKAEDTA VYYCARGNWDDDYWGOGTTVTVSS 200 CD123 hzCAR-14 MAL PVT AL L L P LAL L L HAAR P EWL T Q S PAT L S L S P G E RAT L S C RASK SISKDLAWYQQKPGQAPRLLIYSGSTLQSGIPARFS GSGSGTDFTLTI SSLEPEDFAVYYCQQHNKYPYTFGGGTKVEIKGGGGSGGGGSGGGGSG GGGSOVOLVOSGSELKKPGASVKVSCKASGYTFTSYWMNWVROAPGOG LEWMGRIDPYDSETHYNQKFKDRFVFSVDKSVSTAYLOISSLKAEDTA VYYCARGNWDDYWGOGTTVTVSS 201

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CD123 hzCAR-15 MALPVTALLLPLALLLHAARPDWMTQSPAFLSVTPGEKVTITCRASK SISKDLAWYOOKP DQAP KL LIYSGSTLQSGVPSRFSGSGSGTDFTFTI SSLEAEDAATYYCQQHNKYPYTFGGGTKVEIKGGGGSGGGGSGGGGSG GGGSOVOLVOSGSELKKPGASVKVSCKASGYTFTSYWMNWVROAPGOG LEWMGRIDPYDSETHYNQKFKDRFVFSVDKSVSTAYLQIS SLKAEDTA VYYCARGNWDDYWGOGTTVTVSS 202 CD123 hzCAR-16 MAL PVT AL L L P LAL L L HAAR P DWMT Q S P D S LAVS L G E RAT IN CRASH SISKDLAWYQQКPGQPPKLLIYSGSTLQSGVPDRFSGSGSGTDFTLTI SSLOAEDVAVYYCOQHNKYPYTFGGGTKVEIKGGGGSGGGGSGGGGSG GGGSQVQLVQSGSELKKPGASVKVSCKASGYTFTSYWMNWVRQAPGQG LEWMGRIDPYDSETHYNQKFKDRFVFSVDKSVSTAYLQIS SLKAEDTA VYYCARGNWDDYWGOGTTVTVSS 203 CD123 hzCAR-17 MALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGESLRISCKGSGY TFTSYWMNWVROMPGKGLEWMGRIDPYDSETHYNQKFKDHVTISVDKS ISTAYLOWSSLKASDTAMYYCARGNWDDYWGOGTTVTVSSGGGGSGGG GSGGGGSGGGGSDVQLTQSPSELSASVGDRVTITCRASKSISKDLAWY OOKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTEFTLTISSLOPEDFA TYYCQQHNKYPYTFGGGTKVEIK 204 CD123 hzCAR-18 MALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGESLRISCKGSGY TFTSYWMNWVROMPGKGLEWMGRIDPYDSETHYNQKFKDHVTISVDKS ISTAYLOWSSLKASDTAMYYCARGNWDDYWGOGTTVTVSSGGGGSGGG GSGGGGSGGGGSEWLTQSPATLSLSPGERATLSCRASKSISKDLAWY OOKPGOAPRLLIYSGSTLQSGIPARFSGSGSGTDFTLTISSLEPEDFA VYYCQQHNKYPYTFGGGTKVEIК 205 CD123 hzCAR-19 MALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGESLRISCKGSGY TFTSYWMNWVROMPGKGLEWMGRIDPYDSETHYNQKFKDHVTISVDKS ISTAYLQWSSLKASDTAMYYCARGNWDDYWGQGTTVTVSSGGGGSGGG GS GGGGS GGGGS DWMTQS PAFLSVT PGEKVTI TCRASKSISKDLAWY OOKPDOAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTFTISSLEAEDAA TYYCQQHNKYPYTFGGGTKVEIK 206 CD123 hzCAR-20 MALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGESLRISCKGSGY TFTSYWMNWVROMPGKGLEWMGRIDPYDSETHYNQKFKDHVTISVDKS ISTAYLQWSSLKASDTAMYYCARGNWDDYWGQGTTVTVSSGGGGSGGG GSGGGGSGGGGSDWMTOSPDSLAVSLGERATINCRASKSISKDLAWY OOKPGOPPKLLIYSGSTLQSGVPDRFSGSGSGTDFTLTISSLOAEDVA VYYCQQHNKYPYTFGGGTKVEIK 207 CD123 hzCAR-21 MALPVTALLLPLALLLHAARPDVOLTOSPSFLSASVGDRVTITCRASK SISKDLAWYQQKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTEFTLTI SSLQPEDFATYYCQQHNKYPYTFGGGTKVEIKGGGGSGGGGSGGGGSG GGGSEVOLVOSGAEVKKPGESLRISCKGSGYTFTSYWMNWVROMPGKG LEWMGRIDPYDSETHYNQKFKDHVTISVDKSISTAYLQWS S LKAS DTA MYYCARGNWDDYWGOGTTVTVSS 208

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CD123 hzCAR-22 MALPVTALLLPLALLLHAARPEWLTQSPATLSLSPGERATLSCRASK SISKDLAWYOOKPGQAPRLLIYSGSTLQSGIPARES GSGSGTDFTLTI SSLEPEDFAVYYCQQHNKYPYTFGGGTKVEIKGGGGSGGGGSGGGGSG GGGSEVOLVOSGAEVKKPGESLRISCKGSGYTFTSYWMNWVROMPGKG LEWMGRIDPYDSETHYNQKFKDHVTISVDKSISTAYLQWS S LKASDTA MYYCARGNWDDYWGQGTTVTVSS 209 CD123 hzCAR-23 MALPVTALLLPLALLLHAARPDWMTQSPAFLSVTPGEKVTITCRASK SISKDLAWYOOKPDQAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTFTI SSLEAEDAATYYCQQHNKYPYTFGGGTKVEIKGGGGSGGGGSGGGGSG GGGSEVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMNWVRQMPGKG LEWMGRIDPYDSETHYNQKFKDHVTISVDKSISTAYLQWS S LKAS DTA MYYCARGNWDDYWGQGTTVTVSS 210 CD123 hzCAR-24 MAL P VT AL L L P L AL L L H AAR P D WMT Q S P D S L AVS L G E RAT IN C RASK SISKDLAWYOOKPGOPPKLLIYSGSTLQSGVPDRFSGSGSGTDFTLTI SSLOAEDVAVYYCQQHNKYPYTFGGGTKVEIKGGGGSGGGGSGGGGSG GGGSEVQLVQSGAEVKKPGESLRISCKGSGYTFTSYWMNWVRQMPGKG LEWMGRIDPYDSETHYNQKFKDHVTISVDKSISTAYLQWS S LKAS DTA MYYCARGNWDDYWGOGTTVTVSS 211 CD123 hzCAR-25 MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGY TFTSYWMNWVROAPGKGLVWVSRIDPYDSETHYNQKFKDRFTISVDKA KSTAYLOMNSLRAEDTAVYYCARGNWDDYWGOGTTVTVSSGGGGSGGG GSGGGGSGGGGSDVQLTQSPSFLSASVGDRVTITCRASKSISKDLAWY OOKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTEFTLTISSLOPEDFA TYYCQQHNKYPYTFGGGTKVEIK 212 CD123 hzCAR-26 MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGY TFTSYWMNWVROAPGKGLVWVSRIDPYDSETHYNQKFKDRFTISVDKA KSTAYLQMNSLRAEDTAVYYCARGNWDDYWGQGTTVTVSSGGGGSGGG GSGGGGSGGGGSEWLTOSPATLSLSPGERATLSCRASKSISKDLAWY OOKPGOAPRLLIYSGSTLQSGIPARFSGSGSGTDFTLTISSLEPEDFA VYYCQQHNKYPYTFGGGTKVEIK 213 CD123 hzCAR-27 MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGY TFTSYWMNWVROAPGKGLVWVSRIDPYDSETHYNQKFKDRFTISVDKA KSTAYLQMNSLRAEDTAVYYCARGNWDDYWGQGTTVTVSSGGGGSGGG GSGGGGSGGGGSDWMTOSPAFLSVTPGEKVTITCRASKSISKDLAWY OOKPDOAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTFTISSLEAEDAA TYYCQQHNKYPYTFGGGTKVEIK 214 CD123 hzCAR-28 MALPVTALLLPLALLLHAARPEVQLVESGGGLVQPGGSLRLSCAASGY TFTSYWMNWVRQAPGKGLVWVSRIDPYDSETHYNQKFKDRFTISVDKA KSTAYLQMNSLRAEDTAVYYCARGNWDDYWGQGTTVTVSSGGGGSGGG GSGGGGSGGGGSDWMTOSPDSLAVSLGERATINCRASKSISKDLAWY OOKPGOPPKLLIYSGSTLQSGVPDRFSGSGSGTDFTLTISSLOAEDVA VYYCQQHNKYPYTFGGGTKVEIK 215

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CD123 hzCAR-29 MALPVTALLLPLALLLHAARPDVQLTQSPSFLSASVGDRVTITCRASK SISKDLAWYOOKPGKAPKLLIYSGSTLQSGVPSRFSGSGSGTEFTLTI SSLOPEDFATYYCQQHNKYPYTFGGGTKVEIKGGGGSGGGGSGGGGSG GGGSEVOLVESGGGLVOPGGSLRLSCAASGYTFTSYWMNWVROAPGKG LVWVSRIDPYDSETHYNQKFKDRFTISVDKAKSTAYLQMNSLRAEDTA VYYCARGNWDDYWGOGTTVTVSS 216 CD123 hzCAR-30 MALPVTALLLPLALLLHAARPEWLTQSPATLSLSPGERATLSCRASK SISKDLAWYOOKPGQAPRLLIYSGSTLQSGIPARFS GSGSGTDFTLTI SSLEPEDFAVYYCQQHNKYPYTFGGGTKVEIKGGGGSGGGGSGGGGSG GGGSEVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMNWVRQAPGKG LVWVSRIDPYDSETHYNQKFKDRFTISVDKAKSTAYLQMNSLRAEDTA VYYCARGNWDDYWGOGTTVTVSS 217 CD123 hzCAR-31 MALPVTALLLPLALLLHAARPDWMTQSPAFLSVTPGEKVTITCRASK SISKDLAWYOOKPDQAPKLLIYSGSTLQSGVPSRFSGSGSGTDFTFTI SSLEAEDAATYYCQQHNKYPYTFGGGTKVEIKGGGGSGGGGSGGGGSG GGGSEVOLVESGGGLVOPGGSLRLSCAASGYTFTSYWMNWVRQAPGKG LVWVSRIDPYDSETHYNOKFKDRFTISVDKAKSTAYLOMNSLRAEDTA VYYCARGNWDDYWGOGTTVTVSS 218 CD123 hzCAR32 MAL P VT AL L L P L AL L L H AAR P D WMT Q S P D S L AVS L G E RAT IN C RASK SISKDLAWYOOKPGOPPKLLIYSGSTLQSGVPDRFSGSGSGTDFTLTI SSLOAEDVAVYYCOQHNKYPYTFGGGTKVEIKGGGGSGGGGSGGGGSG GGGSEVQLVESGGGLVOPGGSLRLSCAASGYTFTSYWMNWVRQAPGKG LVWVSRIDPYDSETHYNOKFKDRFTISVDKAKSTAYLOMNSLRAEDTA VYYCARGNWDDYWGOGTTVTVSS 219 EGFR III huscFvl Eiqlvqsgaevkkpgatvkisckgsgfniedyyihwvqqapgkglewm gridpendetkygpifqgrvtitadtstntvymelsslrsedtavyyc afrggvywgqgttvtvssggggsggggsggggsggggsdvvmtqspds lavslgeratinckssqslldsdgktylnwlqqkpgqppkrlislvs k Idsgvpdrfsgsgsgtdftltisslqaedvavyycwqgthfpgtfggg tkveik 220 EGFR III huscFv2 Dvvmtqspdslavslgeratinckssqslldsdgktylnwlqqkpgqp pkrlisivskidsgvpdrfsgsgsgtdftltisslqaedvavyycwqg thfpgtfgggtkveikggggsggggsggggsggggseiqlvqsgaevk kpgatvkisckgsgfniedyyihwvqqapgkglewmgridpendetky gpifqgrvtitadtstntvymelsslrsedtavyycafrggvywgqgt tvtvss 221 EGFR III huscFv3 Eiqlvqsgaevkkpgeslrisckgsgfniedyyihwvrqmpgkglewm gridpendetkygpifqghvtisadtsintvylqwssIkasdtamyyc afrggvywgqgttvtvssggggsggggsggggsggggsdvvmtqspls Ipvtlgqpasisckssqslldsdgktylnwlqqrpgqsprrlislvs k Idsgvpdrfsgsgsgtdftikisrveaedvgvyycwqgthfpgtfggg tkveik 222

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EGFR III huscFv4 Dvvmtqsplslpvtlgqpasisckssqslldsdgktylnwlqqrpgqs prrlislvskldsgvpdrfsgsgsgtdftikisrveaedvgvyycwqg thfpgtfgggtkveikggggsggggsggggsggggseiqlvqsgaevk kpgeslrisckgsgfniedyyihwvrqmpgkglewmgridpendetky gpifqghvtisadtsintvylqwsslkasdtamyycafrggvywgqgt tvtvss 223 EGFR III huscFv5 Eiqlvqsgaevkkpgatvkisckgsgfniedyyihwvqqapgkglewm gridpendetkygpifqgrvtitadtstntvymelsslrsedtavyyc afrggvywgqgttvtvssggggsggggsggggsggggsdvvmtqspls Ipvtlgqpasisckssqslldsdgktylnwlqqrpgqsprrlislvs к Idsgvpdrfsgsgsgtdftikisrveaedvgvyycwqgthfpgtfggg tkveik 224 EGFR III huscFv6 Eiqlvqsgaevkkpgeslrisckgsgfniedyyihwvrqmpgkglewm gridpendetkygpifqghvtisadtsintvylqwssIkasdtamyyc afrggvywgqgttvtvssggggsggggsggggsggggsdvvmtqspds lavslgeratinckssqslldsdgktylnwlqqkpgqppkrlislvs к Idsgvpdrfsgsgsgtdftltisslqaedvavyycwqgthfpgtfggg tkveik 225 EGFR III huscFv7 Dvvmtqspdslavslgeratinckssqslldsdgktylnwlqqkpgqp pkrlisivskidsgvpdrfsgsgsgtdftltisslqaedvavyycwqg thfpgtfgggtkveikggggsggggsggggsggggseiqlvqsgaevk kpgeslrisckgsgfniedyyihwvrqmpgkglewmgridpendetky gpifqghvtisadtsintvylqwssIkasdtamyycafrggvywgqgt tvtvss 226 EGFR III huscFv8 Dvvmtqsplslpvtlgqpasisckssqslldsdgktylnwlqqrpgqs prrlislvskldsgvpdrfsgsgsgtdftikisrveaedvgvyycwqg thfpgtfgggtkveikggggsggggsggggsggggseiqlvqsgaevk kpgatvkisckgsgfniedyyihwvqqapgkglewmgridpendetky gpifqgrvtitadtstntvymelsslrsedtavyycafrggvywgqgt tvtvss 227 EGFR III Mu310C eiqlqqsgaelvkpgasvklsctgsgfniedyyihwvkqrteqglewi gridpendetkygpifqgratitadtssntvylqlssltsedtavyyc afrggvywgpgttltvssggggsggggsggggshmdvvmtqspltlsv aigqsasisckssqslldsdgktylnwllqrpgqspkrlislvsklds gvpdrftgsgsgtdftlrisrveaedlgiyycwqgthfpgtfgggtkl eik 228 mesothelin s s1 (mu) QVQLQQSGPELEKPGASVKI SCKA SGYSFTGYTMNWVKQSHGKSLEWI GLITPYNGASSYNQKFRGKATLTV DKSSSTAYMDLLSLTSEDSAVYFC ARGGYDGRGFDYWGQGTTVTVSSG GGGSGGGGSGGGGSDIELTQSPAI 229

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MSASPGEKVTMTCSASSSVSYMHW YQQKSGTSPKRWIYDTSKLASGVP GRFSGSGSGNSYSLTISSVEAEDD ATYYCQQWSGYPLTFGAGTKLEI mesothelium M5 (human) QVQLVQ S GAEVEКR GAS VKVSСKASGYTFTDYYMHWVRQAPGQ GL EWM GWINPNSGGTNYAQKFQGRVTMTRDT SIS TAYMEL S RLRS DDTAVYYC ASGWDFDYWGOGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIVMTOSPS SLSASVGDRVTITCR ASQSIRYYLSWYOOKPGKAPKLLIYTASILQNGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCLQTYTTPDFGPGTKVEIK 234 mesothelium MP (human) QVQLQQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWM GWINPNSGGTNYAQNFQGRVTMTRDTSISTAYMELRRLRSDDTAVYYC ASGWDFDYWGOGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIRMTOSPS S L SASVGDRVTIТ CRASQSIRYYLSWYQQKPGKAPKLLIYTASILQNG VPSRFSGSGSGTDFTLTISSLOPEDFATYYCLQTYTTPDFGPGTKVEI К 240 mesothelium ml (human) QVQLQQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWM GRINPNSGGTNYAQKFQGRVTMTRDT SIS TAYMEL S RLRS EDTAVYYC ARGRYYGMDVWGOGTMVTVSSGGGGSGGGGSGGGGSGGGGSEIVLTOS PAT L S L S P GE RATIS CRASQSVSSNFAWYQQRP GQAPRLLIYDASNRA TGIPPRFSGSGSGTDFTLTISSLEPEDFAAYYCHQRSNWLYTFGOGTK VDIK 230 mesothelium M2 (human) QVQ LVQ S GAEVKK P GASVKVS C KAS GYT FT GYYMHWVROAPGQGLEWM GWINPNSGGTNYAQKFQGRVTMTRDT SIS TAYMEL S RLRS DDTAVYYC ARDLRRTWTPRAYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSGGG GSDIOLTOSPSTLSASVGDRVTITCOASQDISNSLNWYOOKAGKAPKL LIYDASTLETGVPSRFSGSGSGTDFSFTISSLQPEDIATYYCQQHDNL PLTFGOGTKVEIK 231 mesothelium M3 (human) QVQ LVQ S GAEVKK P GAPVKVS C KAS GYT FT GYYMHWVROAPGQGLEWM GWINPNSGGTNYAQKFQGRVTMTRDT SIS TAYMEL S RLRS DDTAVYYC ARGEWDGSYYYDYWGOGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIVL TOTPSSLSASVGDRVTITCRASQSINTYLNWYOHKPGKAPKLLIYAAS SLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSFSPLTFGGG TKLEIK 232 mesothelium M4 (human) OVOLVESGGGLVOPGGSLRLSCAASGFTFSSYWMHWVROVPGKGLVWV SRINTDGSTTTYADSVEGRFTIS RDNAKNTLYLQMNS LRDDDTAVYYC VGGHWAVWGOGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIOMTOSPST L SASVGDRVTIT CRASQSISDRLAWYQQKPGKAPKLLIYKASSLESGV PSRFSGSGSGTEFTLTISSLQPDDFAVYYCQQYGHLPMYTFGQGTKVE IK 233 mesothelium MB (human) QVQ LVQ S GAEVKK P GASVKVS C KAS GYT FT SYYMHWVROAPGQGLEWM GIINPSGGSTSYAQKFQGRVTMTRDT S T S TVYMEL S S LRS EDTAVYYC 235

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ARYRLIAVAGDYYYYGMDVWGOGTMVTVSSGGGGSGGGGSGGGGSGGG GSDIOMTOSPSSVSASVGDRVTITCRASQGVGRWLAWYOOKPGTAPKL LIYAASTLQSGVPSRFSGSGSGTDFTLTINNLQPEDFATYYCQQANSF PLTFGGGTRLEIK mesothelium M7 (human) QVQLVQ S GGGWQ P GRS LRL S CAASGFTFSSYAMHWVRQAP GKGLEWV AVISYDGSNKYYADSVKGRFTIS RDN S KNT LYLQMN S LRAEDTAVYYC ARWKVSSSSPAFDYWGOGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIV LTQSPATLSLSPGERAILSCRASQSVYTKYLGWYQQКPGQAPRLLIYD ASTRATGIPDRFSGSGSGTDFTLTINRLEPEDFAVYYCQHYGGSPLIT FGQGTRLEIK 236 mesothelium M8 (human) QVQLQQSGAEVKKPGASVKVSCKTSGYPFTGYSLHWVRQAPGQGLEWM GWINPNSGGTNYAQKFQGRVTMTRDT SIS TAYMEL S RLRS DDTAVYYC ARDHYGGNSLFYWGOGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIOLT QS PS SISASVGDTVSITCRASQDSGTWLAWYOOKPGKAPNLLMYDAST LEDGVPSRFSGSASGTEFTLTVNRLOPEDSATYYCQQYNSYPLTFGGG TKVDIK 237 mesothelium M9 (human) QVQ LVQ S GAEVKK P GASVEVS C KAS GYT FT SYYMHWVRQAPGQ GL EWM GIINPSGGSTGYAQKFQGRVTMTRDT S T S TVHMEL S S LRS EDTAVYYC ARGGYSSSSDAFDIWGOGTMVTVSSGGGGSGGGGSGGGGSGGGGSDIO MTQSPPSLSASVGDRVTITCRASQDISSALAWYQQКPGTPPKLLIYDA SSLESGVPSRFSGSGSGTDFTLTISSLOPEDFATYYCQQFSSYPLTFG GGTRLEIK 238 mesothelium M10 (human) QVQ LVQ S GAEVKK P GASVKVS C KASGYTFTSYGISWVROAPGQGLEWM GWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYC ARVAGGIYYYYGMDVWGQGTTITVSSGGGGSGGGGSGGGGSGGGGSDI VMT QT P D S LAVS L GE RATIS CKSSHSVLYNRNNKNYLAWYOOKP GQ P P KLLFYWASTRKSGVPDRFSGSGSGTDFTLTISSLQPEDFATYFCQQTQ TFPLTFGOGTRLEIN 239 mesothelium M12 (human) QVQ LVQ S GAEVKK P GASVKVS C KAS GYT FT GYYMHWVRQAPGQGLEWM GRINPNSGGTNYAQKFQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYC ARTTTSYAFDIWGOGTMVTVSSGGGGSGGGGSGGGGSGGGGSDIOLTO SPSTLSASVGDRVTITCRASQSISTWLAWYOOKPGKAPNLLIYKASTL ESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNTYSPYTFGQG TKLEIK 241 mesothelium M13 (human) OVOLVOSGGGLVKPGGSLRLSCEASGFIFSDYYMGWIROAPGKGLEWV SYIGRSGSSMYYADSVKGRFTFSRDNAKNSLYLOMNSLRAEDTAVYYC AASPWAATEDFQHWGOGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIV MTQTPATLSLSPGERATLSCRASQSVTSNYLAWYQQKPGQAPRLLLFG ASTRATGIPDRFSGSGSGTDFTLTINRLEPEDFAMYYCQQYGSAPVTF GQGTKLEIK 242 mesothelium M14 (human) QVQ LVQ S GAEVRAP GASVKIS C KASGFTFRGYYIHWVROAPGQGLEWM GIINPSGGSRAYAQKFQGRVTMTRDT S T S TVYMEL S S LRS DDTAMYYC 243

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ARTASCGGDCYYLDYWGOGTLVTVSSGGGGSGGGGSGGGGSGGGGSDI mesothelin M15 (human) QVQLVQSGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWV SGISWNSGSIGYADSVKGRFTIS RDNAKN S LYLQMN S LRAEDTAVYYC AKDGSSSWSWGYFDYWGOGTLVTVSSGGGGSGGGGSGGGGSSSELTOD PAVSVAL GQTVRT T CQGDALRSYYASWYOOKP GQAPMLVIYGKNNRPS GIPDRFSGSDSGDTASLTITGAOAEDEADYYCNSRDSSGYPVFGTGTK VTVL 244 mesothelin M16 (human) EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWV SGISWNSGSTGYADSVKGRFTIS RDNAKN S LYLQMN S LRAEDTALYYC AKDS S SWYGGGSAFDIWGOGTMVTVS S GGGGS GGGGS GGGGS S S ELTO EPAVSVALGOTVRITCQGDSLRSYYASWYOOKPGOAPVLVIFGRSRRP SGIPDRFSGSSSGNTASLIITGAOAEDEADYYCNSRDNTANHYVFGTG TKLTVL 245 mesothelin M17 (human) EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWV SGISWNSGSTGYADSVKGRFTIS RDNAKN S LYLQMN S LRAEDTALYYC AKDS S SWYGGGSAFDIWGOGTMVTVS S GGGGS GGGGS GGGGS S S ELTO DPAVSVALGOTVRITCQGDSLRSYYASWYOOKPGOAPVLVIYGKNNRP SGIPDRFSGSSSGNTASLTITGAOAEDEADYYCNSRGSSGNHYVFGTG TKVTVL 246 mesothelin M18 (human) QVQLVQ S GGGLVQ P GGS LRL S CAASGFTFSSYWMHWVROAPGKGLVWV SRINSDGSSTSYADSVKGRFTIS RDNAKNT LYLQMN S LRAEDTAVYYC VRTGWVGSYYYYMDVWGKGTTVTVSSGGGGSGGGGSGGGGSGGGGSEI VLTOSPGTLSLSPGERATLSCRASQSVSSNYLAWYOOKPGOPPRLLIY DVSTRATGIPARFSGGGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPW TFGQGTKVEIK 247 mesothelin M19 (human) QVQLVQ S GGGWQ P GRS LRL S CAASGFTFSSYGMHWVROAPGKGLEWV AVISYDGSNKYYADSVKGRFTIS RDN S KNT LYLQMN S LRAEDTAVYYC AKGYSRYYYYGMDVWGOGTTVTVSSGGGGSGGGGSGGGGSGGGGSEIV MTOSPATLSLSPGERAILSCRASQSVYTKYLGWYOOKPGOAPRLLIYD ASTRATGIPDRFSGSGSGTDFTLTINRLEPEDFAVYYCQHYGGSPLIT FGQGTKVDIK 248 mesothelin M2 0 (human) OVOLVOSGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWV SAISGSGGSTYYADSVKGRFTIS RDN S KNT LYLQMN S LRAEDTAVYYC AKREAAAGHDWYFDLWGRGTLVTVS SGGGGSGGGGSGGGGSGGGGSDI RVTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSIPLTF GQGTKVEIK 249 mesothelin M21 (human) OVOLVOSWAEVKKPGASVKVSCKASGYTFTSYYMHWVROAPGOGLEWM GIINPSGGSTSYAQKFQGRVTMTRDT S T S TVYMEL SNLRS EDTAVYYC 250

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ARSPRVTTGYFDYWGOGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIOL TOSPSTLSASVGDRVTITCRASQSISSWLAWYOOKPGKAPKLLIYKAS SLESGVPSRFSGSGSGTEFTLTISSLOPDDFATYYCQQYSSYPLTFGG GTRLEIK mesothelium M2 2 (person) QVQLVQS GAEVRRP GASVKISCRASGDTSTRHYIHWLRQAP GQGP EWM GVINPTTGPATGSPAYAQMLQGRVTMTRDTSTRTVYMELRSLRFEDTA VYYCARSWGRSAPYYFDYWGOGTLVTVSSGGGGSGGGGSGGGGSGGG GSDIOMTOSPSSLSASVGDRVTITCRASQGISDYSAWYOOKPGKAPKL LIYAASTLQSGVPSRFSGSGSGTDFTLTISYLOSEDFATYYCQQYYSY PLTFGGGTKVDIK 251 mesothelium M2 3 (person) QVQLQQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQGLEWM GIINPSGGYTTYAQKFQGRLTMTRDT S T S TVYMEL S S LRS EDTAVYYC ARIRSCGGDCYYFDNWGOGTLVTVSSGGGGSGGGGSGGGGSGGGGSDI QLTQSPSTLSASVGDRVTITCRASENVNTWLAWYQQKPGKAPKLLIYK SSSLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYY CQQYQSYPLTF GGGTKVDIK 252 mesothelium M2 4 (person) QITLKESGPALVKPTQTLTLTCTFSGFSLSTAGVHVGWIRQPPGKALE WLALISWADDKRYRPSLRSRLDIT RVT S KDQWLSMTNMQP EDTATYY CALQGFDGYEANWGPGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIVMT Q S P S S L SASAGD RVTIT CRASRGISSALAWYOOKPGKPPKLLIYDASS LESGVPSRFSGSGSGTDFTLTIDSLEPEDFATYYCQQSYSTPWTFGOG TKVDIK 253 CLL-1 139115 (human) EVOLOOSGAEVKKPGSSVKVSCKASGGTFSSYAISWVROAPGOGLEWM GGIIPIFGTANYAQKFQGRVTITADESTS TAYMELSSLRSEDTAVYYC ARDLEMATIMGGYWGQGTLVTVSSGGGGSGGGGSGGGGSQSALTQPAS VSGSPGQSITIS CTGTSSDVGGYNYVSWYOOHPGKAPKLMIYDVSNRP SGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLDWFGG GTKLTVL 254 CLL-1 139116 (human) EVQLVE S GGGWQ P GGS LRL S CAASGFTFDDYAMHWVROAPGKGLEWV SLISGDGGSTYYADSVKGRFTIS RDN S KNT LYLQMN S LRVEDTAVYYC ARVFDSYYMDVWGKGTTVTVSSGGGGSGGGGSGSGGSEIVLTOSPLSL PVTPGQPASIS CRSSQSLVYTDGNTYLNWFQQRPGQSPRRLIYKVSNR DSGVPDRFSGSGSDTDFTLKISRVEAEDVGIYYCMQGTHWSFTFGQGT RLEIK 255 CLL-1 139118 (human) OVOLOESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIROPPGKGLE WIGSIYYSGSTYYNPSLKSRVSISVDT S KNQFS LKLKYVTAADTAVYY CATPGTYYDFLSGYYPFYWGOGTLVTVSSGGGGSGGGGSGGGGSDIVM TQSPSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAAS TLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQLNSYPYTFGQ GTKLEIK 256 CLL-1 139122 (human) OVOLVESGGGLVOPGGSLRLSCAASGFTFSSYWMSWVROAPGKGLEWV ANINEDGSAKFYVDSVKGRFTIS RDNAKNS LYLQMNS LRAEDTAVYFC 257

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ARDLRSGRYWGOGTLVTVSSGGGGSGGGGSGGGGSEIVLTOSPGTLSL S P GGRAT L S CRASQSISGSFLAWYQQKPGQAPRLLIYGASSRATGIР D RFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSSPPTFGLGTKLEIK CLL-l 139117 (human) EVQLQQSGPGLVRPSETLSLTСТVSGGPVRSGSHYWNWIRQРРGRGLE WIGYIYYSGSTNYNPSLENRVTISIDT SNNHFS LKLS SVTAADTALYF CARGTATFDWNFPFDSWGQGTLVTVSSGGGGSGGGGSGSGGSDIQMTQ SPSSLSASIGDRVTIT CRASQSISSYLNWYQQКP GKAP KL LIYAASSL QSGVPSRFSGSGSGTDFTLTISSLOPEDFATYYCQQSYSTPWTFGOGT KLEIK 258 CLL-1 139119 (human) OVOLOESGAGLLKPSETLSLTCAVYGGSFSGYYWSWIROPPGKGLEWV GEINHSGSTNYNPSLKSRVTISVDT S KNQ FS LKL S SVTAADTAVYYCA RGSGLWYAIRVGSGWFDYWGOGT LVT VS SGGGGSGGGDSGGGGSDIQ MTOSPSSLSASVGDRVTITCRASQSISSYLNWYOOKPGKAPKLLMYAA SSLQSGVPSRFSGSGSGTDFTLTISSLOPEDFATYYCQQSYSTPPWTF GQGTKVDIK 259 CLL-l 139120 (human) EVOLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVROAPGKGLEWV SSISSSSSYIYYADSVKGRFTIS RDNAKN S LYLQMN S LRAEDTAVYYC ARDPSSSGSYYMEDSYYYGMDVWGOGTTVTVSSGGGGSGGGGSGGGGS NFMLTOPHSVSESPGKTVTISCTGSSGSIASNYVQWYOORPGSAPTTV IYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDS SNQWFGGGTKLTVL 260 CLL-1 139121 (human) QVNLRESGGGLVQPGGSLRLSCAASGFTFSSYEMNWVRQAPGKGLEWV SYISSSGSTIYYADSVKGRFTIS RDNAKN S LYLQMN S LRAEDTAVYYC AREALGSSWEWGOGTTVTVSSGGGGSGGGGSGGGGSDIOMTOSPSSLS ASVGDRVTIT CQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPS RFSGSGSGTDFTFTISSLOPEDIATYYCQQYDNLPLTFGGGTKLEIK 261 CLL-l 146259 (human) QVQLVQ S GAEVKE P GASVKVSCKAPANTFSDHVMHWVROAPGQRFEWM GYIHAANGGTHYSQKFQDRVTITRDT SANTVYMDLS S LRS EDTAVYYC ARGGYNSDAFDIWGOGTMVTVSSGGGGSGGGGSGGGGSGGGGSDIVMT Q S P S SVSASVGDRVTIT CRASQDISSWLAWYOOKPGKAPKLLIYAASS LQSGVPSRFNGSGSGTDFTLTISSLOPEDFATYYCQQSYSTPLTFGGG TKVEIK 262 CLL-1 146261 (human) QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWV SYISSSSSTIYYADSVKGRFTIS RDNAKN S LYLQMN S LRAEDTAVYYC ARDLSVRAIDAFDIWGOGTMVTVSSGGGGSGGGGSGGGGSGGGGSDIV LTOSPSSLSASVGDRVTITCQASQDISNYLNWYOOKPGKAPKLLIYDA SNLETGVPSRFSGSGSGTDFTFTISSLOPEDFATYYCQQAYSTPFTFG PGTKVEIK 263 CLL-1 146262 (human) EVQLVQ S GGGWRS GRS LRL S CAASGFTFNSYGLHWVRQAPGKGLEWV ALIEYDGSNKYYGDSVKGRFTIS RDKS KSTLYLQMDNLRAEDTAVYYC AREGNEDLAFDIWGOGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVLT Q S P S S L SASVGDRVTIT CQASQFIKKNLNAPWLOOHKPGK 264

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LQTGVPSRFSGNRSGTTFSFTISSLQPEDVATYYCQQHDNLPLTFGGG TKVEIK CLL-l 146263 (human) OVOLVESGGGLVOPGGSLRLSCAASGFNVSSNYMTWVROAPGKGLEWV SVIYSGGATYYGDSVKGRFTVSRDNSKNTVYLOMNRLTAEDTAVYYCA RDRLYCGNNCYLYYYYGMDVWGQGTLVTVSS GGGGS GGGGS GGGGS GG GGSDIQVTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPK LLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLOPEDFATYYCQQSYS TPPLTFGOGTKVEIK 265 CLL-l 146264 (human) 0VO LVO S GAEVKK S GAS VKVS C KAS GY P FT GYYIQWVROAP GO GL EWM GWIDPNSGNTGYAQKFQGRVTMTRNT SIS TAYMEL S S LRS EDTAVYYC ASDSYGYYYGMDVWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQM TOSPSSLSASVGDRVTFTCRASQGISSALAWYOOKPGKPPKLLIYDAS SLESGVPSRFSGSGSGTDFTLTISSLOPEDFATYYCQQFNNYPLTFGG GTKVEIK 266 CLL-l 181268 (human) EVOLVESGGGLVOPGGSLRLSCAASGFTFSSYEMNWVROAPGKGLEWV SYISSSGSTIYYADSVKGRFTIS RDNAKN S LYLQMN S LRAEDTAVYYC ARDPYSSSWHDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSEIVLTQS P GT L S L S P GE RAT L S CRASQSVSSSYLAWYOOKPGQAPRLLIYGASSR ATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGT KVDIK 267 BCMA 139103 (human) OVOLVESGGGLVOPGRSLRLSCAASGFTFSNYAMSWVROAPGKGLGWV SGISRSGENTYYADSVKGRFTIS RDN S KNT LYLQMN S LRDEDTAVYYC ARSPAHYYGGMDVWGOGTTVTVSSASGGGGSGGRASGGGGSDIVLTOS P GT L S L S P GE RAT L S CRASQSISSSFLAWYOOKPGQAPRLLIYGASRR ATGIPDRFSGSGSGTDFTLTISRLEPEDSAVYYCQQYHSSPSWTFGQG TKLEIK 268 BCMA 139105 (human) OVOLVESGGGLVOPGRSLRLSCAASGFTFDDYAMHWVROAPGKGLEWV SGISWNSGSIGYADSVKGRFTIS RDNAKN S LYLQMN S LRAEDTALYYC SVHSFLAYWGOGTLVTVSSASGGGGSGGRASGGGGSDIVMTOTPLSLP VTPGEPASIS CRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRA SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPYTFGOGTK VEIK 269 BCMA 139111 (human) EVQLLESGGGLVQPGGSLRLSCAVSGFALSNHGMSWVRRAPGKGLEWV SGIVYSGSTYYAASVKGRFTISRDNSRNTLYLQMNSLRPEDTAIYYCS AHGGESDVWGOGTTVTVSSASGGGGSGGRASGGGGSDIVMTOTPLSLS VT P GQ PASIS CKSSQSLLRNDGKTPLYWYLOKAGOP PQLLIYEVSNRF SGVPDRFSGSGSGTDFTLKISRVEAEDVGAYYCMONIQFPSFGGGTKL EIK 270 BCMA 139100 (human) QVQ LVQ S GAEVRKT GASVKVS C KASGYIFDNFGINWVRQAPGQGLEWM GWINPKNNNTNYAQKFQGRVTITADESTNTAYMEVS S LRS EDTAVYYC ARGPYYYQSYMDVWGOGTMVTVSSASGGGGSGGRASGGGGSDIVMTOT PLSLPVTPGEPASIS CRSSQSLLIHSNGYNYLNWYLQKPGQSPQLQYL 271

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GSKRASGVPDRFSGSGSGTDFTLHITRVGAEDVGVYYCMQALQTPYTF GQGTKLEIK VSMA 139101 (human) OVOLOESGGGLVOPGGSLRLSCAASGFTFSSDAMTWVROAPGKGLEWV SVISGSGGTTYYADSVKGRFTIS RDN S KNT LYLQMN S LRAEDTAVYYC AKLDSSGYYYARGPRYWGQGT LVTVS SAS GGGGS GGRAS GGGGSDIQL TQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYGAS TLASGVPARFSGSGSGTHFTLTINSLOSEDSATYYCQQSYKRASFGOG TKVEIK 272 VSMA 139102 (human) Q VQ LVQ S GAEVKK P GAS VKVS C KASGYTFSNYGITWVROAPGQGLEWM GWISAYNGNTNYAQKFQGRVTMTRNT SIS TAYMELSSLRSEDTAVYYC ARGPYYYYMDVWGKGTMVTVSSAS GGGGS GGRAS GGGGS EIVMTQ S P L SLPVTPGEPASIS CRSSQSLLYSNGYNYVDWYLOKPGQSPQLLIYLGS NRASGVPDRFSGSGSGTDFKLOISRVEAEDVGIYYCMQGRQFPYSFGO GTKVEIK 273 VSMA 139104 (human) EVOLLETGGGLVOPGGSLRLSCAVSGFALSNHGMSWVRRAPGKGLEWV SGIVYSGSTYYAASVKGRFTIS RDN S RNT LYLQMN S LRP EDTAIYYC S AHGGESDVWGQGTTVTVSSASGGGGSGGRASGGGGSEIVLTQSPATLS VSPGESATLS CRASQSVSSNLAWYOOKPGQAPRLLIYGASTRASGIP D RFSGSGSGTDFTLTISSLOAEDVAVYYCQQYGSSLTFGGGTKVEIK 274 VSMA 139106 (human) EVQLVETGGGLVQPGGSLRLSCAVSGFALSNHGMSWVRRAPGKGLEWV SGIVYSGSTYYAASVKGRFTIS RDN S RNT LYLQMN S LRP EDTAIYYC S AHGGESDVWGQGTTVTVSSASGGGGSGGRASGGGGSEIVMTQSPATLS VS P GE RAT L S CRASQSVSSKLAWYOOKP GQAP RLLMYGASIRATGIP D RFSGSGSGTEFTLTISSLEPEDFAVYYCQQYGSSSWTFGOGTKVEIK 275 VSMA 139107 (human) EVQLVET GGGWQ P GGS LRL S CAVSGFALSNHGMSWVRRAPGKGLEWV SGIVYSGSTYYAASVKGRFTIS RDN S RNT LYLQMN S LRP EDTAIYYC S AHGGESDVWGOGTTVTVSSASGGGGSGGRASGGGGSEIVLTOSPGTLS L S P GE RAT L S CRASQSVGSTNLAWYQQKPGQAPRLLIYDASNRATGIP DRFSGGGSGTDFTLTISRLEPEDFAVYYCQQYGSSPPWTFGOGTKVEI К 276 VSMA 139108 (human) OVOLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIROAPGKGLEWV SYISSSGSTIYYADSVKGRFTIS RDNAKNS LYLQMNS LRAEDTAVYYC ARESGDGMDVWGQGTTVTVSSASGGGGSGGRASGGGGSDIQMTQSPSS L SASVGDRVTIT CRASQSISSYLNWYQQKPGKAP KLLIYAASSLQSGV PSRFSGSGSGTDFTLTISSLOPEDFATYYCQQSYTLAFGOGTKVDIK 277 VSMA 139109 (human) EVOLVESGGGLVOPGGSLRLSCAVSGFALSNHGMSWVRRAPGKGLEWV SGIVYSGSTYYAASVKGRFTIS RDN S RNT LYLQMN S LRP EDTAIYYC S AHGGESDVWGOGTTVTVSSASGGGGSGGRASGGGGSDIOLTOSPSSLS ASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLOPEDFATYYCQQSYSTPYTFGOGTKVEIK 278 VSMA 139110 (human) OVOLVOSGGGLVKPGGSLRLSCAASGFTFSDYYMSWIROAPGKGLEWV SYISSSGNTIYYADSVKGRFTIS RDNAKNS LYLQMNS LRAEDTAVYYC 279

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ARSTMVREDYWGOGTLVTVSSASGGGGSGGRASGGGGSDIVLTOSPLS LPVTLGQPASIS CKSSESLVHNSGKTYLNWFHQRPGQSPRRLIYEVSN RDSGVPDRETGSGSGTDFTLCISRVEAEDVGVYYCMQGTHWPGTFGQG TKLEIK VSMA 139112 (human) QVQLVESGGGLVQPGGSLRLSCAVSGFALSNHGMSWVRRAPGKGLEWV SGIVYSGSTYYAASVKGRFTISRDNSRNTLYLQMNSLRPEDTAIYYCS AHGGESDVWGOGTTVTVSSASGGGGSGGRASGGGGSDIRLTOSPSPLS ASVGDRVTIT CQASEDINKFLNWYHOTP GKAP KLLIYDASTLQTGVPS RFSGSGSGTDFTLTINSLOPEDIGTYYCQQYESLPLTFGGGTKVEIK 280 VSMA 139113 (human) EVOLVETGGGLVOPGGSLRLSCAVSGFALSNHGMSWVRRAPGKGLEWV SGIVYSGSTYYAASVKGRFTISRDNSRNTLYLQMNSLRPEDTAIYYCS AHGGESDVWGOGTTVTVSSASGGGGSGGRASGGGGSETTLTOSPATLS VS P GE RAT L S CRASQSVGSNLAWYOOKPGQGPNDRLLIYGASTRATATGIPA RFSGSGSGTEFTFSAVLTVYPA 281 VSMA 139114 (human) EVOLVESGGGLVOPGGSLRLSCAVSGFALSNHGMSWVRRAPGKGLEWV SGIVYSGSTYYAASVKGRFTISRDNSRNTLYLOMNSLRPEDTAIYYCS AHGGESDVWGQGTTVTVSSASGGGGSGGRASGGGGSEIVLTQSPGTLS LSPGERATLSCRASQSIGSSSLAWYOOKPGOCAPRLLMYGASSRASGIP DRAGSSTGTFDTFTLTYK 282 VSMA 149362 (human) Qvq1qesgpg1vkpset1s1tctvsqqsisssvvvwqwirqppgkg1e wi gsiyysgsavvnpslksrvti s vdt sknqfslrlssvt a adt avyу carhwqewpdafdiwgqgtmvtvssggggsggggsggggsettltqsp a fms atpgdkviis ckasqdiddamnwyqqkp ge ap1fi i qsatspvp gipprfsgsgfgtdfsltinniesedaayyfclqhdnfpltfgqgtkl eik 283 VSMA 149363 (human) vn1resgpa1vkptqt1t1tctfsqfslrtsqmcvswirqppgka1ew 1aridwdedkfystslktrItiskdtsdnqvvlrmtnmdp adtatyyc arsqaqqtsatafdiwgpgtmvtvssggggsggggsggggsdiqmtqs p s s1s a s vgdrvt i t crasqdiynnlawfqlkp g s ap r sImyaanksq sqvpsrfsqsasqtdftltisslqpedfatyvcqhvvrfpys fqqqtk leik 284 VSMA 149364 (human) evqlvesqqqlvkpqqslrlscaasqftfssysmnwvrqapqkqlewv s sisssssyivyadsvkgrftisrdnaknslylqmnslraedtavyyc aktiaavyafdiwgqgttvtvssggggsggggsggggseivltqspls Ipvtpeepasis crssqsllhsnqynvldwylqkpgqspqlli ylqsn rasqvpdrfsgsgsgtdftikisrveaedvqvvvcmqalqtpytfggg tkleik 285 VSMA 149365 (human) evqlvesggglvkpggslrlscaasgftfsdyymswirqapgkglewv s yisssqstivvadsvkqrftisrdnaknslylqmnslraedtavyyc ardlrqafdiwqqqtmvtvssqqqqsqqqqsqqqqssyvltqspsvsa apgytatiscqqnniqtksvhgwyqs gkprdqs 286

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fsgsnsgnmatltisgvqagdeadfycqvwdsdsehvvfgggtkltvl VSMA 149366 (human) qvqlvqsqaevkkpqasvkvsckpsqytvtshvihwvrrapqqqlewm qminpsqqvtaysqtlqqrvtmts dt s s s tvyme1s s1r s edt amyуc areqsqsqwyfdfwqrqtlvtvssqqqqsqqqqsqqqqssyvltqpps vsvspgqtasit csgdglskkyvswyqqkagqspvvlis rdkerpsgi pdrfsgsnsadtatltisgtqamdeadvycqawddttvvfgggtkltv 1 287 VSMA 149367 (human) qvqlqesqpqlvkpsqtlsltctvsqqsissqqyvwswirqhpqkqle wi qyivvsqstvvnpslksrvti s vdt sknqfslklssvt a adt avyу caraqiaarlrqafdiwqqqtmvtvssqqqqsqqqqsqqqqsdivmtq sps s vs as vgdrvii t crasqqirnwlawyqqkpqkapnlliyaasnl qsqvpsrfsqsqsqadftltisslqpedvatyvcqkvnsapftfqpqt kvdik 288 VSMA 149368 (human) qvqlvqsqaevkkpqssvkvsckasqqtfssvaiswvrqapqqqlewm qqiipifqtanyaqkfqqrvtifadeststaymelss1rs edtavyyc arrqqyqllrwdvqllrsafdiwqqqtmvtvss ggggs ggggs ggggs syvltqppsvsvapqqtaritcqqnniqsksvhwyqqkpqqapvlvlу qknnrpsqvpdrfsgsrsgttas1titgaqaedeadууcssrdssqdh Irvfqtqtkvtvl 289 VSMA 149369 (human) evqlqqsqpqlvkpsqtlsltcaisqdsvssnsaawnwirqspsrqle w1grtvyrskwysfyaislksriiinpdtsknqfslqlksvtpedtav yvcarsspeqlflvwfdpwqqqtlvtvssqqdqsqqqqsqqqqsssel tqdpavsvalqqtiritcqqdslqnvvatwvqqkpqqapvlviyqtnn rpsqipdrfsasssqntasltitqaqaedeadyvcnsrdssqhhllfq tgtkvtvl 290 VSMA EBB-S1978- A4 (human) EVOLVESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWV SAISGSGGSTYYADSVKGRFTIS RDN S KNT LYLQMN S LRAEDTAVYYC AKVEGSGSLDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVMTQSPGTL S L S P GE RAT L S CRASQSVSSAYLAWYQQКPGQPPRLLISGASTRATGI PDRFGGSGSGTDFTLTISRLEPEDFAVYYCQHYGSSFNGSSLFTFGOG TRLEIK 291 VSMA EBB-S1978- G1 (human) EVQLVETGGGLVQPGGSLRLSCAASGITFSRYPMSWVRQAPGKGLEWV SGISDSGVSTYYADSAKGRFTIS RDN S KNT L FLQMS S LRDEDTAVYYC VTRAGSEASDIWGQGTMVTVSSGGGGSGGGGSGGGGSEIVLTQSPATL S L S P GE RAT L S CRASQSVSNSLAWYQQКPGQAPRLLIYDASSRATGIP DRFSGSGSGTDFTLTISRLEPEDFAIYYCQQFGTSSGLTFGGGTKLEI К 292 VSMA EBB-S1979- C1 (human) OVOLVESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWV SAISGSGGSTYYADSVKGRFTIS RDNAKN S LYLQMN S LRAEDTAIYYC ARATYKRELRYYYGMDVWGOGTMVTVSSGGGGSGGGGSGGGGSEIVMT QSPGTVSLS P GE RAT L S CRASQSVSSSFLAWYOOKPGQAPRLLIYGAS SRATGIPDRFSGSGSGTDFTLTISRLEPEDSAVYYCQQYHSSPSWTFG 293

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QGTRLEIK VSMA EBB-1978- C7 (human) EVOLVETGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWV SAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNTLKAEDTAVYYC ARATYKRELRYYYGMDVWGOGTTVTVSSGGGGSGGGGSGGGGSEIVLT QSРSТLSLSРGESATLSСRASQSVSTTFLAWYQQКРGQAPRLLIYGSS NRATGIР DRFS GS GS GT D FT LTIRRLE P EDFAVYYCQQYHSSPSWTFG QGTKVEIK 294 VSMA EBB-1978- D10 EVOLVETGGGLVOPGRSLRLSCAASGFTFDDYAMHWVROAPGKGLEWV SGISWNSGSIGYADSVKGRFTIS RDNAKNS LYLQMNS LRDEDTAVYYC ARVGKAVPDVWGOGTTVTVSSGGGGSGGGGSGGGGSDIVMTOTPSSLS ASVGDRVTIT CRASQSISSYLNWYQQKPGKAP KLLIYAASSLQSGVP S RFSGSGSGTDFTLTISSLOPEDFATYYCQQSYSTPYSFGOGTRLEIK 295 VSMA EBB-1979- C12 (human) EVOLVESGGGLVOPGRSLRLSCTASGFTFDDYAMHWVRORPGKGLEWV ASINWKGNSLAYGDSVKGRFAIS RDNAKNTVFLQMN S LRT EDTAVYYC ASHQGVAYYNYAMDVWGRGTLVTVSSGGGGSGGGGSGGGGSEIVLTQS P GT L S L S P GE RAT L S CRATQSIGSSFLAWYQQ RP GQAPRLLIYGASQR ATGIPDRFSGRGSGTDFTLTISRVEPEDSAVYYCQHYESSPSWTFGQG TKVEIK 296 VSMA EBB-1980- G4 (human) EVOLVESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWV SAISGSGGSTYYADSVKGRFTIS RDN S KNT LYLQMN S LRAEDTAVYYC AKWRDGMDVWGOGTTVTVSSGGGGSGGGGSGGGGSEIVLTOSPATLS L S P GE RAT L S CRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIP DRFSGNGSGTDFTLTISRLEPEDFAVYYCQQYGSPPRFTFGPGTKVDI К 297 VSMA EBB-1980- D2 (human) EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWV SAISGSGGSTYYADSVKGRFTIS RDN S KNT LYLQMN S LRAEDTAVYYC AKIPQTGTFDYWGOGTLVTVSSGGGGSGGGGSGGGGSEIVLTOSPGTL S L S P GE RAT L S CRASQSVSSSYLAWYQQRPGQAPRLLIYGASSRATGI PDRFSGSGSGTDFTLTISRLEPEDFAVYYCQHYGSSPSWTFGOGTRLE IK 298 VSMA EBB-1978- A10 (human) EVOLVETGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWV SAISGSGGSTYYADSVKGRFTMSRENDKNSVFLOMNSLRVEDTGVYYC ARANYKRELRYYYGMDVWGQGTMVTVSSGGGGSGGGGSGGGGSEIVMT QSPGTLSLSPGESATLSCRASQRVASNYLAWYQHKPGQAPSLLISGAS SRATGVPDRFSGSGSGTDFTLAISRLEPED SAVYYCQHYDSSPSWTFG QGTKVEIK 299 VSMA EBB-1978- D4 EVOLLETGGGLVOPGGSLRLSCAASGFSFSSYAMSWVROAPGKGLEWV SAISGSGGSTYYADSVKGRFTIS RDN S KNT LYLQMN S LRAEDTAVYYC AKALVGATGAFDIWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPG T L S L S P GE RAT L S CRASQSLSSNFLAWYOOKPGQAPGLLIYGASNWAT GTPDRFSGSGSGTDFTLTITRLEPEDFAVYYCQYYGTSPMYTFGOGTK VEIK 300

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VSMA EBB-1980- A2 (human) EVOLLESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWV SAISGSGGSTYYADSVKGRFTIS RDN S KNT LYLQMN S LRAEDTAVYYC VLWFGEGFDPWGQGTLVTVSSGGGGSGGGGSGGGGSDIVLTQSPLSLP VTPGEPASIS CRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRA SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPLTFGGGTК VDIK 301 VSMA EBB-1981- NW OVOLVESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWV SAISGSGGSTYYADSVKGRFTIS RDN S KNT LYLQMN S LRAEDTAVYYC AKVGYDSSGYYRDYYGMDVWGOGTTVTVSSGGGGSGGGGSGGGGSEIV LTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYG TSSRATGISDRFSGSGSGTDFTLTISRLEPEDFAVYYCQHYGNSPPKF TFGPGTKLEIK 302 VSMA EBB-1978- G4 (human) EVOLVESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWV SAISGSGGSTYYADSVKGRFTIS RDN S KNT LYLQMN S LRAEDTAVYYC AKMGWSSGYLGAFDIWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQS P GT L S L S P GE RAT L S CRASQSVASSFLAWYQQКPGQAPRLLIYGASGR ATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQHYGGSPRLTFGGG TKVDIK 303 VSMA Humanized huscFvBCMA 1 QVQLVQSGAEVKKPGSSVKVSCKA SGGTFSNYWMHWVRQAPGQGLEWM GATYRGHSDTYYNQKFKGRVTITA DKSTSTAYMELSSLRSEDTAVYYC ARGAIYNGYDVLDNWGQGTLVTVS SGGGGSGGGGSGGGGSGGGGSDIQ MTQSPSSLSASVGDRVTITCSASQ DISNYLNWYQQKPGKAPKLLIYYT SNLHSGVPSRFSGSGSGTDFTLTI SSLQPEDFATYYCQQYRKLPWTFG QGTKLEIKR 304 VSMA Humanized huscFvBCMA 2 DIQMTQSPSS LSASVGDRVT ITCSASQDIS NYLNWYQQKP GKAPKLLIYY TSNLHSGVPSRFSGSGSGTD FTLTISSLQP EDFATYYCQQ YRKLPWTFGQ GTKLEIKRGG GGSGGGGSGGGGSGGGGSQV QLVQSGAEVK KPGSSVKVSC KASGGTFSNY WMHWVRQAPG QGLEWMGATYRGHSDTYYNQ KFKGRVTITA DKSTSTAYME LSSLRSEDTA VYYCARGAIYNGYDVLDNWGQGTLVTVSS 305 CD33 141643 (human) OVOLVOSGAEVKKPGESLKISCKGSGYSFTSYWIGWVROMPGKGLEWM GIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWS S LKAS DTAMYYC ARLGGSLPDYGMDVWGOGTMVTVSSASGGGGSGGGGSGGGGSEIVLTO SPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLOKPGOSPOLLIY LGSNRASGVPDRFSGSGSGTDFTLKIS RVEAEDVGVYYCMQALQTLIT FGQGTKVDIK 306

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CD33 141644 (human) QVQ LVQ S GAEVKK Р GASVRVS С KASGYIFTNYYVHWVROAPGQ GL EWM GIISPSGGSPTYAORLQGRVTMTRDL S Т S ТVYMEL S S LT S EDTAVYFC ARESRLRGNRLGLQSSIFDHWGOGTLVTVSSASGGGGSGGGGSGGGGS DIRMTQSPPSLSASVGDRVTIPCQASQDINNHLNWYQQКPGKAPQLLI YDTSNLEIGVPSRFSGSGSGTDFTLTISSLQPEDIATYYCQQYENLPL TFGGGTKVEIK 307 CD33 141645 (human) OVOLVOSGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWV SAISGSGGSTYYADSVKGRFTIS RDN S KNT LYLQMN S LRAEDTAVYYC AKEDTIRGPNYYYYGMDVWGOGTTVTVSSASGGGGSGGGGSGGGGSET TLTQSPSSVSASVGDRVSITCRASQDIDTWLAWYQLKPGKAPKLLMYA ASNLQGGVPSRFSGSGSGTDFILTISSLQPEDFATYYCQQASIFPPTF GGGTKVDIK 308 CD33 141646 (human) OVOLVOSGAEVKKPGESLKISCKGSGYSFTSYWIGWVROMPGKGLEWM GIIYPGDSDTRYSPSFQGQVTISADKSITTAYLQWS S LRAS DSAMYYC ARGGYSDYDYYFDFWGOGTLVTVSSASGGGGSGGGGSGGGGSEIVMTO SPLSLPVTPGEPASIS CRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIY LGSNRASGVPDRFSGSGSGTDFTLKIS RVEAEDVGVYYCMQALQT P FT FGGGTKVEIK 309 CD33 141647 (human) QVQLVQ S GGDLAQ P GRS LRL S CAASGFTFDDYAMHWVROAPGKGLEWV AVIWPDGGQKYYGDSVKGRFTVSRDN P KNT LYLQMN S LRAEDTAIYYC VRHFNAWDYWGOGTLVTVSSASGGGGSGGGGSGGGGSDIOLTOSPSSL SAYVGGRVTITCQASQGISQFLNWFQQKPGKAPKLLISDASNLEPGVP SRFSGSGSGTDFTFTITNLOPEDIATYYCQQYDDLPLTFGGGTKVEIK 310 CD33 141648 (human) QVQLVQ S GGGWQ P GKS LRL S CAASGFTFSIFAMHWVROAPGKGLEWV ATISYDGSNAFYADSVEGRFTIS RDN S KD S LYLQMD S LRP EDTAVYYC VKAGDGGYDVFDSWGOGTLVTVSSASGGGGSGGGGSGGGGSEIVMTOS PLSLPVTPGEPASIS CRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYL GSNRASGVPDRFSGSGSGTDFTLKIS RVEAEDVGVYYCMQALQTPTFG PGTKVDIK 311 CD33 141649 (human) EVOLVESGGGLVOPGGSLRLSCAASGFTFSSYAMSWVROAPGKGLEWV SAISGSGGSTYYADSVKGRFTIS RDN S KNT LYLQMN S LRAEDTAVYYC AKETDYYGSGTFDYWGOGTLVTVSSASGGGGSGGGGSGGGGSDIOMTO S P S S LSASVGDRVTIS CRASQGIGIYLAWYQQRSGKPPQLLIHGASTL QSGVPSRFSGSGSGTDFTLTISSLOPEDFASYWCQQSNNFPPTFGOGT KVEIK 312 CD33 141650 (human) QVQ LVQ S GAEVKK P GASVRVS C KASGYMFTDFFIHWVROAPGQGLEWM GWINPNSGVTKYAQKFQGRVTMTRNT SIS TAYMEL S S LRS EDTAVYYC ATWYSSGWYGIANIWGQGTMVTVSSASGGGGSGGGGSGGGGSDIQLTQ S P S S L SASVGDRVTIT CQASHDISNYLHWYQQKPGKAPKLLIYDASNL ETGVPSRFTGSGSGTDFTLTIRSLOPEDVAAYYCQQSDDLPHTFGOGT KVDIK 313 CD33 141651 OVOLVOSGAEVKKPGESLKISCKGSGYSFTNYWIGWVROMPGKGLEWM 314

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(human) GIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWS S LKAS DTAMYYC ARHGPSSWGEFDYWGOGTLVTVSSASGGGGSGGGGSGGGGSDIRLTOS P S S L SASVGDRVTIT CRASQSISSYLNWYOOKPGKAP KLLIYAASSLQ SGVPSRFSGSGSGTDFTLTISSLOPEDFATYYCQQSYSTPLTFGGGK CD33 2213 (humanized) NIMLTQSPSSLAVSAGEKVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQ SPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQSEDLAIYYCHQ YLSSRTFGGGTKLEIKRGGGGSGGGGSSGGGSQVQLQQPGAEWKPGA SVKMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKF KGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYFDVWGAGT TVTVSS 315 CD33 Mu 9 6 (humanized) EIVLTQSPGSLAVSPGERVTMSCKSSQSVFFSSSQKNYLAWYQQIPGQ SPRLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQPEDLAIYYCHQ YLSSRTFGQGTKLEIKRGGGGSGGGGSSGGGSQVQLQQPGAEWKPGA SVKMSCKASGYTFTSYYIHWIKQTPGQGLEWVGVIYPGNDDISYNQKF QGKATLTADKSSTTAYMQLSSLTSEDSAVYYCAREVRLRYFDVWGQGT TVTVSS 316 Claudia6 muMAB 64A EVOLOOSGPELVKPGASMKISCKASGYSFTGYTMNWVKOSHGKNLEWI GLINPYNGGTIYNQKFKGKATLTVDКS S S ΤAYMELLSLTSED SAVYYC ARDYGFVLDYWGOGTTLTVSSGGGGSGGGGSGGGGSGGGGSOIVLTOS PSIMSVSPGEKVTITCSASSSVSYMHWFOOKPGTSPKLCIYSTSNLAS GVPARFSGRGSGTSYSLTISRVAAEDAATYYCQQRSNYPPWTFGGGTK LEIK 317 Claudia6 mAb206-LCC EVOLOOSGPELVKPGASMKISCKASGYSFTGYTMNWVKOSHGKNLEWI GLINPYNGGTIYNQKFKGKATLTVDКS S S TAYMELLSLTSED SAVYYC ARDYGFVLDYWGOGTTLTVSSGGGGSGGGGSGGGGSGGGGSOIVLTOS PAIMSASPGEKVTITCSASSSVSYLHWFQQКPGTSPKLWVYSTSNLPS GVPARFGGSGSGTSYSLTISRMEAEDAATYYCQQRSIYPPWTFGGGTK LEIK 318 Claudia6 rpAL206- SUBG EVOLOOSGPELVKPGASMKISCKASGYSFTGYTMNWVKOSHGKNLEWI GLINPYNGGTIYNQKFKGKATLTVDКS S S TAYMELLSLTSED SAVYYC ARDYGFVLDYWGOGTTLTVSSGGGGSGGGGSGGGGSGGGGSOIVLTOS P SIMSVS PGEKVTITCSASSSVSYMHWFQQKPGTSPKLGIYSTSNLAS GVPARFSGRGSGTSYSLTISRVAAEDAATYYCQQRSNYPPWTFGGGTK LEIK 319 WT1 ESK-1 QAWTQPPSA SGTPGQRVTI SCSGSSSNIG SNTVNWYQQV PGTAPKLLIY SNNQRPSGVPDRFSGSKSSGT SASLAISGLQ SEDEADYYCA AWDDSLNGWV FGGGTKLTVL GSRGGGGSGG GGSGGGGSLE MAQMQLVQSG AEVKEPGESL RISCKGSGYS FTNFWISWVR QMPGKGLEWM 320 WT1 WT1-2 QTWTQPPSA SGTPGQRVTI SCSGSSSNIG SNYVYWYQQL PGTAPKLLIYRSNQRPSGVP DRFSGSKSGT SASLAISGPR 321

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SVDEADYYCA AWDDSLNGW FGGGTKLTVL GSRGGGGSGG GGSGGGSLEM AQVQLVQSGA EVKKPGSSVK VSCKASGGTF SSYAISWVRQ APGQGLEWMG GIIPIFGTAN YAQKFQGRVT ITADESTSTA YMELSSLRSE DTAVYYCARR IPPYYGMDVW GQGTTVTVSS WT1 WT1-3 DIQMTQSPSS LSASVGDRVT ITCRASQSIS SYLNWYQQKP GKAPKLLIYA ASSLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ SYSTPLTFGG GTKVDIKRSR GGGGSGGGGS GGGGSLEMAQ VQLQQSGPGL VKPSQTLSLT CAISGDSVSS NSAAWNWIRQ SPSRGLEWLG RTYYGSKWYN DYAVSVKSRI TINPDTSKNQ FSLQLNSVTP EDTAVYYCAR GRLGDAFDIW GQGTMVTVSS 322 WT1 WT1-4 DIQMTQSPST LSASVGDRVT ITCRASQNIN KWLAWYQQRP GKAPQLLIYK ASSLESGVPS RFSGSGSGTE YTLTISSLQP DDFATYYCQQ YNSYATFGQG TKVEIKRSRG GGGSGGGGSG GGGSLEMAQV QLVQSGAEVK KPGESLKISC KGSGYNFSNK WIGWVRQLPG RGLEWIAIIY PGYSDITYSP SFQGRVTISA DTSINTAYLH WHSLKASDTA MYYCVRHTAL AGFDYWGLGT LVTVSS 323 WT1 WT1-5 QSWTQPPSV SVAPGKTARI TCGRNNIGSK SVHWYQQKPG QAPVLWYDD SDRPSGIPER FSGSNSGNTA TLTISRVEAG DEADYYCQVW DSSSDHWFG GGTKLTVLGS RGGGGSGGGG SGGSLEMAEV QLVQSGGGW RPGGSLRLSC AASGFTFDDY GMSWVRQAPG KGLEWVSGIN WNGGSTGYAD SVRGRFTISR DNAKNSLYLQ MNSLRAEDTA LYYCARERGY GYHDPHDYWG QGTLVTVSS 324 WT1 WT1-6 QSVLTQPPSV SGAPGQRVTI SCTGSSSNIG AGYDVHWYQQ LPGTAPKLLI YGNSNRPSGV PDRFSGSKSG TSASLAISGL QSEDEADYYC AAWDDSLNGY VFGTGTKLTV LGSRGGGGSG GGGSGGGGSL EMAEVQLVET GGGLLQPGGS LRLSCAASGF SVSGTYMGWV RQAPGKGLEW VALLYSGGGT YHPASLQGRF IVSRDSSKNM VYLQMNSLKA EDTAVYYCAK GGAGGGHFDS WGQGTLVTVS S 325 WT1 WT1-7 EVQLLESGGG LVQPGGSLRL SCAASGFTFS SYAMSWVRQA PGKGLEWVSQ IDPWGQETLY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAKLT GRFDYWGQGT LVTVSSGGGG SGGGGSGGGG STDIQMTQSP SSLSASVGDR VTITCRASQS ISSYLNWYQQ KPGKAPKLLI YSASQLQSGV PSRFSGSGSG TDFTLTISSL QPEDFATYYC QQGPGTPNTF GQGTKVEIKR A 326

In one embodiment, the anti-mesothelin antigen-binding domain is an antigen-binding site, for example, a CDR, the anti-mesothelin antibody binding domain described in Table 1. 4, such as ss1, M1, M2, M3, M4, M5, M6, M7, M8, M9, 10, M11, M12, M13, M14,

M15, M16, M17, M18, M19, M20, M21, M22, M23 or M24, e.g. ss1, M5 or M11. In one embodiment, the anti-mesothelin antigen-binding domain is a human antigen-binding site, such as the CDR, of the binding domain of a human anti-mesothelin antibody described in Table 1. 4, such as Ml, M2, M3, M4, M5, M6, M7, M8, M9, 10, M11, M12, M13, M14, M15, M16, M17, M18, M19, M20, M21, M22, M23, or M24, e.g. M5 or M11. In one embodiment, the antigen-binding domain that binds to mesothelin is an antigen-binding domain according to Table. 2, 3 WO 2015/090230, incorporated herein by reference.

In one embodiment, the human antigen-binding domain that binds to mesothelin binds to the same epitope as the epitope bound by the mouse SS1 antigen-binding domain (provided in Table 4). In one embodiment, the human antigen-binding domain that binds to mesothelin binds to a different epitope than the epitope bound by the mouse SS1 antigen-binding domain (provided in Table 4). As described in WO

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2015/090230, thus incorporated by reference, the binding domains of human antibodies against mesothelin M-5 and M-11, described in table. 4 bind to an epitope other than SS1.

In one embodiment, the antigen-binding domain against CLL-1 is an antigen-binding site, eg CDR, of an antibody available from R&D, Ebiosciences, Abcam, eg PE-CLL1-hu cat. No. 353604 (BioLegend) and PE-CLL1 (CLEC12A) Cat. No. 562566 (BD). In one embodiment, the antigen-binding site against CLL-1 is an antigen-binding site, for example, CDR, the binding domain of the antibody against CLL-1 described in table. 4, e.g. 139115, 139116, 139117, 139118, 139119, 139120, 139121, 139121, 139122, 146259, 146261, 146262, 146263, 146264 or 181286.

In one embodiment, the antigen-binding domain against CD123 is an antigen-binding site, for example, the CDR, of the binding domain of an anti-CD123 antibody described in Table 1. 4, for example, CD123-1, CD123-2, CD123-3, CD123-4 or hzCAR1-32. In one of the embodiments antigennegative domain against CD123 is an antigennegative domain in accordance with table. 1, 2 of WO 2014/130635, incorporated herein by reference. In one embodiment, the antigen-binding domain against CD33 is an antigen-binding site, eg, the CDR, of an antibody, as described in, eg, Bross et al., Clin. Cancer Res., 7(6):1490-1496 (2001) (gemtuzumab ozogamicin, hP67.6), Caron et al., Cancer Res., 52(24):67616767 (1992) (lintuzumab, HuM195), Lapusan et al., Invest. New Drugs, 30(3):1121-1131 (2012) (AVE9633), Aigner et al., Leukemia, 27(5):1107-1115 (2013) (AMG330, CD33 BiTE), Dutour et al., Adv. hematol., 2012:683065 (2012), and Pizzitola et al., Leukemia, doi:10.1038/Lue.2014,62 (2014). In one embodiment, the CD33 antigen-binding site is an antigen-binding site, such as the CDR, of the anti-CD33 antibody binding domain described in Table 1. 4, such as 141643, 141644, 141645, 141646, 141647, 141648, 141649, 141650, 141651, 2213 or My96.

In one embodiment, the antigen-binding domain against GD2 is an antigen-binding site, eg CDR, of an antibody as described in, eg, Mujoo et al., Cancer Res., 47(4):1098-1104 (1987); Cheung et al., Cancer Res., 45(6):2642-2649 (1985), Cheung et al., J. Clin. Oncol., 5(9):1430-1440 (1987), Cheung et al., J. Clin. Oncol., 16(9):3053-3060 (1998), Handgretinger et al., Cancer Immunol. Immunother., 35(3):199-204 (1992). In some embodiments, the antigen-binding domain against GD2 is the antigen-binding region of an antibody selected from mAbs 14.18, 14G2a, ch14.18, hu14.18, 3F8, hu3F8, 3G6, 8B6, 60C3, 10B8, ME36.1, and 8H9, see e.g. , Wo 2012033885, Wo 2013040371, Wo 2013192294, Wo 2013061273, Wo 2013123061, Wo 2013074916 and Wo 201385552. In some embodimenting domain against GD2, the anti -taging area of the antibodel described in the publication of US No. 20100150910 or publication PCT No. WO 20111616016161616161616116116116116116116116116116116116116116116116116116116116116160161616160160161601616016161161601ALSE.

In one embodiment, the anti-BCMA antigen-binding domain is an antigen-binding site, e.g., the CDR, of an antibody as described in, for example, WO 2012163805, WO 200112812 and WO 2003062401. CDR binding domain against bcma, described in table. 4, например, 139100, 139101, 139102, 139103, 139104, 139105, 139106, 139107, 139108, 139109, 139110, 139111, 139112, 139113, 139114, 149362, 149363, 149364, 149365, 149366, 149367, 149368, 149369, EB c1978-A4, EB C1978-G1, EBB C1978-C7, EBB C1978-D10, EBB C1978-A10, EBB C1978-D4, EBB C1978-G4, EBB C1979-C1, EBB C1979-C12, EBB C1980-G4, EBB C1980-D2, EBB C1980-A2, EBB C1981-C3, huscFvBCMA1 or huscFvBCMA2.

In one embodiment, the antigen-binding domain against WT-1 is an antigen-binding site, eg the CDR, of an antibody as described in, eg, Dao et al., Sci. Transl. Med., 5(176):176ra33 (2013) or in WO 2012/135854. In one embodiment, the antigen-binding domain against WT1 is an antigen-binding site, for example, the CDR, of the binding domain of an anti-WT1 antibody described in Table 1. 4, such as ESK1, WT1-2, WT1-3, WT1-4, WT1-5, WT1-6 or WT1-7.

In one embodiment, the anti-CLDN6 antigen-binding domain is an antigen-binding site, eg CDR, of the IMAB027 antibody (Ganymed Pharmaceuticals), see eg clinicaltrial.gov/show/NCT02054351. In one embodiment, the antigen-binding domain against CLDN6 is an antigen-binding site, for example, the CDR, of the binding domain of an anti-CLDN6 antibody described in Table 1. 4, for example, muMAB64A, mAb206-LCC or mAb206-SUBG.

In one embodiment, the antigen binding domain comprises one, two, three (e.g., all three) of the heavy chain CDRs, CDR1 HC, CDR2 HC, and CDR3 HC from the antibody above, and/or one, two, three (e.g., all three ) light chain CDR, CDR1 LC, CDR2 LC and CDR3 LC from the antibody above. In one embodiment, the antigen binding domain comprises the heavy chain variable region and/or the light chain variable region of the antibody as defined above.

Frameworks that do not belong to the antibody.

In embodiments, the antigen-binding domain contains a non-antibody

- 89 040145 scaffold, eg fibronectin, ankyrin, domain antibody, lipocalin, small modular protein immunopharmaceutical, maxi-antibody, protein A or affilin. The framework that does not belong to the antibody has the ability to bind to the target antigen on the cell. In embodiments, the antigen-binding domain is a polypeptide, or a natural protein fragment thereof, expressed by the cell. In some embodiments, the antigen-binding domain contains a non-antibody backbone. A wide variety of non-antibody scaffolds can be used, provided that the resulting polypeptide contains at least one binding region that specifically binds to the target antigen on the target cell.

Non-antibody scaffolds include fibronectin (Novartis, MA), ankyrin (Molecular Partners AG, Zurich, Switzerland), domain antibodies (Domantis, Ltd., Cambridge, MA and Ablynx nv, Zwijnaarde, Belgium), lipocalin (Pieris Proteolab AG, Freising , Germany), small modular protein immunopharmaceuticals (Trubion Pharmaceuticals Inc., Seattle, WA), maxiantibodies (Avidia, Inc., Mountain View, CA), protein A (Affibody AG, Sweden) and affilin (gamma-crstallin or ubiquitin) (Scil Proteins GmbH, Halle, Germany).

Fibronectin-based scaffolds can be based on a type III fibronectin domain (eg, type III fibronectin tenth module (Fn3 domain ¹⁰ )). The type III fibronectin domain contains 7 or 8 beta chains, which are located between two beta sheets, which are themselves stacked opposite each other to form the core of the protein, and additionally contain loops (similar to CDRs) that connect the beta chains to each other and provide solvent. There are at least three such loops on each edge of the beta sheet sandwich, where the edge is a protein boundary perpendicular to the direction of the beta chains (see US 6,818,418). Because of this structure, such a non-antibody scaffold mimics antigen-binding properties that are similar in nature and antibody binding affinity. Such scaffolds can be used in an in vitro randomization strategy and loop swapping, which is analogous to the in vivo antibody affinity maturation process.

Ankyrin technology is based on the use of proteins with ankyrin-derived repeating modules as a scaffold for containing variable regions that can be used to bind various targets. The ankyrin repeating module is a 33 amino acid long polypeptide consisting of two anti-parallel α-helices and a β-fold. Binding of variable regions is generally optimized using ribosome display.

Avimers are derived from a naturally occurring A-domain containing protein such as HER3. These domains are used in nature for protein-protein interactions, and in humans over 250 proteins are structurally based on A-domains. Avimers are composed of a number of different (2-10) A-domain monomers linked by amino acid linkers. It is possible to design avimers that bind to a target antigen using the methodology described, for example, in US Patent Application Publications Nos.

Affinity affinity ligands are small, simple proteins composed of a three-stranded bundle based on the backbone of one of the IgG-binding domains of protein A. Protein A is a surface protein from the bacterium Staphylococcus aureus. This framework domain consists of 58 amino acids, 13 of which are randomized to form a library of affitels with a large number of ligand variants (see, for example, US 5831012). Affitel molecules mimic antibodies, they have a molecular weight of 6 kD compared to the molecular weight of antibodies which is 150 kD. Despite its small size, the binding site of the affitel molecules is similar to the binding site of the antibody.

Epitope mimetic (PEM) proteins are medium-sized cyclic peptide-like molecules (MW 1-2 kDa) that mimic beta-hairpin secondary structures of proteins, the main secondary structure involved in protein-protein interactions.

Antigen-binding domains paired contrary to the principle of complementarity.

It has been found that cells containing a constellation of chimeric membrane-embedded receptors each containing an antigen-binding domain (CMER) for which interactions between the CMER antigen-binding domain may be undesirable, for example, because it inhibits the ability of one or more antigen-binding domains to bind to its own cognate antigen. Thus, the present description discloses a first and second non-natural CMER containing antigen-binding domains that minimize such interactions when expressed in the same cell. In one embodiment, the CMER population contains two TCARs. In one embodiment, the implementation of the set of CMER contains TCAR and another CMER. In one embodiment, the CMER population contains two NKR-CARs. In one embodiment, the implementation of the set of CMER contains NKR-CAR and another CMER. In one embodiment, the implementation of the set of CMER contains TCAR and NKR-CAR.

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In some embodiments, the described invention comprises a first and a second CMER, wherein the antigen-binding domain of one of said first CMER and said second CMER does not contain a light chain variable domain and a heavy chain variable domain. In some embodiments, the antigen binding domain of one of said first CMER and said second CMER is scFv and the other is not scFv. In some embodiments, the antigen-binding domain of one of said first CMER and said second CMER comprises a single VH domain, e.g., a single camel, shark, or lamprey VH domain, or a single VH domain derived from a human or mouse sequence, or a non-antibody framework. In some embodiments, the antigen-binding domain of one of said first CMER and said second CMER comprises a nanobody. In some embodiments, the antigen binding domain of one of said first CMER and said second CMER comprises a camelid VHH domain.

In some embodiments, the antigen binding domain of one of said first CMER and said second CMER contains scFv and the other contains a single VH domain, such as a single camel, shark, or lamprey VH domain, or a single VH domain derived from a human or mouse sequence. In some embodiments, the antigen-binding domain of one of said first CMER and said second CMER contains scFv and the other contains a nanobody. In some embodiments, the antigen binding domain of one of said first CMER and said second CMER contains scFv and the other contains a camelid VHH domain.

In some embodiments, when present on the cell surface, the binding of the antigen-binding domain of said first CMER to its cognate antigen is not substantially reduced by the presence of said second CMER. In some embodiments, the binding of the antigen-binding domain of said first CMER to its cognate antigen in the presence of said second CMER is 85%, 90, 95, 96, 97, 98, or 99% of the binding of the antigen-binding domain of said first CMER to its cognate antigen in the absence of said second CMER .

In some embodiments, when present on the cell surface, the antigen-binding domains of said first CMER and said second CMER interact with each other to a lesser extent than if both were scFv antigen-binding domains. In some embodiments, the antigen-binding domains of said first CMER and said second CMER interact with each other 85%, 90%, 95%, 96%, 97%, 98%, or 99% less than if both were scFv antigen-binding domains.

In some embodiments, the invention comprises a first and a second KIRCAR, wherein the antigen-binding domain of one of said first KIR-CAR and said second KIRCAR does not contain a light chain variable domain and a heavy chain variable domain. In some embodiments, the antigen binding domain of one of said first KIR-CAR and said second KIR-CAR is scFv and the other is not scFv. In some embodiments, the antigen-binding domain of one of said first KIR-CAR and said second KIR-CAR comprises a single VH domain, such as a single camel, shark, or lamprey VH domain, or a single VH domain derived from a human or mouse sequence, or not framework belonging to the antibody. In some embodiments, the antigen-binding domain of one of said first KIR-CAR and said second KIR-CAR comprises a nanobody. In some embodiments, the antigen binding domain of one of said first KIR-CAR and said second KIR-CAR comprises a camelid VHH domain.

In some embodiments, the antigen binding domain of one of said first KIR-CAR and said second KIR-CAR contains scFv and the other contains a single VH domain, such as a single camel, shark, or lamprey VH domain, or a single VH domain derived from a human sequence. or mice. In some embodiments, the antigen-binding domain of one of said first KIR-CAR and said second KIR-CAR contains scFv and the other contains a nanobody. In some embodiments, the antigen binding domain of one of said first KIR-CAR and said second KIR-CAR contains scFv and the other contains a camelid VHH domain.

In some embodiments, when present on the cell surface, the binding of the antigen-binding domain of said first KIR-CAR to its cognate antigen is not substantially reduced by the presence of said second KIR-CAR. In some embodiments, the binding of the antigen-binding domain of said first KIR-CAR to its cognate antigen in the presence of said second KIR-CAR is 85%, 90, 95, 96, 97, 98, or 99% of the binding of the antigen-binding domain of said first KIR-CAR to its cognate antigen. antigen in the absence of the specified second KIR-CAR.

In some embodiments, when present on the cell surface, the antigen-binding domains of said first KIR-CAR and said second KIR-CAR interact with each other to a lesser extent than if both were scFv antigen-binding domains. In some

- 91 040145 embodiments, the antigen-binding domains of said first KIR-CAR and said second KIR-CAR interact with each other 85%, 90%, 95%, 96%, 97%, 98%, or 99% less than if both were scFv antigen-binding domains.

In some embodiments, the invention comprises a first and a second TCAR, wherein the antigen-binding domain of one of said first TCAR and said second TCAR does not contain a light chain variable domain and a heavy chain variable domain. In some embodiments, the antigen-binding domain of one of said first TCAR and said second TCAR is scFv and the other is not scFv. In some embodiments, the antigen-binding domain of one of said first TCAR and said second TCAR contains a single VH domain, such as a single camel, shark, or lamprey VH domain, or a single VH domain derived from a human or mouse sequence, or a non-antibody framework. In some embodiments, the antigen-binding domain of one of said first TCAR and said second TCAR comprises a nanobody. In some embodiments, the antigen-binding domain of one of said first TCAR and said second TCAR contains a camelid VHH domain.

In some embodiments, the antigen-binding domain of one of said first TCAR and said second TCAR contains scFv and the other contains a single VH domain, such as a single camel, shark, or lamprey VH domain, or a single VH domain derived from a human or mouse sequence. In some embodiments, the antigen-binding domain of one of said first TCAR and said second TCAR contains scFv and the other contains a nanobody. In some embodiments, the antigen-binding domain of one of said first TCAR and said second TCAR contains scFv and the other contains a camelid VHH domain.

In some embodiments, when present on the cell surface, the binding of the antigen-binding domain of said first TCAR to its cognate antigen is not substantially reduced by the presence of said second TCAR. In some embodiments, the binding of the antigen-binding domain of said first TCAR to its cognate antigen in the presence of said second TCAR is 85%, 90, 95, 96, 97, 98, or 99% of the binding of the antigen-binding domain of said first TCAR to its cognate antigen in the absence of said second TCAR .

In some embodiments, when present on the cell surface, the antigen-binding domains of said first TCAR and said second TCAR interact with each other to a lesser extent than if both were scFv antigen-binding domains. In some embodiments, the antigen-binding domains of said first TCAR and said second TCAR interact with each other 85%, 90%, 95%, 96%, 97%, 98%, or 99% less than if both were scFv antigen-binding domains.

Bispecific CARs.

In one embodiment, an antigen-binding domain as described herein, eg for NKR-CAR or TCAR as described herein, comprises a multispecific antibody molecule, eg a bispecific antibody molecule. A bispecific antibody has specificity for no more than two antigens. The bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. In one embodiment, the first and second epitopes are on the same antigen, eg, the same protein (or subunit of a multimeric protein). In one embodiment, the first and second epitopes overlap. In one embodiment, the first and second epitopes do not overlap. In one embodiment, the first and second epitopes are on different antigens, such as different proteins (or different subunits of a multimeric protein). In one embodiment, the bispecific antibody molecule comprises a heavy chain variable domain sequence and a light chain variable domain sequence that have binding specificity for a first epitope, and a heavy chain variable domain sequence and a light chain variable domain sequence that have binding specificity for a second epitope. In one embodiment, the bispecific antibody molecule comprises a semi-antibody having binding specificity for a first epitope and a semi-antibody having binding specificity for a second epitope. In one embodiment, the bispecific antibody molecule comprises a semi-antibody or fragment thereof having binding specificity for a first epitope and a semi-antibody or fragment thereof having binding specificity for a second epitope. In one embodiment, the bispecific antibody molecule comprises an scFv or fragment thereof that has binding specificity for a first epitope and an scFv or fragment thereof that has binding specificity for a second epitope.

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In certain embodiments, the antibody molecule is a polyspecific (eg, bispecific or trispecific) antibody molecule. Protocols for the production of bespecifically or heterodimeric antibody molecules are known in the art, including, but not limited to, for example, the ledge-in-the-trough approach described, for example, in US 5,731,168; Fc pairing by electrostatic interaction as described, for example, in WO 09/089004, WO 06/106905 and WO 2010/129304; the formation of heterodimers with domains designed using standard chain replacement (SEED), as described, for example, in WO 07/110205; substitution of Fab fragments as described, for example, in WO 08/119353, WO 2011/131746 and WO 2013/060867; a double antibody conjugate, for example, by cross-linking antibodies to form a bispecific structure using a heterobifunctional reagent containing a reactive amino group and a reactive sulfhydryl group, as described, for example, in US 4433059; bispecific antibody determinants obtained by recombination of semi-antibodies (pairs of heavy-light chains or Fab) from different antibodies using a cycle of reduction and oxidation of disulfide bonds between two heavy chains, as described, for example, in US 4444878; trifunctional antibodies, eg three Fab' fragments, cross-linked via reactive sulfhydryl groups as described, eg, in US5273743; biosynthetic binding proteins, eg a pair of scFvs, tail-linked at the C-terminus, preferably via disulfide or amino chemical cross-linking, as described, for example, in US5534254; bifunctional antibodies, eg Fab fragments with different binding specificities, dimerized by leucine zippers (eg c-fos and c-jun), which contain a substituted constant domain, as described in eg US5582996; bispecific and oligospecific mono- and oligovalent receptors, e.g., the VH-CH1 regions of two antibodies (two Fab fragments) linked via a polypeptide spacer between the CH1 region of one antibody and the VH region of the other antibody, typically with linked light chains as described, for example , in US5591828; bispecific DNA-antibody conjugates, for example, linking antibodies or Fab fragments through a double-stranded DNA region, as described, for example, in US5635602; bispecific fusion proteins, for example, an expression construct containing two scFvs with a hydrophilic helical peptide linker between them and a complete constant region, as described, for example, in US5637481; polyvalent and binding proteins, e.g., a dimer of polypeptides containing a first domain with an Ig heavy chain variable region binding region and a second domain with an Ig light chain variable region binding region, generally referred to as diabodies (also provide for higher order structures to obtain bispecific, trispecific or tetraspecific molecules, as described, for example, in US5837242; mini-antibody constructs with linked VL and VH chains, further connected by peptide spacers to the antibody hinge region and the CH3 region, which can be dimerized to form bispecific/polyvalent molecules, as described, for example, in US5837821; VH and VL domains associated with a short peptide linker (e.g. 5 or 10 amino acids) or no linker in any orientation, which can form dimers to form bispecific diabodies; trimers and tetramers as described in, for example, US5844094; thread VH domains (or VL domains in the members of the family) linked by peptide bonds with cross-linking groups at the C-terminus, further linked to VL domains to form a series of FVs (or several scFvs), as described, for example, in US5864019, and single-stranded binding polypeptides with VH and VL domains linked through the peptide linker are combined into polyvalent structures by non-covalent or chemical linkage to form, for example, homobivalent, heterobivalent, trivalent and tetravalent structures using an scFV-type format or a diabody, as described, for example, in US5869620. Additional illustrative polyspecific and bispecific molecules and methods for their preparation can be found, for example, in US 5910573, US 5932448, US 5959083, US 5989830, US 6005079, US 6239259, US 6294353, US 6333396, US 6476198, US 6511663 6743896, US 6809185, US 6833441, US 7129330, US 7183076, US 7521056, US 7527787, US 7534866, US 7612181, US

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In each antibody or antibody fragment (eg, scFv) of a bispecific antibody molecule, the VH may be above or below the VL. In some embodiments, in an upstream antibody or antibody fragment (e.g., scFv), VH (VH1) is located upstream of VL (VL1), and in an upstream antibody or antibody fragment (e.g., scFv), VL (VL ₂ ) is upstream of VH (VH2 ), so that the complete bispecific antibody molecule is characterized by the order VH1-VL1-VL2-VH2. In other embodiments, in an upstream antibody or antibody fragment (e.g., scFv) the VL (VL1) is located upstream of the VH (VH1), and in an upstream antibody or antibody fragment (e.g., scFv) the VH (VH ₂ ) is upstream of the VL (VL ₂ ), so that the complete bispecific antibody molecule is characterized by the order VL1-VH1VH2-VL2.

Optionally, a linker is placed between two antibodies or antibody fragments (e.g. scFvs), e.g. between VL1 and VL2 if the construct is positioned as VH1-VL1-VL2-VH2, or between VH1 and VH2 if the construct is positioned as VL1-VH1-VH2- VL2. The linker may be a linker as described herein, for example a (Gly ₄ -Ser)n linker where n is 1, 2, 3, 4, 5 or 6, preferably 4 (SEQ ID NO: 63). In general, the linker between two scFvs should be of sufficient length to eliminate the possibility of mismatch between the domains of the two scFvs. Optionally, a linker is placed between the VL and VH of the first scFv. Optionally, a linker is placed between the VL and VH of the second scFv. In constructs that contain multiple linkers, any two or more linkers may be the same or different. Thus, in some embodiments, a bispecific CAR comprises VL, VH, and optionally one or more linkers, in the order as described herein.

In one aspect, the bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence, e.g., scFv, that has binding specificity for the tumor antigen of the present invention, e.g., contains an scFv as described herein, e.g., as described in Table 1. 4 or contains a light chain CDR and/or a heavy chain CDR from the scFv described herein and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope on another antigen. In some aspects, the second immunoglobulin variable domain sequence has binding specificity for an antigen expressed on a tumor, eg, a tumor antigen as described herein.

Exemplary CAR sequences.

In embodiments, the implementation of the NKR-CAR or TCAR described in the present invention may contain one or more of the sequences provided in table. 5.

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Table 5

Sequences of various CAR components (aa - amino acid sequence, na - nucleic acid sequence)

SEQ Description Subsequence ID NO CGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGT CCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGA AGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCC TTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGT GAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCC GTGTGTGGTTCCCGCGGGCCTGGCCCTCTTTACGGGTTATGGCCCTTGCG TGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGA GCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGA GCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCC GCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGA TAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTT TTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTA eleven EF-1 promoter (pa) TTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCG CACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGA CGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGC CGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACC AGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGC TCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCA CACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTC CACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTT TGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGT TTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTT GATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTC ATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTTCCATTTCAGG TGTCGTGA 1 Leader (ah) MALPVTALLLPLALLLHAARP ATGGCCCTGCCTGTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCTGC 12 Leader (pa) ATGCCGCTAGACCC 2 Hinge CD 8 (aa) TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGT 13 Hinge CD8 (pa) CGCAGCCCCTGTCCCCTGCGCCCAGAGGCGTGCCGGCCAAGCGGCGGGGGG CGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVS QEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNG 3 Hinge Ig4 (aa) KEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDK SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKM GAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCC 14 Hinge 1d4 (pa) TGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCT GATGATCAGCCGGACCCCCGAGGTGACCTTGTGTGGTGGTGGACGTGTCC

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CAGGAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGG TGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTA CCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGC AAGGAATACAAGTGTAAGGTGTCCAACAAGGGCCTGCCCAGCAGCATCG AGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTA CACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTG ACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGG AGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCT GGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAG AGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGG CCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAA GATG 4 Hinge IgD (aa) RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKE KEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFWGSD LKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGT SVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLC EVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVP APPSPQPATYTCWSHEDSRTLLNASRSLEVSYVTDH 15 Hinge IgD (pa) AGGTGGCCCGAAAGTCCCAAGGCCCAGGCATCTAGTGTTCCTACTGCAC AGCCCCAGGCAGAAGGCAGCCTAGCCAAAGCTACTACTGCACCTGCCAC TACGCGCAATACTGGCCGTGGCGGGGAGGAGAAGAAAAAGGAGAAAGAG AAAGAAGAACAG GAAGAGAG G GAGAC СAAGAC С С С Т GAAT GT С CAT С С С ATACCCAGCCGCTGGGCGTCTATCTCTTGACTCCCGCAGTACAGGACTT GTGGCTTAGAGATAAGGCCACCTTTACATGTTTCGTCGTGGGCTCTGAC CTGAAGGATGCCCATTTGACTTGGGAGGTTGCCGGAAAGGTACCCACAG GGGGGGTTGAGGAAGGGTTGCTGGAGCGCCATTCCAATGGCTCTCAGAG CCAGCACTCAAGACTCACCCTTCCGAGATCCCTGTGGAACGCCGGGACC TCTGTCACATGTACTCTAAATCATCCTAGCCTGCCCCCACAGCGTCTGA TGGCCCTTAGAGAGCCAGCCGCCCAGGCACCAGTTAAGCTTAGCCTGAA TCTGCTCGCCAGTAGTGATCCCCCAGAGGCCGCCAGCTGGCTCTTATGC GAAGTGTCCGGCTTTAGCCCGCCCAACATCTTGCTCATGTGGCTGGAGG ACCAGCGAGAAGTGAACACCAGCGGCTTCGCTCCAGCCCGGCCCCCACC CCAGCCGGGTTCTACCACATTCTGGGCCTGGAGTGTCTTAAGGGTCCCA GCACCACCTAGCCCCCAGCCAGCCACATACACCTGTGTTGTGTCCCATG AAGATAGCAGGACCCTGCTAAATGCTTCTAGGAGTCTGGAGGTTTCCTA CGTGACTGACCATT 6 Transmembrane CD8 (aa) IYIWAPLAGTCGVLLLSLVITLYC 17 Transmembrane CD8 (pa) ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGT CACTGGTTATCACCCTTTACTGC 7 Intracellular domain 4-1BB (aa) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

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18 Intracellular domain 4-1BB (pa) AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGA GACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTCC AGAAGAAGAAGAAGGAGGAT GT GAACT G 8 CD27 (aa) QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP 19 CD27 (pa) AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTC CCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACC ACGCGACTTCGCAGCCTATCGCTCC 9 POPs-zeta (aa) (Q/K mutant) RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPR 20 CDS-zeta (pa) (Q/K mutant) AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAGGGCC AGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGA TGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCG AGAAGGAAGAACCCT CAGGAAGGCCT GTACAAT GAACT GCAGAAAGATA AGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAG GGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAG GACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC 10 POP-zeta (aa) (reference subsequence NCBI NM_000734.3) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPR 21 POP-zeta (pa) (reference subsequence NCBI NM_000734.3) AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCC AGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGA TGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCG AGAAGGAAGAACCCT CAGGAAGGCCT GTACAAT GAACT GCAGAAAGATA AGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAG GGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAG GACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC 36 Intracellular domain of CD28 (amino acid sequence) RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS 37 Intracellular domain of CD28 (nucleotide sequence) AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTC CCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACC ACGCGACTTCGCAGCCTATCGCTCC 38 Intracellular ICOS domain (amino acid sequence) TKKKYSSSVHDPNGEYMFMRAVNTA KKSRLTDVTL 39 Intracellular ICOS domain ACAAAAAAGAAGTAT T CAT C CAGT GT GCAC GAC C CTAAC GGT GAATACA T GTT CAT GAGAGCAGT GAACACAGCCAAAAAAT CCAGACT CACAGAT GT

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(nucleotide subsequence) GACCCTA 5 hinge/linker GS (ah) GGGGSGGGGS 16 hinge/linker GS (pa) GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC 356 hinge/linker GS (pa) GGTGGCGGAGGTTCTGGAGGTGGGGGTTCC 25 linker GGGGS 26 linker (Gly-Gly-Gly-Gly-Ser)n, where n=1-6, for example, GGGGSGGGGS GGGGSGGGGS GGGGSGGGGS 27 linker (Gly4 Ser)4 28 linker (Gly4 Ser)3 29 linker (Gly3Ser) 40 linker (Gly-Gly-Gly-Ser)n, where n is positive integer equal to or greater than 1 41 linker (Gly-Gly-Gly-Ser)n, where n=1-10, for example, GGGSGGGSGG GSGGGSGGGS GGGSGGGSGG GSGGGSGGGS 42 linker GSTSGSGKPGSGEGSTKG thirty polyA (A) 5000 · This sequence may contain 50-5000 adenines. 31 polit (T) yuo 32 poliT (T) ₅₀ oo This sequence can contain 50-5000 thymines. 33 polyA (A) 5000 · This sequence may contain 100-5000 adenines. 34 polyA (A) 100 This sequence may contain 100-400 adenines. 35 polyA (A) 2000 This sequence may contain 50-2000 adenines. 22 PD1 CAR (aa) pqwfIdspdrpwnpptfspallvvteqdnatftcs fsntses fvlnwyr mspsnqtdklaafpedrsqpcfqdcrf rvtqlpncfrdfhmsvvrarrnds cftylccfaislapkaqikeslraelrvterraevptahpspsprpacfqf q tlvtttpaprpptpaptiasqplslrpeacrpaaqqavhtrqldfacdi yiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeed gcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeyd vldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrr gkghdglyqglstatkdtydalhmqalppr 23 PD-1 CAR (pa) (PD1 ECD underlined) atggccctccctgtcactgccctgcttctccccctcgcactcctgctcc acgccgctagacc acccqqatqqtttctqqactctccqqatcqcccqtq qaatcccccaaccttctcaccqqcactcttqqttqtqactqaqqqcqat

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aatcfccfaccttcaccftcfctccfttctccaacacctcccfaatcattccftcfc tcfaactcfcftacccfcatcfacfccccftcaaaccacfacccfacaacfctccfcccfc cftttcccfcfaacfatccfcftccfcaacccfcfcfacacfcfattcftccfcfttcccfccftcf actcaactqccqaatqqcaqaqacttccacatqaqcqtqqtccqcqcta qqcqaaacqactccqqqacctacctqtqcqqaqccatctcqctqqcqcc taacfcfcccaaatcaaacfacfacfcttcfacfcfcfcccfaactcfacfacftcfacccfacf ccfcacfacfctcfacfcftcfccaactcfcacatccatccccatccfcctccfcfcctcf cqqqqcaqtttcaqaccctqqtcacqaccactccqqcqccqcqcccacc gactccggccccaactatcgcgagccagcccctgtcgctgaggccggaa gcatgccgccctgccgccggaggtgctgtgcatacccggggattggact tcgcatgcgacatctacatttgggctcctctcgccggaacttgtggcgt gctccttctgtccctggtcatcaccctgtactgcaagcggggtcggaaa aagcttctgtacattttcaagcagcccttcatgaggcccgtgcaaacca cccaggaggaggacggttgctcctgccggttccccgaagaggaagaagg aggttgcgagctgcgcgtgaagttctcccggagcgccgacgcccccgcc tataagcagggccagaaccagctgtacaacgaactgaacctgggacggc gggaagagtacgatgtgctggacaagcggcgcggccgggaccccgaaat gggcgggaagcctagaagaaagaaccctcaggaaggcctgtataacgag ctgcagaaggacaagatggccgaggcctactccgaaattgggatgaagg gagagcggcggaggggaaaggggcacgacggcctgtaccaaggactgtc caccgccaccaaggacacatacgatgccctgcacatgcaggcccttccc cctcgc 24 PD-1 CAR (aa) with signal sequence. (PD1 ECD underlined) Malpvtalllplalllhaa rppqwfldspdrpwnpptfspallvvteqd natftcs fsntsesfvlnwyrmspsngtdklaafpedrsgpggdcrfrv tglpngrdfhmsvvrarrndsgtylcgaislapkagikeslraelrvte rraevptahpspsprpaggfgtlvtttpaprpptpaptiasqplslrpe acrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrk kllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapa ykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglyne Iqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalp pr 338 cytoplasmic PD1 domain (amino acids 192- 288) CSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVP CVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL 339 cytoplasmic CTLA-4 domain (amino acids 18 3- 223) AVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN 340 Human CD8 Hinge TTTPAPRPPT PAPTIASQPL SLRPEACRPA AGGAVHTRGL DFA

Exemplary NKR-CAR sequences and NKR-CAR components are additionally provided herein.

Given below is the nucleic acid sequence of KIR-CAR containing an antigen-binding domain that binds to mesothelin and a cytoplasmic domain containing the KIR2DS2 domain. Any of the antigen-binding domains described herein can be replaced by the mesothelin antigen-binding domain provided below.

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Sequence of the KIR2DS2 SS1 gene (SEQ ID NO: 327).

gtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagtttt cgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattat cccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggt tgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagt gctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccga aggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaacc ggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaaca acgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagact ggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttat tgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagat ggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaa atagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagttta ctcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatc ctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagacc ccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgca aacaaaaaaaccaccgctaccagcggtggt ttgtttgccggatcaagagctaccaactcttttt ccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagt taggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgetaatcctgttacc agtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccg gataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacga cctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggag aaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttcca gggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgat ttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacg gttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtg gataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgca gcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttg gccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaac gcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctc

- 100 040145 gtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgatta cgccaagcgcgcaattaaccctcactaaagggaacaaaagctggagctgcaagcttaatgtagt cttatgcaatactcttgtagtcttgcaacatggtaacgatgagttagcaacatgccttacaagg agagaaaaagcaccgtgcatgccgattggtggaagtaaggtggtacgatcgtgccttattagga aggcaacagacgggtctgacatggattggacgaaccactgaattgccgcattgcagagatattg tatttaagtgcctagctcgatacaataaacgggtctctctggttagaccagatctgagcctggg agctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttca agtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtea gtgtggaaaatctctagcagtggcgcccgaacagggacctgaaagcgaaagggaaaccagagct ctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtg agtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagta ttaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaa atataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggc ctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacag gatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggat agagataaaaga caccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagacc accgcacagcaagcggccgctgatcttcagacctggaggaggagatatgagggacaattggaga agtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaa agagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttctt gggagcagcaggaagcactatgggcgcagcctcaatgacgctgacggtacaggccagacaatta ttgtctggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgt tgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatacct aaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtg ccttggaatgctagttggagtaataaatctctggaacagattggaatcacacgacctggatgga gtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaac cagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggt ttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtagg tttaagaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccatta tcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaagaag gtggagagagagacagagacagatccattcgattagtgaacggatctcgacggtatcgattaga ctgtagcccaggaatatggcagctagattgtacacat ttagaaggaaaagttatcttggtagca gttcatgtagccagtggatatatagaagcagaagtaattccagcagagacagggcaagaaacag catacttcctcttaaaattagcaggaagatggccagtaaaaacagtacatacagacaatggcag caatttcaccagtactacagttaaggccgcctgttggtgggcggggatcaagcaggaatttggc attccctacaatccccaaagtcaaggagtaatagaatctatgaataaagaattaaagaaaatta taggacaggtaagagatcaggctgaacatcttaagacagcagtacaaatggcagtattcatcca

- 101 040145 caattttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataata gcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttcgggttt attacagggacagcagagatccagtttggctgcatacgcgtcgtgaggctccggtgcccgtcag tgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaaccg gtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttt tcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaac gggtttgccgccagaacacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgg gttatggcccttgcgtgccttgaattacttccacctggctgcagtacgtgattcttgatcccga gcttcgggttggaagtgggtgggagagttcgaggccttgcgcttaaggagccccttcgcctcgt gcttgagttgaggcctggcctgggcgctggggccgccgcgtgcgaatctggtggcaccttcgcg cctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctgctgcgacgct ttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtatttcggttttt ggggccgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgc gagcgcggccaccgagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctgg cctcgcgccgccgtgtatcgccccgccctgggcggcaaggctggcccggtcggcaccagttgcg tgagcggaaaga tggccgcttcccggccctgctgcagggagctcaaaatggaggacgcggcgct cgggagagcgggcgggtgagtcacccacacaaaggaaaagggcctttccgtcctcagccgtcgc ttcatgtgactccacggagtaccgggcgccgtccaggcacctcgattagttctcgtgcttttgg agtacgtcgtctttaggttggggggaggggttttatgcgatggagtttccccacactgagtggg tggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttga gtttggatcttggttcattctcaagcctcagacagtggttcaaagtttttttcttccatttcag gtgtcgtgagctagaATGGGGGGACTTGAACCCTGCAGCAGGCTCCTGCTCCTGCCTCTCCTGC TGGCTGTAAGTGGTCTCCGTCCTGTCCAGGCCCAGGCCCAGAGCGATTGCAGTTGCTCTACGGT GAGCCCGGGCGTGCTGGCAGGGATCGTGATGGGAGACCTGGTGCTGACAGTGCTCATTGCCCTG GCCGTGTACTTCCTGGGCCGGCTGGTCCCTCGGGGGCGAGGGGCTGCGGAGGCAGCGACCCGGA AACAGCGTATCACTGAGACCGAGTCGCCTTATCAGGAGCTCCAGGGTCAGAGGTCGGATGTCTA CAGCGACCTCAACACACAGAGGCCGTATTACAAAgTCGAGGGCGGCGGAGAGGGCAGAGGAAGT CTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTAGGatggccttaccagtgaccgcct tgctcctgccgctggccttgctgctccacgccgccaggccgggatcccaggtacaactgcagca gtctgggcctgagctggagaagcctggcgcttcagtgaagatatcctgcaaggcttctggttac tcattcactggctacaccatgaactgggtgaagcaga gccatggaaagagccttgagtggattg gacttattactccttacaatggtgcttctagctacaaccagaagttcaggggcaaggccacatt aactgtagacaagtcatccagcacagcctacatggacctcctcagtctgacatctgaagactct gcagtctatttctgtgcaagggggggttacgacgggaggggttttgactactggggccaaggga ccacggtcaccgtctcctcaggtggaggcggttcaggcggcggtggctctagcggtggtggatc ggacatcgagctcactcagtctccagcaatcatgtctgcatctccaggggagaaggtcaccatg

- 102 040145 acctgcagtgccagctcaagtgtaagttacatgcactggtaccagcagaagtcaggcacctccc ccaaaagatggatttatgacacatccaaactggcttctggagtcccaggtcgcttcagtggcag tgggtctggaaactcttactctctcacaatcagcagcgtggaggctgaagatgatgcaacttat tactgccagcagtggagtaagcaccctctcacgtacggtgctgggacaaagttggaaatcaaag ctagcACGCGTggtggcggaggttctggaggtgggggttcccagggggcctggccacatgaggg agtccacagaaaaccttccctcctggcccacccaggtcccctggtgaaatcagaagagacagtc atcctgcaatgttggtcagatgtcaggtttgagcacttccttctgcacagagaggggaagtata aggacactttgcacctcattggagagcaccatgatggggtctccaaggccaacttctccatcgg tcccatgatgcaagaccttgcagggacctacagatgctacggttctgttactcactccccctat cagttgtcagctcccagtgaccctctggacatcgtcatcacaggtctatatgagaaaccttctc tctcagcccagccgggccccacggttttggcaggagagagcgtgaccttgtcctgcagctcccg gagctcctatgacatgtaccatctatccagggagggggaggcccatgaacgtaggttctctgca gggcccaaggtcaacggaacattccaggccgactttcctctgggccctgccacccacggaggaa cctacagatgcttcggctctttccgtgactctccctatgagtggtcaaactcgagtgacccact gcttgtttctgtcacaggaaacccttcaaatagttggccttcacccactgaaccaagctccaaa accggtaacccc agacacctgcatgttctgattgggacctcagtggtcaaaatccctttcacca tcctcctcttctttctccttcatcgctggtgctccaacaaaaaaaatgctgctgtaatggacca agagcctgcagggaacagaacagtgaacagcgaggattctgatgaacaagaccatcaggaggtg tcatacgcataaGtcgacaatcaacctctggattacaaaatttgtgaaagattgactggtattc ttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctat tgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgag gagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaaccccca ctggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctat tgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggc actgacaattccgtggtgttgtcggggaagctgacgtcctttccatggctgctcgcctgtgttg ccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggacct tccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacg agtcggatctccctttgggccgcctccccgcctggaattcgagctcggtacctttaagaccaat gacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaagggcta attcactcccaacgaagacaagatctgctttttgcttgtactgggtctctctggttagaccaga tctgagcctgggagctctctggctaactagggaaccc actgcttaagcctcaataaagcttgcc ttgagtgettcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctсада cccttttagtcagtgtggaaaatctctagcagtagtagttcatgtcatcttattattcagtatt tataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttattgcagcttataatgg ttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagt tgtggtttgtccaaactcatcaatgtatcttatcatgtctggctctagctatcccgcccctaac

- 103 040145 tccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggcc gaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctacgct tttgcgtcgagacgtacccaattcgccctatagtgagtcgtattacgcgcgctcactggccgtc gttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatc cccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcg cagcctgaatggcgaatggcgcgacgcgccctgtagcggcgcattaagcgcggcgggtgtggtg gttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcc cttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagg gttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgt agtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaata gtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttata agggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcg aattttaacaaaatattaacgtttacaatttcccaggtggcacttttcggggaaatgtgcgcgg aacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccc tgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgccc ttattccctttt ttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagt aaaagatgctgaagatcagttgg

Below is the nucleic acid sequence of KIR-CAR containing an antigen-binding domain that binds to mesothelin and a cytoplasmic domain containing a KIRS2 domain. Any of the antigen-binding domains described herein can be replaced by the mesothelin antigen-binding domain provided below.

Последовательность гена KIRS2 SS1 (SEQ ID NO: 328) gtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagtttt cgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattat cccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggt tgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagt gctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccga aggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaacc ggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaaca acgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagact ggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttat tgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagat ggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaa atagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagttta

- 104 040145 ctcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatc ctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagacc ccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgca aacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactcttttt ccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagt taggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgetaatcctgttacc agtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccg gataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacga cctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggag aaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttcca gggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgat ttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacg gttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtg gataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgca gcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttg gccgattcatta atgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaac gcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctc gtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgatta cgccaagcgcgcaattaaccctcactaaagggaacaaaagctggagctgcaagcttaatgtagt cttatgcaatactcttgtagtcttgcaacatggtaacgatgagttagcaacatgccttacaagg agagaaaaagcaccgtgcatgccgattggtggaagtaaggtggtacgatcgtgccttattagga aggcaacagacgggtctgacatggattggacgaaccactgaattgccgcattgcagagatattg tatttaagtgcctagctcgatacaataaacgggtctctctggttagaccagatctgagcctggg agctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttca agtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtea gtgtggaaaatctctagcagtggcgcccgaacagggacctgaaagcgaaagggaaaccagagct ctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtg agtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagta ttaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaa atataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggc ctgttagaaacatcagaaggctgtagacaaatactgg gacagctacaaccatcccttcagacag gatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggat agagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagacc accgcacagcaagcggccgctgatcttcagacctggaggaggagatatgagggacaattggaga agtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaa agagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttctt

- 105 040145 gggagcagcaggaagcactatgggcgcagcctcaatgacgctgacggtacaggccagacaatta ttgtctggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgt tgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatacct aaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtg ccttggaatgctagttggagtaataaatctctggaacagattggaatcacacgacctggatgga gtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaac cagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggt ttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtagg tttaagaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccatta tcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaagaag gtggagagagagacagagacagatccattcgattagtgaacggatctcgacggtatcgattaga ctgtagcccaggaatatggcagctagattgtacacatttagaaggaaaagttatcttggtagca gttcatgtagccagtggatatatagaagcagaagtaattccagcagagacagggcaagaaacag catacttcctcttaaaattagcaggaagatggccagtaaaaacagtacatacagacaatggcag caatttcaccagtactacagttaaggccgcctgttggtgggcggggatcaagcaggaatttggc attccctacaat ccccaaagtcaaggagtaatagaatctatgaataaagaattaaagaaaatta taggacaggtaagagatcaggctgaacatcttaagacagcagtacaaatggcagtattcatcca caattttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataata gcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttcgggttt attacagggacagcagagatccagtttggctgcatacgcgtcgtgaggctccggtgcccgtcag tgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaaccg gtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttt tcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaac gggtttgccgccagaacacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgg gttatggcccttgcgtgccttgaattacttccacctggctgcagtacgtgattcttgatcccga gcttcgggttggaagtgggtgggagagttcgaggccttgcgcttaaggagccccttcgcctcgt gcttgagttgaggcctggcctgggcgctggggccgccgcgtgcgaatctggtggcaccttcgcg cctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctgctgcgacgct ttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtatttcggttttt ggggccgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgc gagcgcggccaccgagaatcggacgggggtagtctca agctggccggcctgctctggtgcctgg cctcgcgccgccgtgtatcgccccgccctgggcggcaaggctggcccggtcggcaccagttgcg tgagcggaaagatggccgcttcccggccctgctgcagggagctcaaaatggaggacgcggcgct cgggagagcgggcgggtgagtcacccacacaaaggaaaagggcctttccgtcctcagccgtcgc ttcatgtgactccacggagtaccgggcgccgtccaggcacctcgattagttctcgtgcttttgg agtacgtcgtctttaggttggggggaggggttttatgcgatggagtttccccacactgagtggg

- 106 040145 tggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttga gtttggatcttggttcattctcaagcctcagacagtggttcaaagtttttttcttccatttcag gtgtcgtgagctagaATGGGGGGACTTGAACCCTGCAGCAGGCTCCTGCTCCTGCCTCTCCTGC TGGCTGTAAGTGGTCTCCGTCCTGTCCAGGCCCAGGCCCAGAGCGATTGCAGTTGCTCTACGGT GAGCCCGGGCGTGCTGGCAGGGATCGTGATGGGAGACCTGGTGCTGACAGTGCTCATTGCCCTG GCCGTGTACTTCCTGGGCCGGCTGGTCCCTCGGGGGCGAGGGGCTGCGGAGGCAGCGACCCGGA AACAGCGTATCACTGAGACCGAGTCGCCTTATCAGGAGCTCCAGGGTCAGAGGTCGGATGTCTA CAGCGACCTCAACACACAGAGGCCGTATTACAAAgTCGAGGGCGGCGGAGAGGGCAGAGGAAGT CTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTAGGatggccttaccagtgaccgcct tgctcctgccgctggccttgctgctccacgccgccaggccgggatcccaggtacaactgcagca gtctgggcctgagctggagaagcctggcgcttcagtgaagatatcctgcaaggcttctggttac tcattcactggctacaccatgaactgggtgaagcagagccatggaaagagccttgagtggattg gacttattactccttacaatggtgcttctagctacaaccagaagttcaggggcaaggccacatt aactgtagacaagtcatccagcacagcctacatggacctcctcagtctgacatctgaagactct gcagtctatttctgtgcaagggggggttacgacgggaggggttttgactactggggccaaggga ccacggtcaccg tctcctcaggtggaggcggttcaggcggcggtggctctagcggtggtggatc ggacatcgagctcactcagtctccagcaatcatgtctgcatctccaggggagaaggtcaccatg acctgcagtgccagctcaagtgtaagttacatgcactggtaccagcagaagtcaggcacctccc ccaaaagatggatttatgacacatccaaactggcttctggagtcccaggtcgcttcagtggcag tgggtctggaaactcttactctctcacaatcagcagcgtggaggctgaagatgatgcaacttat tactgccagcagtggagtaagcaccctctcacgtacggtgctgggacaaagttggaaatcaaag ctagcggtggcggaggttctggaggtgggggttcctcacccactgaaccaagctccaaaaccgg taaccccagacacctgcatgttctgattgggacctcagtggtcaaaatccctttcaccatcctc ctcttctttctccttcatcgctggtgctccaacaaaaaaaatgctgctgtaatggaccaagagc ctgcagggaacagaacagtgaacagcgaggattctgatgaacaagaccatcaggaggtgtcata cgcataaGtcgacaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaac tatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgctt cccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagtt gtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggt tggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgcca cggcggaactcatcgccgcctgccttgcccgctgctg gacaggggctcggctgttgggcactga caattccgtggtgttgtcggggaagctgacgtcctttccatggctgctcgcctgtgttgccacc tggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttcctt cccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcg gatctccctttgggccgcctccccgcctggaattcgagctcggtacctttaagaccaatgactt acaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattca

- 107 040145 ctcccaacgaagacaagatctgctttttgcttgtactgggtctctctggttagaccagatctga gcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgag tgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagaccctt ttagtcagtgtggaaaatctctagcagtagtagttcatgtcatcttattattcagtatttataa cttgcaaagaaatgaatatcagagagtgagaggaacttgtttattgcagcttataatggttaca aataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtgg tttgtccaaactcatcaatgtatcttatcatgtctggctctagctatcccgcccctaactccgc ccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggc cgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctacgcttttgc gtcgagacgtacccaattcgccctatagtgagtcgtattacgcgcgctcactggccgtcgtttt acaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccct ttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcc tgaatggcgaatggcgcgacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttac gcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcc tttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttcc gatttagtgctt tacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgg gccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtgga ctcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataaggga ttttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattt taacaaaatattaacgtttacaatttcccaggtggcacttttcggggaaatgtgcgcggaaccc ctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgata aatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttatt cccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaag atgctgaagatcagttgg

Below is the nucleic acid sequence of KIR-CAR containing an antigen-binding domain that binds to mesothelin and a cytoplasmic domain containing the KIR2DL3 domain. Any of the antigen-binding domains described herein can be replaced by the mesothelin antigen-binding domain provided below.

Последовательность гена KIR2DL3 SS1 (SEQ ID NO: 329) gtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagtttt cgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattat cccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggt tgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagt gctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccga

- 108 040145 aggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaacc ggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaaca acgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagact ggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttat tgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagat ggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaa atagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagttta ctcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatc ctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagacc ccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgca aacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactcttttt ccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagt taggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgetaatcctgttacc agtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccg gataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacga cctacaccgaac tgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggag aaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttcca gggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgat ttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacg gttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtg gataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgca gcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttg gccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaac gcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctc gtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgatta cgccaagcgcgcaattaaccctcactaaagggaacaaaagctggagctgcaagcttaatgtagt cttatgcaatactcttgtagtcttgcaacatggtaacgatgagttagcaacatgccttacaagg agagaaaaagcaccgtgcatgccgattggtggaagtaaggtggtacgatcgtgccttattagga aggcaacagacgggtctgacatggattggacgaaccactgaattgccgcattgcagagatattg tatttaagtgcctagctcgatacaataaacgggtctctctggttagaccagatctgagcctggg agctctctggctaactagggaacccactgcttaagcc tcaataaagcttgccttgagtgcttca agtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtca gtgtggaaaatctctagcagtggcgcccgaacagggacctgaaagcgaaagggaaaccagagct ctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtg agtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagta ttaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaa

- 109 040145 atataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggc ctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacag gatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggat agagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagacc accgcacagcaagcggccgctgatcttcagacctggaggaggagatatgagggacaattggaga agtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaa agagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttctt gggagcagcaggaagcactatgggcgcagcctcaatgacgctgacggtacaggccagacaatta ttgtctggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgt tgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatacct aaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtg ccttggaatgctagttggagtaataaatctctggaacagattggaatcacacgacctggatgga gtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaac cagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggt ttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtagg tttaagaatagt ttttgctgtactttctatagtgaatagagttaggcagggatattcaccatta tcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaagaag gtggagagagagacagagacagatccattcgattagtgaacggatctcgacggtatcgattaga ctgtagcccaggaatatggcagctagattgtacacatttagaaggaaaagttatcttggtagca gttcatgtagccagtggatatatagaagcagaagtaattccagcagagacagggcaagaaacag catacttcctcttaaaattagcaggaagatggccagtaaaaacagtacatacagacaatggcag caatttcaccagtactacagttaaggccgcctgttggtgggcggggatcaagcaggaatttggc attccctacaatccccaaagtcaaggagtaatagaatctatgaataaagaattaaagaaaatta taggacaggtaagagatcaggctgaacatcttaagacagcagtacaaatggcagtattcatcca caattttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataata gcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttcgggttt attacagggacagcagagatccagtttggctgcatacgcgtcgtgaggctccggtgcccgtcag tgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaaccg gtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttt tcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaac gggtttgccgccagaacacaggtaagtgccgtgtgtg gttcccgcgggcctggcctctttacgg gttatggcccttgcgtgccttgaattacttccacctggctgcagtacgtgattcttgatcccga gcttcgggttggaagtgggtgggagagttcgaggccttgcgcttaaggagccccttcgcctcgt gcttgagttgaggcctggcctgggcgctggggccgccgcgtgcgaatctggtggcaccttcgcg cctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctgctgcgacgct ttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtatttcggttttt

- 110 040145 ggggccgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgc gagcgcggccaccgagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctgg cctcgcgccgccgtgtatcgccccgccctgggcggcaaggctggcccggtcggcaccagttgcg tgagcggaaagatggccgcttcccggccctgctgcagggagctcaaaatggaggacgcggcgct cgggagagcgggcgggtgagtcacccacacaaaggaaaagggcctttccgtcctcagccgtcgc ttcatgtgactccacggagtaccgggcgccgtccaggcacctcgattagttctcgtgcttttgg agtacgtcgtctttaggttggggggaggggttttatgcgatggagtttccccacactgagtggg tggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttga gtttggatcttggttcattctcaagcctcagacagtggttcaaagtttttttcttccatttcag gtgtcgtgagctagaATGGGGGGACTTGAACCCTGCAGCAGGCTCCTGCTCCTGCCTCTCCTGC TGGCTGTAAGTGGTCTCCGTCCTGTCCAGGCCCAGGCCCAGAGCGATTGCAGTTGCTCTACGGT GAGCCCGGGCGTGCTGGCAGGGATCGTGATGGGAGACCTGGTGCTGACAGTGCTCATTGCCCTG GCCGTGTACTTCCTGGGCCGGCTGGTCCCTCGGGGGCGAGGGGCTGCGGAGGCAGCGACCCGGA AACAGCGTATCACTGAGACCGAGTCGCCTTATCAGGAGCTCCAGGGTCAGAGGTCGGATGTCTA CAGCGACCTCAACACACAGAGGCCGTATTACAAAgTCGAGGGCGGCGGAGAGGGCAGAGGAAGT CTTCTAACATGC GGTGACGTGGAGGAGAATCCCGGCCCTAGGatggccttaccagtgaccgcct tgctcctgccgctggccttgctgctccacgccgccaggccgggatcccaggtacaactgcagca gtctgggcctgagctggagaagcctggcgcttcagtgaagatatcctgcaaggcttctggttac tcattcactggctacaccatgaactgggtgaagcagagccatggaaagagccttgagtggattg gacttattactccttacaatggtgcttctagctacaaccagaagttcaggggcaaggccacatt aactgtagacaagtcatccagcacagcctacatggacctcctcagtctgacatctgaagactct gcagtctatttctgtgcaagggggggttacgacgggaggggttttgactactggggccaaggga ccacggtcaccgtctcctcaggtggaggcggttcaggcggcggtggctctagcggtggtggatc ggacatcgagctcactcagtctccagcaatcatgtctgcatctccaggggagaaggtcaccatg acctgcagtgccagctcaagtgtaagttacatgcactggtaccagcagaagtcaggcacctccc ccaaaagatggatttatgacacatccaaactggcttctggagtcccaggtcgcttcagtggcag tgggtctggaaactcttactctctcacaatcagcagcgtggaggctgaagatgatgcaacttat tactgccagcagtggagtaagcaccctctcacgtacggtgctgggacaaagttggaaatcaaag CTAGCggtggcggaggttctggaggtgggggttccCAGGGGGCCTGGCCACATGAGGGAGTCCA CAGAAAACCTTCCCTCCTGGCCCACCCAGGTCCCCTGGTGAAATCAGAAGAGACAGTCATCCTG CAATGTTGGTCAGATGTCAGGTTTCAGCACTTCCTTC TGCACAGAGAAGGGAAGTTTAAGGACA CTTTGCACCTCATTGGAGAGCACCATGATGGGGTCTCCAAGGCCAACTTCTCCATCGGTCCCAT GATGCAAGACCTTGCAGGGACCTACAGATGCTACGGTTCTGTTACTCACTCCCCCTATCAGTTG TCAGCTCCCAGTGACCCTCTGGACATCGTCATCACAGGTCTATATGAGAAACCTTCTCTCTCAG CCCAGCCGGGCCCCACGGTTCTGGCAGGAGAGAGCGTGACCTTGTCCTGCAGCTCCCGGAGCTC CTATGACATGTACCATCTATCCAGGGAGGGGGAGGCCCATGAACGTAGGTTCTCTGCAGGGCCC

- 111 040145

AAGGTCAACGGAACATTCCAGGCCGACTTTCCTCTGGGCCCTGCCACCCACGGAGGAACCTACA GATGCTTCGGCTCTTTCCGTGACTCTCCATACGAGTGGTCAAACTCGAGTGACCCACTGCTTGT TTCTGTCACAGGAAACCCTTCAAATAGTTGGCTTTCACCCACTGAACCAAGCTCCGAAACCGGT AACCCCAGACACCTGCATGTTCTGATTGGGACCTCAGTGGTCATCATCCTCTTCATCCTCCTCC TCTTCTTTCTCCTTCATCGCTGGTGCTGCAACAAAAAAAATGCTGTTGTAATGGACCAAGAGCC T GCAGGGAACAGAACAGT GAACAGGGAGGAC T С T GAT GAACAAGACCC T GAG GAG GT GAG AT AT G GAGAG T T GAAT GAG TGCGTTTT CACACAGAGAAAAAT СAC T СAC С С T T С T CAGAG G С С CAAGA CACCCCCAACAGATATCATCGTGTACACGGAACTTCCAAATGCTGAGCCCTGAGtcgacaatca acctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacg ctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattt tctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggca acgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacc tgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccg cctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtc ggggaagctgacgtcctttccatggctgctcgcctgtgttgccacctggattctg cgcgggacg tccttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccgg ctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctccctttgggccgc ctccccgcctggaattcgagctcggtacctttaagaccaatgacttacaaggcagctgtagate ttagccactttttaaaagaaaaggggggactggaagggctaattcactcccaacgaagacaaga tctgctttttgcttgtactgggtctctctggttagaccagatctgagcctgggagctctctggc taactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtg cccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaat ctctagcagtagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaata tcagagagtgagaggaacttgtttattgcagcttataatggttacaaataaagcaatagcatca caaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaa tgtatcttatcatgtctggctctagctatcccgcccctaactccgcccagttccgcccattctc cgccccatggctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagct attccagaagtagtgaggaggcttttttggaggcctacgcttttgcgtcgagacgtacccaatt cgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactggga aaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaat agcgaagaggcccgc accgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgcg acgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctac acttgccagcgccctagcgcccgctcctttegetttcttcccttcctttctcgccacgttegee ggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggc acctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagac ggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactgga acaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcct attggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtt tacaatttcccaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttct aaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattg aaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattt tgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgg

- 112 040145

The nucleic acid sequence of KIR-CAR containing an antigen-binding domain that binds to CD19 and a cytoplasmic domain containing a KIR2DS2 domain is provided below.

Последовательность конструкции KIR2DS2 CD19 (SEQ ID NO: 330) gtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagtttt cgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattat cccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggt tgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagt gctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccga aggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaacc ggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaaca acgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagact ggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttat tgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagat ggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaa atagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagttta ctcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatc ctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagacc ccgtagaaaagatcaaaggatcttcttgagatcc tttttttctgcgcgtaatctgctgcttgca aacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactcttttt ccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagt taggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgetaatcctgttacc agtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccg gataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacga cctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggag aaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttcca gggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgat ttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacg

- 113 040145 gttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtg gataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgca gcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttg gccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaac gcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctc gtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgatta cgccaagcgcgcaattaaccctcactaaagggaacaaaagctggagctgcaagcttaatgtagt cttatgcaatactcttgtagtcttgcaacatggtaacgatgagttagcaacatgccttacaagg agagaaaaagcaccgtgcatgccgattggtggaagtaaggtggtacgatcgtgccttattagga aggcaacagacgggtctgacatggattggacgaaccactgaattgccgcattgcagagatattg tatttaagtgcctagctcgatacaataaacgggtctctctggttagaccagatctgagcctggg agctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttca agtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtca gtgtggaaaatctctagcagtggcgcccgaacagggacctgaaagcgaaagggaaaccagagct ctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtg agtacgccaaaa attttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagta ttaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaa atataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggc ctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacag gatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggat agagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagacc accgcacagcaagcggccgctgatcttcagacctggaggaggagatatgagggacaattggaga agtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaa agagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttctt gggagcagcaggaagcactatgggcgcagcctcaatgacgctgacggtacaggccagacaatta ttgtctggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgt tgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatacct aaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtg ccttggaatgctagttggagtaataaatctctggaacagattggaatcacacgacctggatgga gtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaac cagcaagaaaagaatgaacaagaattattggaattag ataaatgggcaagtttgtggaattggt ttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtagg tttaagaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccatta tcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaagaag gtggagagagagacagagacagatccattcgattagtgaacggatctcgacggtatcgattaga ctgtagcccaggaatatggcagctagattgtacacatttagaaggaaaagttatcttggtagca

- 114 040145 gttcatgtagccagtggatatatagaagcagaagtaattccagcagagacagggcaagaaacag catacttcctcttaaaattagcaggaagatggccagtaaaaacagtacatacagacaatggcag caatttcaccagtactacagttaaggccgcctgttggtgggcggggatcaagcaggaatttggc attccctacaatccccaaagtcaaggagtaatagaatctatgaataaagaattaaagaaaatta taggacaggtaagagatcaggctgaacatcttaagacagcagtacaaatggcagtattcatcca caattttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataata gcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttcgggttt attacagggacagcagagatccagtttggctgcatacgcgtcgtgaggctccggtgcccgtcag tgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaaccg gtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttt tcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaac gggtttgccgccagaacacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgg gttatggcccttgcgtgccttgaattacttccacctggctgcagtacgtgattcttgatcccga gcttcgggttggaagtgggtgggagagttcgaggccttgcgcttaaggagccccttcgcctcgt gcttgagttgaggcctggcctgggcgctggggccgccgcgtgcgaatctggtggcaccttcgcg cctgtctcgctg ctttcgataagtctctagccatttaaaatttttgatgacctgctgcgacgct ttttttctggcaagatagtcttgtaaatgcgggccaagatctgcacactggtatttcggttttt ggggccgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgc gagcgcggccaccgagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctgg cctcgcgccgccgtgtatcgccccgccctgggcggcaaggctggcccggtcggcaccagttgcg tgagcggaaagatggccgcttcccggccctgctgcagggagctcaaaatggaggacgcggcgct cgggagagcgggcgggtgagtcacccacacaaaggaaaagggcctttccgtcctcagccgtcgc ttcatgtgactccacggagtaccgggcgccgtccaggcacctcgattagttctcgtgcttttgg agtacgtcgtctttaggttggggggaggggttttatgcgatggagtttccccacactgagtggg tggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttga gtttggatcttggttcattctcaagcctcagacagtggttcaaagtttttttcttccatttcag gtgtcgtgagctagaATGGGGGGACTTGAACCCTGCAGCAGGCTCCTGCTCCTGCCTCTCCTGC TGGCTGTAAGTGGTCTCCGTCCTGTCCAGGCCCAGGCCCAGAGCGATTGCAGTTGCTCTACGGT GAGCCCGGGCGTGCTGGCAGGGATCGTGATGGGAGACCTGGTGCTGACAGTGCTCATTGCCCTG GCCGTGTACTTCCTGGGCCGGCTGGTCCCTCGGGGGCGAGGGGCTGCGGAGGCAGCGACCCGGA AACAGCGTATCACTGAGACCGAGTCGCCTTATCAGGA GCTCCAGGGTCAGAGGTCGGATGTCTA CAGCGACCTCAACACACAGAGGCCGTATTACAAAgTCGAGGGCGGCGGAGAGGGCAGAGGAAGT CTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCTAGGatggccttaccagtgaccgcct tgctcctgccgctggccttgctgctccacgccgccaggccgggatccGACATCCAGATGACACA GACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAG GAGAT TAG TAAATAT T TAAAT T G G TAT GAG CAGAAAC CAGAT G GAAC T G T TAAAC TCCTGATCT

- 115 040145

ACCATACATCAAGATTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGA TTATTCTCTCACCATTAGCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGT AATACGCTTCCGTACACGTTCGGAGGGGGGACTAAGTTGGAAATAACAGGTGGCGGTGGCTCGG GCGGTGGTGGGTCGGGTGGCGGCGGATCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGT GGCGCCCTCACAGAGCCTGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGT GTAAGCTGGATTCGCCAGCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTG AAAC GAGATAC TATAAT T GAG С T С T СAAAT С CAGAC T GAG CAT CAT CAAG GACAAC T С CAAGAG С CAAG Τ Τ Τ T C Τ ΤAAAAAT GAACAG T С T G CAAAC T GAT GACACAG CCATTTACTACTGTGC CAAA CATTATTACTACGGTGGTAGCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCT CCTCAgctagcACGCGTggtggcggaggttctggaggtgggggttcccagggggcctggccaca tgagggagtccacagaaaaccttccctcctggcccacccaggtcccctggtgaaatcagaagag acagtcatcctgcaatgttggtcagatgtcaggtttgagcacttccttctgcacagagagggga agtataaggacactttgcacctcattggagagcaccatgatggggtctccaaggccaacttctc catcggtcccatgatgcaagaccttgcagggacctacagatgctacggttctgttactcactcc ccctatcagttgtcagctcccagtgaccctctggacatcgtcatcacaggt ctatatgagaaac cttctctctcagcccagccgggccccacggttttggcaggagagagcgtgaccttgtcctgcag ctcccggagctcctatgacatgtaccatctatccagggagggggaggcccatgaacgtaggttc tctgcagggcccaaggtcaacggaacattccaggccgactttcctctgggccctgccacccacg gaggaacctacagatgcttcggctctttccgtgactctccctatgagtggtcaaactcgagtga cccactgcttgtttctgtcacaggaaacccttcaaatagttggccttcacccactgaaccaagc tccaaaaccggtaaccccagacacctgcatgttctgattgggacctcagtggtcaaaatccctt tcaccatcctcctcttctttctccttcatcgctggtgctccaacaaaaaaaatgctgctgtaat ggaccaagagcctgcagggaacagaacagtgaacagcgaggattctgatgaacaagaccatcag gaggtgtcatacgcataaGtcgacaatcaacctctggattacaaaatttgtgaaagattgactg gtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatca tgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctt tatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaa cccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccct ccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctg ttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctttccatggctgctcgcct gtgttgccacc tggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagc ggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccct cagacgagtcggatctccctttgggccgcctccccgcctggaattcgagcteggtacctttaag accaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaa gggctaattcactcccaacgaagacaagatctgctttttgcttgtactgggtctctctggttag accagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaag

- 116 040145 cttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatcc ctcagacccttttagtcagtgtggaaaatctctagcagtagtagttcatgtcatcttattattc agtatttataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttattgcagctta taatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcat tctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggctctagctatcccgcc cctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgca gaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcc tacgcttttgcgtcgagacgtacccaattcgccctatagtgagtcgtattacgcgcgctcactg gccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcag cacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaaca gttgcgcagcctgaatggcgaatggcgcgacgcgccctgtagcggcgcattaagcgcggcgggt gtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctt tcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccc tttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggt tcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttct ttaatagtggac tcttgttccaaactggaacaacactcaaccctatctcggtctattcttttga tttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaattt aacgcgaattttaacaaaatattaacgtttacaatttcccaggtggcacttttcggggaaatgt gcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaa taaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtg tcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggt gaaagtaaaagatgctgaagatcagttgg

Below is the nucleic acid sequence of an inhibitory CAR containing an antigen-binding domain that binds to CD19 and a cytoplasmic domain containing a PD1 domain.

Chimeric CAR CD19-PD1 sequence (SEQ ID NO: 331)

TGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGTGCACT CTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTG ACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGG GAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTG ATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTT TTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCC G С T CAT GAGACAATAACСС T GATAAAT GС T T СAATAATAT T GAAAAAGGAAGAGTAT GAGTAT T CAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACC CAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGA

- 117 040145

ACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATG AGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAAC TCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCA TCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACT GCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACA TGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGA CGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAA CTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGAC CACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCG TGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATC TACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCT САС T GAT TAAG CAT T G G TAAC T G T CAGAC CAAG TTTACTCATATATACTT TAGAT T GAT T TAAA ACTTCATTTTTAATT TAAAAG GAT С TAG G T GAAGAT CCTTTTTGATAATCTCAT GAG СAAAAT C CCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTT GAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAG CGGT GGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCG CAGATAC СAAATAC TGTTCTTCTAGTGTAGCCGTAGTTAGGC СAC СAC T T CAAGAAC T С T G TAG CACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTC GTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACG GGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGC GTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGG CAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGT CCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGA GCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGC TCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGA GCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAG AGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGA CAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCAT TAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGAT AACAAT T T CACACAG GAAACAG СТАТ GAC CAT GAT TAG G С CAAG С T С TAATAC GAC T CAC TATA GGGAGACAAGCTTGCATGCCTGCAGGTCGACATGGCCTTACCAGTGACCGCCTTGCTCCTGCCG CTGGCCTTGCTGCTCCACGCCGCCAGGCCGGACATCCAGATGACACAGACTACATCCTCCCTGT CTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAATATTT AAAT T GG T AT CAGCAGAAAC CAGAT GGAAC T GT T AAAC T С С T GAT C TAG CAT ACAT CAAGAT TA CACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGAACAGATTATTCTCTCACCATTA GCAACCTGGAGCAAGAAGATATTGCCACTTACTTTTGCCAACAGGGTAATACGCTTCCGTACAC

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GTTCGGAGGGGGGACTAAGTTGGAAATAACAGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGT GGCGGCGGATCTGAGGTGAAACTGCAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCC TGTCCGTCACATGCACTGTCTCAGGGGTCTCATTACCCGACTATGGTGTAAGCTGGATTCGCCA GCCTCCACGAAAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTAGTGAAACCACATACTATAAT T CAG С T С T СAAAT С CAGAC T GAG CAT CAT CAAG GACAAC T С CAAGAG С CAAG T T T T С T TAAAAA TGAACAGTCTGCAAACTGATGACACAGCCATTTACTACTGTGCCAAACATTATTACTACGGTGG TAGCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAgctagcACGCGT ggtggcggaggttctggaggtgggggttccaccctggtggttggtgtcgtgggcggcctgctgg gcagcctggtgctgctagtctgggtcctggccgtcatctgctcccgggccgcacgagggacaat aggagccaggcgcaccggccagcccctgaaggaggacccctcagccgtgcctgtgttctctgtg gactatggggagctggatttccagtggcgagagaagaccccggagccccccgtgccctgtgtcc ctgagcagacggagtatgccaccattgtctttcctagcggaatgggcacctcatcccccgcccg caggggctcagctgacggccctcggagtgcccagccactgaggcctgaggatggacactgctct tggсссctctgaGGATСССCGGGTACCGAGСTCGAATTСAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACTAGTGGCGCC

The present invention also relates to nucleic acid molecules and encoded amino acid sequences comprising an NKR-CAR as described herein, e.g. , for example, to increase the proliferation or cytotoxic activity of a CAR-expressing cell. Any of the antigen-binding domains described herein can be replaced by an antigen-binding domain in the NKR-CAR constructs below.

The nucleic acid sequence of DAP12 is as follows:

ATGGGGGGACTTGAACCCTGCAGCAGGTTCCTGCTCCTGCCTCTCCTGCTGGCTGTAAG TGGTCTCCGTCCTGTCCAGGTCCAGGCCCAGAGCGATTGCAGTTGCTCTACGGTGAGCCCGGGC GTGCTGGCAGGGATCGTGATGGGAGACCTGGTGCTGACAGTGCTCATTGCCCTGGCCGTGTACT TCCTGGGCCGGCTGGTCCCTCGGGGGCGAGGGGCTGCGGAGGCAGCGACCCGGAAACAGCGTAT CACTGAGACCGAGTCGCCTTATCAGGAGCTCCAGGGTCAGAGGTCGGATGTCTACAGCGACCTC AACACACAGAG G С С G ТАТ ТACAAAT GA (SEQ ID NO: 367)

The amino acid sequence of DAP12 is as follows:

MGGLEPCSRFLLLPLLLAVSGLRPVQVQAQSDCSCSTVSPGVLAGIVMGDLVLTVLIAL

AVYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK (SEQ ID NO: 368)

The nucleic acid sequence of FcεRγ is as follows:

ATGATTCCAGCAGTGGTCTTGCTCTTACTCCTTTTGGTTGAACAAGCAGCGGCCCTGGG AGAGCCTCAGCTCTGCTATATCCTGGAATGCCATCCTGTTTCTGTATGGAATTGTCCTCACCCTC

The amino acid sequence of FceRy is as follows:

MIPAVVLLLLLVEQAAALGEPQLCYILDAILFLYGIVLTLLYCRLKIQVRKAAITSYE

KSDGVYTGLSTRNQETYETLKHEKPPQ (SEQ ID NO: 370)

Also provided herein are nucleic acid sequences for multi-stranded NKR-CARs comprising an NKR-CAR and an adapter molecule. In such constructs, the NKR-CAR nucleic acid sequence and the adapter molecule are linked by a nucleic acid sequence encoding a peptide cleavage site, eg T2A. Such nucleic acid molecules are translated as a single-stranded polypeptide prior to cleavage, and the amino acid sequence of the single-stranded polypeptide is also provided herein.

The following is the nucleic acid sequence and amino acid sequence

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NKR-CAR containing an antigen-binding domain that binds to mesothelin, e.g.

SS1, and the cytoplasmic domain of KIRS2, as well as the DAP12 adapter molecule linked through the T2A peptide cleavage site.

DAP12-T2A-SS1-KIRS2 (SEQ ID NO: 332). DNA 1464 bp signs Localization DAP 12 1...339 T2A sequence 352...408 ssl-scFv 481...1200 GS linker 1207...1236

Derived from KIR2DS2 sequence 1237 ...1464 Subsequence. ATGGGGGGAC TTGAACCCTG CAGCAGGTTC CTGCTCCTGC CTCTCCTGCT GGCTGTAAGT GGTCTCCGTC CTGTCCAGGT CCAGGCCCAG AGCGATTGCA GTTGCTCTAC GGTGAGCCCG GGCGTGCTGG CAGGGATCGT GATGGGAGAC CTGGTGCTGA CAGTGCTCAT TGCCCTGGCC GTGTACTTCC TGGGCCGGCT GGTCCCTCGG GGGCGAGGGG CTGCGGAGGC AGCGACCCGG AAACAGCGTA TCACTGAGAC CGAGTCGCCT TATCAGGAGC TCCAGGGTCA GAGGTCGGAT GTCTACAGCG ASSTSAACAS ACAGAGGCCG TATTACAAAG TCGAGGGCGG CGGAGAGGGC AGAGGAAGTC TTCTAACATG CGGTGACGTG GAGGAGAATC CCGGCCCTAG GATGGCCTTA CCAGTGACCG CCTTGCTCCT GCCGCTGGCC TTGCTGCTCC ACGCCGCCAG GCCGGGATCC CAGGTACAAC TGCAGCAGTC TGGGCCTGAG CTGGAGAAGC CTGGCCGCTTC AGTGAAGATA TCCTGCAAGG CTTCTGGTTA STSATTSACT GGCTACACCA TGAACTGGGT GAAGCAGAGC CATGGAAAGA GCCTTGAGTG GATTGGACTT ATTASTSSTT ACAATGGTGC TTCTAGCTAC AACCAGAAGT TCAGGGGCAA GGCCACATTA ACTGTAGACA AGTCATCCAG CACAGCCTAC ATGGACCTCC TCAGTCTGAC ATCTGAAGAC TCTGAGTCT ATTTTCTGTGC AAGGGGGGGT TACGACGGGA GGGGTTTTGA CTACTGGGGC CAAGGGACCA CGGTCACCGT CTCCTCAGGT GGAGGCGGTT CAGGCGGCGG TGGCTCTAGC GGTGGTGGAT CGGACATCGA GCTCACTCAG TCTCCAGCAA TCATGTCTGC ATCTCCAGGG GAGAAGGTCA CCATGACCTG CAGTGCCAGC TCAAGTGTAA GTTACATGCA CTGGTACCAG CAGAAGTCAG GCACCTCCCC CAAAAGATGG ATTTATGACA SATSSAAAAST GGCTTTCTGGA GTCCAGGTC GCTTCAGTGG CAGTGGGTCT GGAAACTCTT ASSTTSTSAS AATCAGCAGC GTGGAGGCTG AAGATGATGC AASTTATTAS TGCCAGCAGT GGAGTAAGCA CCCTCTCACG TACGGTGCTG GGACAAAAGTT GGAAATCAAA GCTAGCGGTG GCGGAGGTTC TGGAGGTGGG GGTTCCTCAC CCACTGAACC AAGCTCCAAA ACCGGTAACC CCAGACACCT GCATGTTCTG ATTGGGACCT CAGTGGTCAA AATSSSTTTTS ASSATSSTSS TSTTTTTTST CCTTCATTCGC TGGTGCTCCA ASAAAAAAAAA TGCTGCTGTA ATGGACCAAG AGCCTGCAGG GAACAGAACA GTGAACAGCG AGGATTCTGA TGAACAAGAC CATCAGGAGG TGTCATACGC ATAA DAP12-T2A-SS1-KIRS2 (SEQ ID NO: 333). Protein 488 a.k. signs Localization DAP 12 1...113 Subsequence T2A 118.. .136 Signal peptide from CD8 alpha 138.. .158 ssl-scFv 161.. .400 GS linker 403.. .412 Subsequence, derived from KIR2DS2 413.. .487

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Subsequence.

MGGLEPCSRF LLLPLLLAVS GLRPVQVQAQ SDCSCSTVSP GVLAGIVMGD

LVLTVLIALA VYFLGRLVPR GRGAAEAATR KQRITETESP YQELQGQRSD VYSDLNTQRP YYKVEGGGEG RGSLLTCGDV EENPGPRMAL PVTALLLPLA LLLHAARPGS QVQLQQSGPE LEKPGASVKI SCKASGYSFT GYTMNWVKQS HGKSLEWIGL ITPYNGASSY NQKFRGKATL TVDKSSSTAY MDLLSLTTSED SAVYFCARGG YDGRGFDYWG QGTTVTVSSG GGGSGGGGSS GGGSDIELTQ SPAIMASPG EKVTMTCSAS SSVSYMHWYQ QKSGTSPKRW IYDTSKLASG VPGRFSGSGS GNSYSLTISS VEAEDDATYY CQQWSKHPLT YGAGTKLEIK ASGGGGSGGG GSSPTEPSSK TGNPRHLHVL IGTSWKIPF TILLFFLLHR

WCSNKKNAAV MDQEPAGNRT VNSEDSDEQD HQEVSYA

Below is the nucleic acid sequence and amino acid sequence of NKR-CAR containing an antigen-binding domain that binds to mesothelin, such as SS1, and a cytoplasmic domain of TNKp46, as well as an FcsRy adapter molecule linked through the T2A peptide cleavage site.

FCERG-T2A-SS1-TNKp46 (SEQ ID NO: 334).

DNA 1365 bp

signs

FCERG

T2A

Signal peptide from CD8 alpha

ssl-scFv

GS linker

Localization

1...258 271...327

331...393

400...1119

1126...1155

Subsequence, derived from NKp4 6 11 56...1365 Subsequence. ATGATTCCAG CAGTGGTCTT GCTCTTACTC CTTTTGGTTG AACAAGCAGC GGCCCTGGGA GAGCCTCAGC TCTGCTATAT CCTGGATGCC ATCCTGTTTC TGTATGGAAT TGTCCTCACC CTCCTCTACT GCCGACTGAA GATCCAAGTG CGAAAGGCAG CTATAACCAG CTATGAGAAA TCAGATGGTG TTTACACGGG CCTGAGCACC AGGAACCAGG AGACTTACGA GACTCTGAAG CATGAGAAAC CACCACAGTC CGGAGGCGGC GGAGAGGGCA GAGGAAGTCT TCTAACATGC GGTGACGTGG AGGAGAATCC CGGCCCTAGG ATGGCCTTAC CAGTGACGC CTTGCTCCTG CCGCTGGCCT TGCTGCTCCA CGCCGCCAGG CCGGGATCCC AGGTACAACT GCAGCAGTCT GGGCCTGAGC TGGAGAAGCC TGGCGCTTCA GTGAAGATAT CCTGCAAGGC TTCTGGTTAC TCATTCACTG GCTACACCAT GAACTGGGTG AAGCAGAGCC ATGGAAAAGAG CCTTGAGTGG ATTGGACTTA TTACTCCTTTA CAATGGTGCT TCTAGCTACA ACCAGAAGTT CAGGGGCAAG GCCACATTAA CTGTAGACAAA GTCATCCAGC ACAGCCTACA TGGACCTCCT CAGTCTGAC TCTGAAGACT CTGCAGTCTA TTTCTGTGCA AGGGGGGGTT ACGACGGGAG GGGTTTTGAC TACTGGGGCC AAGGGACCAC GGTCACCGTC TCCTCAGGTG GAGGCGGTTC AGGCGGCGGT GGCCTCTAGCG GTGGTGGATC GGACATCGAG CTCACTCAGT CTCCAGCAAT CATGTCTGCA TCTCCAGGGG AGAAGGTCAC CATGACCTG AGTGCCAGCT CAAGTGTAAG TTACATGCAC TGGTACCAGC AGAAGTCAGG CACCTCCCCC AAAAGATGGA TTTATGACAC ATCCAAACTG GCTTTCTGGAG TCCCAGGTCG CTTCAGTGGC AGTGGGTCTG GAAACTCTTA CTCTCTCACA ATCAGCAGCG TGGAGGCTGA AGATGATGCA ACTTATTACT GCCAGCAGTG GAGTAAGCAC CCTCTCACGT ACGGTGCTGG GACAAAAGTTG GAAATCAAAG CTAGCGGTGG CGGAGGTTCT GGAGGTGGGG GTTCCTTAAC CACAGAGACG GGACTCCAGA AAGACCATGC CCTCTGGGAT CACACTGCCC AGAATCTCCT TCGGATGGGC CTGGCCTTTC TAGTCCTGGT GGCCTCTAGTG TGGTTCCTGG TTGAAGACTG GCTCAGCAGG AAGAGGACTA GAGAGCGAGC CAGCAGAGCT TCCACTTGGG AAGGCAGGAG AAGGCTGAAC

ACACAGACTC TTTGA

FCERG-T2A-SS1-TNKp46 (SEQ ID NO: 335).

Protein 455 a.a.

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Localization

signs

386...454

91...10 9

111...131

134...373

376...385

FCERG

Signal peptide from CD8 alpha

ssl-scFv

GS linker

Subsequence

Subsequence.

derived from NKp46

MIPAWLLLL LLVEQAAALG EPQLCYILDA ILFLYGIVLT LLYCRLKIQV RKAAITSYEK SDGVYTGLST RNQETYETLK HEKPPQSGGG GEGRGSLTC GDVEENPGPR MALPVTALLL PLALLLHAAR PGSQVQLQQS GPELEKPGAS VKISOKASGY SFTGYTMNWV KQSHGKSLEW IGLITPYNGA SSYNQKFRGK ATLTVDKSSS TAYMDLLSLT SEDSAVYFCA RGGYDGRGFD YWGQGTTVTV SSGGGGSGGG GSSGGGSDIE LTQSPAIMSA SPGEKVTMTC SASSSVSYMH WYQQKSGTSP KRWIYDTSKL ASGVPGRFSG SGSGNSYSLT ISSVEAEDDA TYYCQQWSKH PLTYGAGTKL EIKASGGGGS GGGGSLTTTET GLQKDHALWD HTAQNLLRMG LAFLVLVALV WFLVEDWLSR KRTRERASRA STWEGRRRLN TQTL

Below is the nucleic acid sequence and amino acid sequence of NKR-CAR containing an antigen-binding domain that binds to CD19 and a KIRS2 cytoplasmic domain, as well as a DAP12 adapter molecule linked through a T2A peptide cleavage site.

DAP12-T2A-CD19-KIRS2 (SEQ ID NO: 336).

DNA 1470 bp

Signs Localization

DAP 12 1... 3 3 9

T2A sequence 352...408

CD19-scFv 481...481

GS linker 1213...1242

Sequence received from KIR2DS2 1243...1470

Subsequence.

ATGGGGGGAC TTGAACCCTG CAGCAGGTTC CTGCTCCTGC CTCTCCTGCT

GGCTGTAAGT GGTCTCCGTC CTGTCCAGGT CCAGGCCCAG AGCGATTGCA GTTGCTCTAC GGTGAGCCCG GGCGTGCTGG CAGGGATCGT GATGGGAGAC CTGGTGCTGA CAGTGCTCAT TGCCCTGGCC GTGTACTTCC TGGGCCGGCT GGTCCCTCGG GGGCGAGGGG CTGCGGAGGC AGCGACCCGG AAACAGCGTA TCACTGAGAC CGAGTCGCCT TATCAGGAGC TCCAGGGTCA GAGGTCGGAT GTCTACAGCG ACCTCAACAC ACAGAGGCCG TATTACAAAG TCGAGGGCGG CGGAGAGGGC AGAGGAAGTC TTCTAACATG CGGTGACGTG GAGGAGAATC CCGGCCCTAG GATGGCCTTA CCAGTGACCG CCTTGCTCCT GCCGCTGGCC TTGCTGCTCC ACGCCGCCAG GCCGGGATCC GACATCCAGA TGACACAGAC TACATCCTCC CTGTCTGCCT CTCTGGGAGA CAGAGTCACC ATCAGTTGCA GGGCAAGTCA GGACATTAGT AAATATTTAA ATTGGTATCA GCAGAAACCA GATGGAACTG TTAAACTCCT GATCTACCAT ACATCAAGAT TACACTCAGG AGTCCCATCA AGGTTCAGTG GCAGTGGGTC TGGAACAGAT TATTCTCTCA CCATTAGCAA CCTGGAGCAA GAAGATATTG CCACTTACTT TTGCCAACAG GGTAATACGC TTCCGTACAC GTTCGGAGGG GGGACTAAGT TGGAAATAAC AGGTGGCGGT GGCTCGGGCG GTGGTGGGTC GGGTGGCGGC GGATCTGAGG TGAAACTGCA GGAGTCAGGA CCTGGCCTGG TGGCGCCCTC ACAGAGCCTG TCCGTCACAT GCACTGTCTC AGGGGTCTCA TTACCCGACT ATGGTGTAAG CTGGATTCGC CAGCCTCCAC GAAAGGGTCT GGAGTGGCTG GGAGTAATAT GGGGTAGTGA AACCACATAC TATAATTCAG CTCTCAAATC CAGACTGACC ATCATCAAGG ACAACTCCAA GAGCCAAGTT TTCTTAAAAA TGAACAGTCT GCAAACTGAT GACACAGCCA TTTACTACTG TGCCAAACAT TATTACTACG GTGGTAGCTA TGCTATGGAC TACTGGGGTC AAGGAACCTC AGTCACCGTC TCCTCAGCTA GCGGTGGCGG AGGTTCTGGA GGTGGGGGTT CCTCACCCAC TGAACCAAGC TCCAAAACCG GTAACCCCAG ACACCTGCAT GTTCTGATTG GGACCTCAGT GGTCAAAATC CCTTTCACCA TCCTCCTCTT CTTTCTCCTT CATCGCTGGT GCTCCAACAA AAAAAATGCT GCTGTAATGG ACCAAGAGCC TGCAGGGAAC AGAACAGTGA ACAGCGAGGA TTCTGATGAA CAAGACCATC

AGGAGGTGTC ATACGCATAA

DAP12-T2A-CD19-KIRS2 (SEQ ID NO: 337).

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Protein 489 a.a.

Signs Localization

DAP12 1...113

T2A sequence 118...136

Signal peptide from CD8 alpha 138...158

CD19-scFv 161...402

GS-linker 405...414

Sequence 415...489 received from KIR2DS2

Subsequence.

MGGLEPCSRF LLLPLLLAVS GLRPVQVQAQ SDCSCSTVSP GVLAGIVMGD

LVLTVLIALA

VYSDLNTQRP

LLLHAARPGS

DGTVKLLIYH

GNTLPYTFGG

SVTCTVSGVS

IIKDNSKSQV

SSASGGGGSG

VYFLGRLVPR

YYKVEGGGEG DIQMTQTTSS TSRLHSGVPS GTKLEITGGG LPDYGVSWIR FLKMNSLQTD GGGSSPTEPS

GRGAAEAATR

RGSLLTCGDV

LSASLGDRVT

RFSGSGSGTD

GSGGGGSGGG

QPPRKGLEWL

DTAIYYCAKH SKTGNPRHLH

KQRITETESP ENPGPRMAL ISCRASQDIS YSLTISNLEQ GSEVKLQESG GVIWGSETTY YYYGGSYAMD VLIGTSWKI

YQELQGQRSD PVTALLLPLA KYLNWYQQKP EDIATYFCQQ PGLVAPSQSL YNSALKSRLT YWGQGTSVTV PFTILLFFLL

HRWCSNKKNA AVMDQEPAGN RTVNSEDSDE QDHQEVSYA*

In one embodiment, the transmembrane domain is a KIR2DS2 transmembrane domain. The amino acid sequence of the transmembrane domain of KIR2DS2 is as follows:

VLIGTSWKIPFTILLFFLL (SEQ ID NO: 357)

In one embodiment, the transmembrane domain is a KIR2DL3 transmembrane domain. The amino acid sequence of the KIR2DL3 transmembrane domain is as follows:

VLIGTSWIILFILLLFFLL (SEQ ID NO: 358)

In one embodiment, the transmembrane domain is the transmembrane domain of NKp46. The amino acid sequence of the NKp46 transmembrane domain is as follows:

LLRMGLAFLVLVALVWFLVEDWLS (SEQ ID NO: 359)

In one embodiment, the cytoplasmic domain is derived from KIR2DS2, eg native KIRS2DS2, as shown in FIG. 29. The amino acid sequence of the cytoplasmic domain derived from KIR2DS2, also referred to herein as the KIRS2 domain, is as follows:

HRWCSNKKNAAVMDQEPAGNRTVNSEDSDEQDHQEVSYA (SEQ ID NO: 360)

In one embodiment, the cytoplasmic domain is derived from KIR2DL3, eg native KIR2DL3, as shown in FIG. 30. The amino acid sequence of the cytoplasmic domain derived from KIR2DL3, also referred to herein as the KIRL3 domain, is as follows:

HRWCCNKKNAWMDQEPAGNRTVNREDSDEQDPQEVTYAQLNHCVFTQRKITHPSQRPK

TPPTDIIVYTELPNAEP (SEQ ID NO: 361)

In one embodiment, the cytoplasmic domain is derived from NKp46, eg native NKp46, as shown in FIG. 31. The amino acid sequence of the cytoplasmic domain derived from NKp46, also referred to herein as the tNKp46 or TNKp46 domain, is as follows:

RKRTRERASRASTWEGRRRRLNTQTL (SEQ ID NO: 362)

In one embodiment, the transmembrane and cytoplasmic domain, together contains the amino acid sequence provided below:

VLIGTSWKIPFTILLFFLLHRWCSNKKNAAVMDQEPAGNRTVNSEDSDEQDHQEVSYA (SEQ ID NO: 371)

The nucleic acid sequence and amino acid sequence of KIR-CAR containing a human antigen-binding domain that binds to mesothelin are also provided herein. For example, below is the nucleic acid sequence of KIR-CAR containing the leader sequence, the mesothelin-specific human M5 antigen-binding domain, and the cytoplasmic domain of KIRS2. The underlined sequence indicates the sequence of the M5 scFv, which can be easily replaced by any other antigen-binding domain, for example, the scFv of the human anti-mesothelin antibody described in the present invention.

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Atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccag gccgggatc ccaagtccaactcgttcaatcaggcgcagaagtcgaaaagcccggagcatcagtc aaagtctcttgcaaggcttccggctacaccttcacggactactасаtgcactgggtgcgccagg ctccaqqccaqqqactgqaqtQgatqqgatqqatcaacccqaattccgggqqaactaactacgc ccagaagtttcagggccgQQtgactatgactcQcgatacctcgatctcgactgcgtacatggag ctcagccgcctccggtcggacgataccgccQtgtactattgtgcgtcgggatgggacttcgact actgQQggcagQQcactctQgtcactgtgtcaagcggaQQaggtQQatcagQtggaggtggaag cggQggaggaggttccgQcggcggaggatcagatatcQtgatgacgcaatcgccttcctcgttg tccgcatccgtQQgagacagggtgaccattacttgcagagcgtcccagtccattcggtactacc tgtcQtQgtaccagcagaaQccggQQaaagccccaaaactQcttatctatactQcctcQatcct ccaaaacggcgtQccatcaagattcagcggttcgggcagcgggaccgactttaccctgactatc agcagcctgcagccggaagatttcgccacgtactactgcctgcaaacctacaccaccccggact tcggacctggaaccaaggtggagatcaaggctagcggtggcggaggttctggaggtgggggttc ctcacccactgaaccaagctccaaaaccggtaaccccagacacctgcatgttctgattgggacc tcagtggtcaaaatccctttcaecatcctcctcttctttctccttcatcgctggtgetccaaca aaaaaaatgctgctgtaatggaccaagag cctgcagggaacagaacagtgaacagcgaggattc tgatgaacaagaccatcaggaggtgtcatacgcataa (SEQ ID NO: 363)

The amino acid sequence of KIR-CAR containing the leader sequence, the human mesothelin-specific M5 antigen-binding domain, and the cytoplasmic domain of KIRS2 is shown below. The underlined sequence indicates the sequence of the M5 scFv, which can be easily replaced by any other antigen-binding domain, for example, the scFv of the human anti-mesothelin antibody described in the present invention.

MAL РVTAL L L Р LAL L L HAAR PGS OVQLVOSGAEVEKPGASVKVSCKASGYTFTDYYMHW VROAPGOGLEWMGWINPNSGGTNYAQKFOGRVTMTRDTSISTAYMELSRLRSDDTAVYYCASGW DFDYWGOGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIVMTQSPSSLSASVGDRVTITCRASOSI RYYLSWYOOKPGKAPKLLIYTASILONGVPSRFSGSGSGTDFTLTISSLOPEDFATYYCLQTYT TPDFGPGTKVEIKASGGGGSGGGGSSPTEPSSKTGNPRHLHVLIGTSWKIPFTILLFFLLHRW CSNKKNAAVMDQEPAGNRTVNSEDSDEQDHQEVSYA (SEQ ID NO: 364)

In another example, below is the nucleic acid sequence of a multi-stranded KIR-CAR containing a leader sequence, a human mesothelin-specific M5 antigen-binding domain and a KIRS2 cytoplasmic domain, and additionally containing a peptide cleavage site and a DAP12 adapter molecule sequence. The underlined sequence indicates the sequence of the M5 scFv, which can be easily replaced by any other antigen-binding domain, for example, the scFv of the human anti-mesothelin antibody described in the present invention. The sequence in bold indicates the DAP12 sequence and the sequence in italics indicates the T2A peptide cleavage site.

ATGGGGGGACTTGAACCCTGCAGCAGGTTCCTGCTCCTGCCTCTCCTGCTGGCTGTAAG TGGTCTCCGTCCTGTCCAGGTCCAGGCCCAGAGCGATTGCAGTTGCTCTACGGTGAGCCCGGGC GTGCTGGCAGGGATCGTGATGGGAGACCTGGTGCTGACAGTGCTCATTGCCCTGGCCGTGTACT TCCTGGGCCGGCTGGTCCCTCGGGGGCGAGGGGCTGCGGAGGCAGCGACCCGGAAACAGCGTAT CACTGAGACCGAGTCGCCTTATCAGGAGCTCCAGGGTCAGAGGTCGGATGTCTACAGCGACCTC AACACACAGAGGCCGTAT ТАСАААТ с с ддаggcagcggagagggcagaggaagtcttctaacat gcggtgacgtggaggagaatcccggccCTAGGatggccttaccagtgaccgccttgctcctgcc

- 124 040145 g c t g g с c 11 g c t g c t с с a c g с c g с c a g g с c g g G A T С C caaqtccaactcqttcaatcaqqcgca qaagtcgaaaagcccggaqcatcagtcaaagtctcttqcaagqcttccggctacaccttcacgg actactacatqcactqqqtqcqccaqqctccaqqccaqqqactqqaqtqqatqqqatqqatcaa cccqaattccqggqqaactaactacgcccagaaqtttcaggqccggqtgactatgactcgcqat acctcqatctcgactgcgtacatqgagctcagccgcctccggtcggacgataccgccgtgtact attqtqcqtcqqqatqqqacttcqactactqqqqqcaqqqcactctqqtcactqtqtcaaqcqq aqqaqqtqqatcaqqtqqaqqtqqaaqcqqqqqaqqaqqttccqqcqqcqqaqqatcaqatatc qtqatqacqcaatcqccttcctcqttqtccqcatccqtqqqaqacaqqqtqaccattacttqca qaqcqtcccaqtccattcqqtactacctqtcqtqqtaccaqcaqaaqccqqqqaaaqccccaaa actqcttatctatactqcctcqatcctccaaaacqqcqtqccatcaaqattcaqcqqttcqqqc aqcqqqaccqactttaccctqactatcaqcaqcctqcaqccqqaaqatttcqccacqtactact qcctqcaaacctacaccaccccqqacttcqqacctqqaaccaaqqtqqaqatcaaqGc t a g c gg tggcggaggttctggaggtgggggttcctcacccactgaaccaagctccaaaaccggtaacccc agacacctgcatGTTCTGATTGGGACCTCAGTGGTCAAAATCCCTTTCACCATCCTCCTCTTCT TTCTCCTTcatcgctggtgctccaacaaaaaaaatg ctgctgtaatggaccaagagcctgcagg gaacagaacagtgaacagcgaggattctgatgaacaagaccatcaggaggtgtcatacgcataa (SEQ ID NO: 365)

Below is the amino acid sequence of a multi-chain KIR-CAR containing a leader sequence, a human mesothelin-specific M5 antigen-binding domain and a KIRS2 cytoplasmic domain, and additionally containing a peptide cleavage site and a DAP12 adapter molecule sequence. The underlined sequence denotes the M5 scFv sequence, which can easily be replaced by any other antigen-binding domain, such as the human anti-mesothelin scFv described herein. The sequence in bold indicates the DAP12 sequence and the sequence in italics indicates the T2A peptide cleavage site.

MGGLEPCSRFLLLPLLLAVSGLRPVQVQAQSDCSCSTVSPGVLAGIVMGDLVLTVLIAL AVYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYKSGGSGEGRGS LLTCGDVEENPGPRMALPVTALLLPLALLLHAARPGSOVOLVQSGAEVEKPGASVKVSCKASGY TFTDYYMHWVROAPGOGLEWMGWINPNSGGTNYAQKFOGRVTMTRDTSISTAYMELSRLRSDDT AVYYCASGWDFDYWGOGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIVMTQSPSSLSASVGDRVT ITCRASQSIRYYLSWYOOKPGKAPKLLIYTASILONGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCLQTYTTPDFGPGTKVEIKASGGGGSGGGGSSPTEPSSKTGNPRHLHVLIGTSWKIPFTI LLFFLLHRWCSNKKNAAVMDQEPAGNRTVNSEDSDEQDHQEVSYA (SEQ ID NO: 366) Цитоплазматический домен.

The cytoplasmic domain of a CAR as described herein, such as NKR-CAR or TCAR, contains an intracellular signaling domain. The intracellular signaling domain is capable of activating at least one of the normal effector functions of the immune system effector cell into which CAR has been administered.

Examples of intracellular signaling domains for use in the CARs of the invention include cytoplasmic T cell receptor (TCR) sequences and co-receptors that act together to initiate signal transduction upon antigen receptor binding, as well as any derivative or variant of these sequences, and any recombinant sequence that has the same functionality.

It is known that the signals generated by a single TCR are not sufficient to fully activate an effector cell of the immune system, eg T cells or NK cells, and that a secondary and/or costimulatory one is also necessary. Thus, it can be hypothesized that immune system effector cell activation, such as T cell activation or NK cell activation, is mediated by two different classes of cytoplasmic signaling sequences: sequences that initiate antigen-dependent primary activation via TCRs (primary intracellular signaling domains), and sequences that act in an antigen-dependent manner to provide a secondary or costimulatory signal (secondary cytoplasmic domain, eg costimulatory domain).

Primary intracellular signaling domain.

In some embodiments, a primary intracellular signaling domain produces an intracellular signal when an extracellular domain, such as an antigen-binding domain with which it is fused, binds to a cognate antigen. It is derived from the primary excitatory molecule, for example it contains the intracellular sequence of the primary excitatory molecule. It contains a primary stimulatory molecule sequence sufficient to produce an intracellular signal, for example, when the antigen-binding domain to which it is fused binds to a cognate antigen.

A primary stimulatory molecule is a molecule that, upon binding a cognate ligand, mediates an immune effector response, eg, in the cell in which it is expressed. Typically, it generates an intracellular signal that depends on binding to a cognate ligand that contains the antigen. The TCR/CD3 complex is an exemplary primary stimulatory molecule; it generates an intracellular signal when bound to a cognate ligand, such as an MHC molecule loaded with a peptide. Typically, for example, in the case of a TCR/CD3 primary stimulatory molecule, the generation of an intracellular signal by the primary intracellular signaling domain depends on binding of the primary stimulatory molecule to an antigen.

Primary stimulation may mediate altered expression of certain molecules, such as decreased expression of TGF-β, and/or reorganization of the cytoskeletal structure, and the like. Stimulation, for example in the presence of costimulation, can lead to optimization, for example, an increase in the immune effector function of the T cell. Stimulation against, for example, a T cell may mediate a T cell response, such as proliferation, activation, differentiation, and the like.

In one embodiment, the primary intracellular signaling domain contains a signaling motif, such as an immunoreceptor tyrosine activating motif or ITAM. The primary intracellular signaling domain may contain an ITAM containing cytoplasmic signal sequences from TCR zeta (CD3 zeta), FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as ICOS) , FcεRI, DAP10, DAP12 and CD66d.

Exemplary ITAM-containing primary intracellular signaling domains are shown in Table 1. 2.

table 2

Primary intracellular signaling domains (for example, containing ITAM domains) CD3 zeta CD5 FcR gamma CD22 FcR beta CD278 (ICOS) CD3 gamma Fc epsilon RI (FceRI) CD3 delta CD66d CD3 epsilon DAP 10 CD79a DAP 12 CD79b

In one embodiment, the primary intracellular signaling domain comprises a modified ITAM domain, eg, a mutant ITAM domain, that has altered (eg, increased or decreased) activity compared to the native ITAM domain. In one embodiment, the primary intracellular signaling domain comprises a modified ITAM-containing primary intracellular signaling domain, such as an optimized and/or truncated ITAM-containing primary intracellular signaling domain. In one embodiment, the primary intracellular signaling domain contains one, two, three, four or more ITAM motifs.

The primary intracellular signaling domain contains a functional fragment or analog of the primary stimulatory molecule (eg CD3 zeta GenBank accession number BAG36664.1). It may contain the entire intracellular region or a fragment of the intracellular region that is sufficient to generate an intracellular signal when the antigen-binding domain to which it is fused or paired via a dimerization switch binds to a cognate antigen. In embodiments, the primary intracellular signaling domain has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with a natural primary stimulatory molecule, e.g., the primary stimulatory molecule described in Table 1. 2, for example, an intracellular primary stimulatory molecule from a human (GenBank accession number BAG36664.1) or from another mammal, eg a non-human species, eg a rodent, monkey, primate or mouse. In embodiments, the primary intracellular signaling domain shares at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 9 or SEQ ID NO: 10.

In embodiments, the primary intracellular signaling domain shares at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity, or differs by no more than 30%, 25%, 20%,

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15, 10, 5, 4, 3, 2 or 1 amino acid residues from the corresponding residues of a natural human primary stimulatory molecule, for example, a natural human primary stimulatory molecule described in the present invention.

The TCAR intracellular signaling domain may comprise a primary intracellular signaling domain, eg, the CD3-zeta signaling domain by itself, or it may be combined with any other desired intracellular signaling domain(s) used in relation to TCAR. For example, a TCAR intracellular signaling domain may comprise a primary intracellular signaling domain, such as a region of the CD3 zeta chain, and one or more co-stimulatory signaling domains. Examples of costimulatory signaling domains are provided below.

costimulatory signaling domain.

In one embodiment, the co-stimulatory signaling domain produces an intracellular signal when an extracellular domain, such as an antigen-binding domain with which it is fused or paired via a dimerization switch, binds to a cognate ligand. It is derived from a costimulatory molecule. It contains sufficient sequence of a primary costimulatory molecule to produce an intracellular signal, for example, when the antigen-binding domain to which it is fused or paired via a dimerization switch binds a cognate ligand.

Costimulatory molecules are cell surface molecules, other than antigen receptors or counterligands thereof, that promote an immune effector response. In some cases, they are essential for an effective or enhanced immune response. Typically, a co-stimulatory molecule generates an intracellular signal that depends on binding to a cognate ligand that, in embodiments, is other than an antigen, eg, an antigen recognized by the antigen-binding domain of a T cell. Typically, signaling from the primary stimulatory molecule and the co-stimulatory molecule contribute to the response of the immune effector, and in some cases, both are necessary for the effective or increased generation of the response of the immune effector.

The costimulatory signaling domain contains a functional fragment or analogue of a costimulatory molecule (eg, 4-1BB, CD28, CD27, and ICOS). It may contain the entire intracellular region or a fragment of the intracellular region of a co-stimulatory molecule that is sufficient to generate an intracellular signal, for example, when the antigen-binding domain to which it is fused or paired via a dimerization switch binds a cognate antigen. In embodiments, the costimulatory signaling domain has at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with a naturally occurring costimulatory molecule of the present invention, e.g., a human or other mammalian intracellular costimulatory molecule, e.g., non-human species such as rodents, monkeys, macaws or mice. In embodiments, the co-stimulatory domain shares at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 36, or SEQ ID NO: 38.

Exemplary co-stimulatory signaling domains (intracellular signaling domains) are provided in Table. 3.

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Table 3

Costimulatory signaling domains (identified by the costimulatory molecules from which they are derived)

CD27 MHC class I molecule SLAMF7 ITGA4 CDllc CD2 8 TNF receptor protein NKp8 0 (KLRF1) IA4 ITGB1 4-1BB (CD137) Immunoglobulin-like protein NKp4 4 CD49D CD2 9 OH4 0 Cytokine receptor NKp3 0 ITGA6 ITGB2 CD30 Integrin NKp4 6 VLA-6 CD18 CD40 Lymphocyte signaling activating molecule (SLAM protein) CD19 CD49f ITGB7 ICOS (CD278) NK cell activating receptors CD4 IT GAD NKG2D ICAM-1 Toll-like receptor CD8 alpha CDlld TNFR2 LFA-1 (CDlla/CD18) BTLA CD8 beta IT GAE TRANCE/ RANKL CD2 CDS IL2R beta CD103 DNAM1 (CD226) CD7 ICAM-1 IL2R gamma IT GAL SLAMF4 (CD244, 2B4) LIGHT GITR IL7R alpha CDlla CD84 NKG2C BAFFR ITGA4 ITGAM CD96 (Tactile) B7-H3 HVEM (LIGHTR) VLA1 CDllb CEACAM1 A ligand that specifically binds to CD83 KIRDS2 CD49a ITGAX CRT AM Ly9 (CD229) CD160 (BY55) PSGL1 CD100 (SEMA4D) CD69 SLAMF6 (NTB-A, Lyl08) SLAM (SLAMF1, CD150, IPO3) BLAME (SLAMF8) SELPLG (CD162) LTBR LAT GADS SLP-76 PAG/Cbp CD19a

In embodiments, the co-stimulatory signaling domain has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity, or differs by no more than 30%, 25%, 20%, 15%, 10%, 5%, 4, 3, 2, or 1 amino acid residue from the corresponding residues of a natural human primary stimulatory molecule, for example, a natural human costimulatory molecule described in the present invention, for example, provided in table. 3.

Intracellular signal sequences in the cytoplasmic domain of TCAR may be linked to each other in a random sequence or in a specific order. Optionally, a short oligo- or polypeptide linker, eg 2 to 10 amino acids long (eg 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) can form a link between intracellular signal sequences. In one embodiment, a glycine-serine doublet can be used as a suitable linker. In one embodiment, a single amino acid, eg alanine, glycine, can be used as a suitable linker.

In one aspect, an intracellular signaling domain is designed to contain two or more, eg 2, 3, 4, 5 or more co-stimulatory signaling domains. In one embodiment, two or more, eg 2, 3, 4, 5 or more co-stimulatory signaling domains are separated by a linker molecule, eg a linker molecule as described herein. In one embodiment, the intracellular signaling domain contains two costimulatory signaling domains. In some embodiments, the linker molecule is

- 128 040145 is a glycine residue. In some embodiments, the linker is an alanine residue.

In one aspect, the intracellular signaling domain is designed to contain the CD3-zeta signaling domain and the 4-1BB signaling domain. In one aspect, the 4-1BB signal domain is a signal domain comprising the amino acid sequence of SEQ ID NO: 7. In one aspect, the 4-1BB signal domain is encoded by the nucleic acid sequence of SEQ ID NO: 18. In one aspect, the CD3- zeta is a signal domain containing the amino acid sequence of SEQ ID NO: 9 (mutant CD3 zeta) or SEQ ID NO: 10 (wild-type human CD3 zeta).

In one aspect, an intracellular signaling domain is designed to contain a CD3-zeta signaling domain and a CD27 signaling domain. In one aspect, the CD27 signal domain comprises the amino acid sequence of SEQ ID NO: 8. In one aspect, the CD27 signal domain is encoded by the nucleic acid sequence of SEQ ID NO: 19. In one aspect, the CD3 zeta signal domain is a signal domain comprising the amino acid sequence of SEQ ID NO: 9 (mutant CD3 zeta) or SEQ ID NO: 10 (wild-type human CD3 zeta).

In one aspect, an intracellular signaling domain is designed to contain a CD3-zeta signaling domain and a CD28 signaling domain. In one aspect, the CD28 signal domain comprises the amino acid sequence of SEQ ID NO: 36. In one aspect, the CD28 signal domain is encoded by the nucleic acid sequence of SEQ ID NO: 37. In one aspect, the CD3-zeta signal domain is a signal domain containing the amino acid sequence SEQ ID NO: 9 (mutant CD3 zeta) or SEQ ID NO: 10 (wild-type human CD3 zeta).

In one aspect, an intracellular signaling domain is designed to contain a CD3-zeta signaling domain and an ICOS signaling domain. In one aspect, the ICOS signal domain comprises the amino acid sequence of SEQ ID NO: 38. In one aspect, the ICOS signal domain is encoded by the nucleic acid sequence of SEQ ID NO: 39. In one aspect, the CD3 zeta signal domain is a signal domain comprising the amino acid sequence of SEQ ID NO: 9 (mutant CD3 zeta) or SEQ ID NO: 10 (wild-type human CD3 zeta).

transmembrane domain.

With respect to the transmembrane domain, in various embodiments, the implementation of the CAR can be designed to contain a transmembrane domain that is attached to the extracellular domain of the CAR. As previously described, the NKR-CAR transmembrane domain described herein can be an NKR transmembrane domain, such as a KIR transmembrane domain, an NCR transmembrane domain, an SLAMF transmembrane domain, an FcR transmembrane domain, such as a CD16 transmembrane domain or a CD64 transmembrane domain, or transmembrane domain of Ly49. Alternatively, the NKR-CAR or TCAR transmembrane domain described in the present invention may be any of the transmembrane domains described below.

The transmembrane domain can be obtained from a natural or recombinant source. When the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. In one aspect, the transmembrane domain is capable of signaling to the intracellular domain(s) whenever the CAR has bound to the target. A transmembrane domain for a particular use in the present invention may comprise at least a transmembrane region(s), e.g., T cell receptor alpha, beta, or zeta chains, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 CD8 alpha, CD8 beta), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In some embodiments, a transmembrane domain may comprise at least a transmembrane region(s), e.g., KIRDS2, OX40, CD2, CD27, LFA-1 (CD 11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR , CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2R gamma, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA -6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 ( CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO- 3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG/Cbp, NKG2D, NKG2C and CD19.

The transmembrane domain may contain one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acids associated with the extracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 to 15 amino acids of the extracellular region) and/or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 to 15 amino acids of the intracellular region). In one aspect, the transmembrane domain is a protein that is associated with one of the other domains of the CAR used.

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In some instances, the transmembrane domain can be selected or modified by amino acid substitutions to eliminate the binding of such domains to transmembrane domains of the same proteins or other membrane surface proteins, for example, to minimize interactions with other members of the receptor complex. In one aspect, the transmembrane domain is capable of homodimerizing with another CAR on the cell surface of a CAR expressing cell, such as a CART cell. In another aspect, the amino acid sequence of the transmembrane domain can be modified or substituted in such a way as to minimize interactions with the binding domains of a native binding partner present in the same CAR expressing cell, eg, a CART cell.

In some cases, the transmembrane domain can be connected to the extracellular region of the CAR, for example, the antigen-binding domain of the CAR through a hinge, for example, a hinge from a human protein. For example, in one embodiment, the hinge may be a human Ig (immunoglobulin) hinge, such as an IgG4 hinge or a CD8 hinge. In one embodiment, the hinge or spacer comprises (e.g., consists of) the amino acid sequence of SEQ ID NO: 2. In one aspect, the transmembrane domain comprises (e.g., consists of) the transmembrane domain of SEQ ID NO: 6.

In one aspect, the hinge or spacer comprises an IgG4 hinge. For example, in one embodiment, the hinge or spacer comprises a hinge of the amino acid sequence SEQ ID NO: 3. In some embodiments, the hinge or spacer comprises a hinge encoded by the nucleotide sequence of SEQ ID NO: 14.

In one aspect, the hinge or spacer comprises an IgD hinge. For example, in one embodiment, the hinge or spacer comprises a hinge of the amino acid sequence SEQ ID NO: 4. In some embodiments, the hinge or spacer comprises a hinge encoded by the nucleotide sequence of SEQ ID NO: 15.

In one aspect, the transmembrane domain may be recombinant, in which case it contains predominantly hydrophobic residues such as leucine and valine. In one aspect, a triplet of phenylalanine, tryptophan and valine can be found at each end of the recombinant transmembrane domain.

Optionally, a short oligo- or polypeptide linker of 2 to 10 amino acids in length may form a link between the transmembrane domain and the cytoplasmic region of the CAR. The glycine-serine doublet is a particularly suitable linker. For example, in one aspect, the linker comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the linker is encoded by the nucleotide sequence of SEQ ID NO: 16.

In one aspect, the hinge or spacer comprises a KIR2DS2 hinge.

Chimeric TCR.

In one aspect, the antigen-binding domain described in the present invention, for example, an antibody or fragments of antibodies described in the present description, for example, as provided in table. 4 can be grafted into one or more T cell receptor (TCR) chain constant domains, eg, TCR alpha chain or TCR beta chain, to produce a chimeric TCR that specifically binds to the desired tumor antigen. Without being bound by theory, it is believed that chimeric TCRs signal through the TCR complex upon antigen binding. Such chimeric TCRs can be prepared by methods known in the art (e.g., Willemsen R.A. et al., Gene Therapy, 2000; 7: 1369-1377; Zhang T. et al., Cancer Gene Ther., 2004; 11: 487-496; Aggen et al., Gene Ther., 2012 Apr; 19(4):365-74).

In one embodiment, the NKR-CAR expressing cell described herein, for example, the KIR-CAR expressing cell described herein, further comprises a chimeric TCR. In an alternative embodiment, the NKR-CAR expressing cell described herein is administered in combination with a chimeric TCR expressing cell. In such embodiments, the antigen-binding domain of the chimeric TCR binds the same tumor antigen as NKR-CAR as described herein, e.g., KIR-CAR, or may bind a different tumor antigen than NKR-CAR as described herein, for example, KIR-CAR.

Split CAR.

In some embodiments, a cleaved CAR is used in a CAR-expressing cell. The split CAR approach is described in more detail in WO 2014/055442 and WO 2014/055657, incorporated herein by reference. Briefly, a cleaved CAR system comprises a cell expressing a first CAR containing a first antigen-binding domain and a costimulatory domain (e.g., 4-1BB), and the cell also expressing a second CAR containing a second antigen-binding domain and an intracellular signaling domain (e.g., CD3 zeta) . When the cell encounters the first antigen, the co-stimulatory domain is activated and the cell proliferates. When the cell encounters the second antigen, the intracellular signaling domain is activated and cytolytic activity against the cell begins to appear. Thus, a CAR-expressing cell is fully activated only in the presence of both antigens. In embodiments, the first antigen-binding domain recognizes a tumor antigen of the present invention, and the second antigen-binding domain recognizes another tumor antigen of the present invention.

Strategies for the regulation of chimeric antigen receptors.

There are many ways to regulate CAR activities. In some embodiments, to optimize the safety and efficacy of CAR therapy, a regulated CAR is desired, where the activity of the CAR can be controlled. For example, inducing apoptosis using, for example, a caspase fused to a dimerization domain (see, for example, Di et al., N. Engl. J. Med., 2011 Nov. 3; 365(18):1673-1683), can be used as a safety switch in the CAR therapy of the present invention. In another example, CAR-expressing cells can also express an inducible caspase 9 molecule (i-caspase 9) that, when administered with a dimerization-promoting drug (e.g. caspase 9 and cell apoptosis The n-caspase 9 molecule contains a chemical inducer dimerization (CID) binding domain that mediates dimerization in the presence of CID. acid other than the CAR-coding vector(s).In some cases, the n-caspase 9 molecule is encoded by the same nucleic acid molecule as the coding CAR vector.n-caspase 9 can provide a safe switch, eliminating any toxicity of CAR-expressing cells.See, e.g., Song et al., Cancer Gene Ther., 2008;15(10):667-75; Trial ibid. NCT02107963 and Di Stasi et al., N. Engl. J. Med., 2011; 365:1673-83.

Alternative strategies for regulating CAR therapy of the present invention include the use of small molecule compounds or antibodies that inactivate or turn off CAR activity, eg by removing CAR expressing cells, eg by displaying antibody-dependent cellular cytotoxicity (ADCC). For example, the CAR-expressing cells of the present invention can also express an antigen that recognizes molecules capable of inducing cell death, such as ADCC or complement-induced cell death. For example, the CAR-expressing cells of the present invention can also express a receptor that can be targeted by an antibody or antibody fragment. Examples of such receptors include EpCAM, VEGFR, integrins (e.g., ave3, α4, αΓ3/ _4β3 , α4β7, α5β1, ave3, av integrins), members of the TNF receptor superfamily (e.g., TRAIL-R1, TRAIL-R2), PDGF receptor, interferon receptor, folate receptor, GPNMB, ICAM-1, HLA-DR, CEA, CA-125, MUC1, TAG-72, IL-6 receptor, 5T4, GD2, GD3, CD2, CD3, CD4, CD5, CD11, CD11a /LFA-l, CD15, CD18/ITGB2, CD19, CD20, CD22, CD23/IgE receptor, CD25, CD28, CD30, CD33, CD38, CD40, CD41, CD44, CD51, CD52, CD62 l, CD74, CD80, CD125 , CD147/basidgin, CD152/CTLA-4, CD154/CD40L, CD195/CCR5, CD319/SLAMF7, and EGFR, and truncated variants thereof (e.g., variants that retain one or more extracellular epitopes but lack one or more regions in the cytoplasmic domain).

For example, the CAR-expressing cell described herein can also express a truncated epidermal growth factor receptor (EGFR) that lacks signal transduction capability but retains an epitope that recognizes molecules capable of inducing ADCC, such as cetuximab (ERBITUX®), thus, administration of cetuximab induces ADCC and subsequent depletion of CAR-expressing cells (see, for example, WO 2011/056894 and Jonnalagadda et al., Gene Ther., 2013; 20(8)853-860). Another strategy involves the expression of a very compact marker/suicide gene that combines target epitopes from CD32 and CD20 antigens in the CAR expressing cells described herein, which binds to rituximab resulting in selective depletion of CAR expressing cells, e.g., via ADCC ( see, for example, Philip et al., Blood 2014; 124(8)1277-1287). Other methods of depleting CAR-expressing cells described herein include administering campas, an anti-CD52 monoclonal antibody that selectively binds and directs mature lymphocytes, eg, CAR-expressing cells, down a pathway of destruction, eg, by induction of ADCC. In other embodiments, a CAR-expressing cell can be targeted using a CAR ligand, such as an anti-idiotypic antibody. In some embodiments, the anti-idiotypic antibody can induce effector cell activity, such as ADCC or ADC activity, thereby reducing the number of CAR-expressing cells. In other embodiments, a CAR ligand, eg, an anti-idiotypic antibody, can be coupled to an agent that induces cell cytolysis, eg, a toxin, thereby reducing the number of CAR-expressing cells. Alternatively, the CAR molecules themselves can be configured in such a way that activity can be controlled, such as being turned on and off, as described below.

In other embodiments, the CAR expressing cell described herein can also express a target protein recognized by the T cell depleting agent. In one embodiment, the target protein is CD20 and the T cell depleting agent is an anti-CD20 antibody such as rituximab. In such an embodiment, the T cell depleting agent is administered as a single dose if reduction or elimination of a CAR expressing cell is desired, for example, to attenuate CAR-induced toxicity. In other embodiments, the T cell depleting agent is an anti-CD52 antibody, such as alemtuzumab, as described in the examples herein.

In other embodiments, a regulated CAR (RCAR) comprises a set of polypeptides, typically two in the simplest embodiments, in which the components of a standard CAR as described herein, such as an antigen-binding domain and an intracellular signaling domain, are distributed on separate polypeptides or representatives. In some embodiments, the set of polypeptides includes a dimerization switch that, in the presence of a dimerization-inducing molecule, can bind polypeptides to each other, for example, can bind an antigen-binding domain to an intracellular signaling domain. Further description and illustrative configurations of such adjustable CARs are provided herein and in WO 2015/090229, which is hereby incorporated by reference in its entirety.

In one aspect, RCAR comprises two polypeptides or components: 1) an intracellular signaling component comprising an intracellular signaling domain, eg, a primary intracellular signaling domain as described herein, and a first switching domain; 2) an antigen-binding component comprising an antigen-binding domain, for example, that specifically binds to a tumor antigen of the present invention as described herein, and a second switch domain. Optionally, RCAR contains a transmembrane domain described in the present invention. In one embodiment, the transmembrane domain may be located on an intracellular signaling component, on an antigen binding component, or on both. (Unless otherwise noted, when RCAR components or elements are described herein, the order may be as provided, but other kinds of order are also included. In other words, in one embodiment, the order is as specified in the text, but in in other embodiments, the order may be different, for example, the order of elements on one side of the transmembrane region may be different from the example, for example, the location of the switch domain relative to the intracellular signaling domain may be different, for example, be reversed).

In one embodiment, the first and second switch domains may form an intracellular or extracellular dimerization switch. In one embodiment, the dimerization switch may be a homodimerization switch, such as when the first and second switch domain are the same, or a heterodimerization switch, such as when the first and second switch domain are different from each other.

In embodiments, an RCAR may comprise a multiple switch. The multiple switch may comprise heterodimerization switch domains or homodimerization switch domains. A multiple switch contains a plurality of, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 switch domains, independent of a first component, such as an antigen-binding component, and a second component, such as an intracellular signaling component. In one embodiment, the first component may comprise a plurality of first switching domains, eg, FKBP-based switching domains, and the second component may comprise a plurality of second switching domains, eg, FRB-based switching domains. In one embodiment, the first component may comprise a first and second switch domain, such as an FKBP-based switch domain and an FRB-based switch domain, and the second component may comprise a first and second switch domain, such as an FKBP-based switch domain and a switch domain. FRB based.

In one embodiment, the intracellular signaling component comprises one or more intracellular signaling domains, such as a primary intracellular signaling domain and one or more costimulatory signaling domains.

In one embodiment, the antigen binding component may comprise one or more intracellular signaling domains, such as one or more co-stimulatory signaling domains. In one embodiment, the antigen binding component comprises a constellation of, e.g., 2 or 3 co-stimulatory signaling domains as described herein, e.g., selected from 4-1BB, CD28, CD27, ICOS, and OX40, and in embodiments does not contain a primary intracellular signaling domain . In one embodiment, the antigen-binding component comprises the following co-stimulatory signaling domains in the extracellular to intracellular direction: 4-1BB-CD27; 4-1BB-CD27; CD27-4-1BB; 4-1BB-CD28; CD28-4-1BB; OX40CD28; CD28-OX40; CD28-4-1BB or 4-1BB-CD28. In such embodiments, the intracellular binding component comprises a CD3 zeta domain. In one such embodiment, the RCAR comprises (1) an antigen-binding component comprising an antigen-binding domain, a transmembrane domain and two costimulatory domains, and a first switch domain, and (2) an intracellular signaling domain containing a transmembrane domain or a membrane anchoring domain, and at least one primary intracellular signaling domain, and a second switching domain.

An embodiment provides a CAR where the antigen binding component is not anchored to the surface of the CAR cell. This allows the cell to contain an intracellular signaling component that is suitably paired with one or more antigen-binding domains without transforming the cell with a sequence encoding the antigen-binding component. In such embodiments, the RCAR comprises: 1) an intracellular signaling component comprising: a first switching domain, a transmembrane domain, an intracellular signaling domain, such as a primary intracellular signaling domain and a first switching domain, and 2) an antigen-binding component comprising: an antigen-binding domain and a second switching domain. a domain where the antigen-binding component does not contain a transmembrane domain or a membrane anchoring domain, and optionally does not contain an intracellular signaling domain. In some embodiments, the RCAR may further comprise 3) a second antigen-binding component comprising: a second antigen-binding domain, e.g., a second antigen-binding domain that binds an antigen other than an antigen that binds the antigen-binding domain, and a second switch domain.

Also disclosed herein are RCARs wherein the antigen-binding component has bispecific activation and the ability to target. In this embodiment, the antigen-binding component may comprise a plurality of, e.g., 2, 3, 4, or 5 antigen-binding domains, e.g. scFvs, where each antigen-binding domain binds to a target antigen, e.g. different antigens or the same antigen, e.g. the same or different epitopes on the same antigen. In one embodiment, a plurality of antigen-binding domains are arranged in series, and optionally a linker or hinge region is located between each of the antigen-binding domains. Suitable linkers and hinge regions are described in the present description.

An embodiment relates to an RCAR with a configuration that allows proliferation to be switched. In this embodiment, the RCAR contains: 1) an intracellular signaling component comprising: optionally a transmembrane domain or a membrane anchoring domain; one or more co-stimulatory signaling domains, e.g., selected from 4-1BB, CD28, CD27, ICOS, and OX40, and a switch domain, and 2) an antigen-binding component comprising: an antigen-binding domain, a transmembrane domain, and a primary intracellular signaling domain, e.g., a CD3 domain -zeta, where the antigen-binding component does not contain a switch domain, or does not contain a switch domain that dimerizes with a switch domain on an intracellular signaling component. In one embodiment, the antigen-binding component does not contain a costimulatory signaling domain. In one embodiment, the intracellular signaling component comprises a switch domain from a homodimerization switch. In one embodiment, the intracellular signaling component comprises a first heterodimerization switch switching domain, and RCAR comprises a second intracellular signaling component that comprises a second heterodimerization switch switching domain. In such embodiments, the second intracellular signaling component contains the same intracellular signaling domains as the intracellular signaling component. In one embodiment, the dimerization switch is intracellular. In one embodiment, the dimerization switch is extracellular.

In any of the RCAR configurations described herein, the first and second switch domains comprise an FKBP-FRB based switch as described herein.

The present description also discloses cells containing the RCAR described in the present invention. Any cell that is engineered to express RCAR can be used as an RCARX cell. In one embodiment, the RCARX cell is a T cell and is referred to as a RCART cell. In one embodiment, the RCARX cell is an NK cell and is referred to as an RCARN cell.

Also disclosed herein are nucleic acids and vectors containing RCAR coding sequences. The sequence encoding the different RCAR elements can be located on the same nucleic acid molecule, eg the same plasmid, or the same vector, eg a viral vector, eg a lentiviral vector. In one embodiment, (i) the sequence encoding the antigen-binding component and (ii) the sequence encoding the intracellular signaling component may be contained on the same nucleic acid, eg, a vector. The production of the corresponding proteins can be obtained, for example, using separate promoters or using a bicistronic transcription product (which can result in the production of two proteins by cleavage of one translation product or by translation of two separate protein products). In one embodiment, the sequence encoding the cleavable peptide, for example, the P2A or F2A sequence is located

- 133 040145 between (i) and (ii). In one embodiment, the sequence encoding the IRES, for example,

EMCV or EV71 IRES, located between (i) and (ii). In these embodiments, (i) and (ii) are transcribed as a single RNA. In one embodiment, the first promoter is operably linked to (i) and the second promoter is operably linked to (ii) such that (i) and (ii) are transcribed as separate mRNAs.

Alternatively, the sequence encoding the different RCAR elements may be located on different nucleic acid molecules, eg different plasmids or vectors, eg a viral vector, eg a lentiviral vector. For example, (i) a sequence encoding an antigen-binding component may be contained in a first nucleic acid, such as a first vector, and (ii) a sequence encoding an intracellular signaling component may be contained in a second nucleic acid, such as a second vector.

Dimerization switches.

Dimerization switches may be non-covalent or covalent. In a non-covalent dimerization switch, the dimerization-inducing molecule promotes non-covalent interaction between the switching domains. In a covalent dimerization switch, the dimerization-inducing molecule promotes covalent interaction between the switching domains.

In one embodiment, the RCAR comprises a dimerization switch based on FKBP/FRAP or FKBP/FRB. FKBP12 (FKBP or FK506 binding protein) is a ubiquitous cytoplasmic protein that serves as the primary intracellular target for the natural immunosuppressive drug product, rapamycin. Rapamycin binds to FKBP and to the large PI3K homologue FRAP (RAFT, mTOR). FRB is a 93 amino acid FRAP region that is sufficient to bind to the FKBP-rapamycin complex (Chen J., Zheng X.F., Brown E.J. and Schreiber S.L., (1995) Identification of an 11-kDa FKBP12-rapamycin-binding domain within the 289-kDa FKBP12-rapamycin-associated protein and characterization of a critical serine residue. Proc. Natl. Acad. Sci. USA 92: 4947-51.)

In embodiments, a dimerization-inducing molecule, such as rapamycin or a rapamycin analog, can be used in a FKBP/FRAP-based switch, eg FKBP/FRB.

The amino acid sequence of FKBP is as follows:

DVPDYASLGGPSSPKKKRKVSRGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFD

SSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDV ELLKLETSY (SEQ ID NO: 43).

In embodiments, the FKBP switch domain may comprise a FKBP fragment having FRB binding ability, or a fragment or analog thereof in the presence of rapamycin or rapalogue, such as the underlined region of SEQ ID NO: 43, which is:

VQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWE

EGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPPHATLVFDVELLKLETS (SEQ ID NO:

44).

The amino acid sequence of the FRB is as follows:

ILWHEMWHEG LEEASRLYFG ERNVKGMFEV LEPLHAMMER GPQTLKETSF

NQAYGRDLME AQEWCRKYMK SGNVKDLTQA WDLYYHVFRRR ISK (SEQ ID NO: 45).

A FKBP/FRAP-based switch, e.g. FKBP/FRB as used herein, refers to a dimerization switch comprising: a first switch domain that contains an FKBP moiety or analogue thereof capable of binding to FRB or a moiety thereof, or analogue in the presence of rapamycin or rapalogue, e.g. RAD001, and has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the FKBP sequence of SEQ ID NO: 54 or 55, or differs by no more than 30, 25, 20, 15, 10, 5, 4, 3, 2 or 1 amino acid residue from it; and a second switch domain that contains a FRB fragment or analog thereof, having the ability to bind to FRB or a fragment or analog thereof in the presence of rapamycin or rapalog, and has at least 70, 75, 80, 85, 90, 95, 96, 97 , 98 or 99% identity with the sequence FRB SEQ ID NO: 56 or differs by no more than 30, 25, 20, 15, 10, 5, 4, 3, 2 or 1 amino acid residue from it. In one embodiment, the RCAR of the present invention contains one switch domain that contains the amino acid residues described in SEQ ID NO: 43 (or SEQ ID NO: 44) and one switch domain that contains the amino acid residues described in SEQ ID NO: 45.

In embodiments, the FKBP/FRB dimerization switch comprises a modified FRB switch domain that detects altered, e.g., increased complexation between an FRB-based switch domain, e.g., a modified FKBP-based switch FRB switch domain, and a dimerization-inducing molecule, e. rapamycin or rapalogue, such as RAD001. In one embodiment, the FRB modifying switch domain contains one or more mutations, e.g., 2, 3, 4, 5, 6,

- 134 040145

7, 8, 9, 10 or more selected from mutations at amino acid position(s) L2031, E2032, S2035, R2036, F2039, G2040, T2098, W2101, D2102, Y2105 and F2108, wherein the wild-type amino acid is mutated to any other natural amino acid. In one embodiment, the mutant FRB contains a mutation in E2032, where E2032 mutates to phenylalanine (E2032F), methionine (E2032M), arginine (E2032R), valine (E2032V), tyrosine (E2032Y), isoleucine (E2032I), e.g., SEQ ID NO: 46 or leucine (E2032L), e.g., SEQ ID NO: 47. In one embodiment, the mutant FRB contains a mutation in T2098, where T2098 mutates to phenylalanine (T2098F) or leucine (T2098L), e.g., SEQ ID NO: 48 In one embodiment, the mutant FRB contains a mutation in E2032 and in T2098, where E2032 mutates to any amino acid, and where T2098 mutates to any amino acid, e.g. T2098L, e.g., SEQ ID NO: 50. In one embodiment, the mutant FRB contains the E2032L and T2098L mutation, e.g., SEQ ID NO: 51.

Table 6

An exemplary FRB mutant having increased affinity for a dimerization-inducing molecule

Mutant FRB Amino acid sequence SEQ ID NO: Mutant E2032I ILWHEMWHEGLIEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGR DLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKTS 46 Mutant E2032L ILWHEMWHEGLLEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGR DLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKTS 47 Mutant T2098L ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGR DLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKTS 48 Mutant E2032, T2098 ILWHEMWHEGLXEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGR DLMEAQEWCRKYMKSGNVKDLXQAWDLYYHVFRRISKTS 49 Mutant E2032I, T2098L ILWHEMWHEGLIEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGR DLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKTS 50 Mutant E2032L, T2098L ILWHEMWHEGLLEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGR DLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKTS 51

Other suitable dimerization switches include a GyrB-GyrB dimerization switch, a gibberellin-based dimerization switch, a tag/binding agent dimerization switch, and a halo-tag/lock-tag (snap-tag) dimerization switch. With the guidance provided herein, one of ordinary skill in the art will appreciate such switches and the corresponding dimerization-promoting molecules.

Dimerization-inducing molecule.

The association between the switching domains is facilitated by a dimerization-inducing molecule. In the presence of a dimerization-inducing molecule, interaction or association between the switch domains provides for signaling between a polypeptide associated, for example, fused to the first switch domain and a polypeptide associated, for example, fused to the second switch domain. In the presence of non-limiting levels of a dimerization-inducing molecule, signal transduction is increased by 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 5 , 10, 50, 100 times, for example, as measured in the system described in the present invention.

Rapamycin and rapamycin analogs (referred to as rapalogues in some cases), for example, RAD001, can be used as dimerization-inducing molecules in the FKBP/FRB-based dimerization switch described herein. In one embodiment, the dimerization-inducing molecule can be selected from rapamycin (sirolimus), RAD001 (everolimus), zotarolimus, temsirolimus, AP-23573 (ridaphorolimus), biolimus, and AP21967. Additional rapamycin analogs suitable for use with FKBP/FRB-based dimerization switches are further described in the section entitled Combination Treatment Methods or in the subsection entitled Combination with a low dose mTOR inhibitor.

Co-expression of CAR with the chemokine receptor.

In embodiments, a CAR expressing cell, eg, an NKR-CAR expressing cell, eg, a KIR CAR expressing cell, as described herein further comprises a chemokine receptor molecule. Transgenic expression of the CCR2b or CXCR2 chemokine receptors in T cells increases migration to CCL2 or CXCL1 secreting solid tumors, including melanoma and neuroblastoma (Craddock et al., J. Immunother., 2010 Oct; 33(8):780-8 and Kershaw et al.,

- 135 040145

Hum. Gene Ther., 2002 Nov 1; 13(16):1971-80). Thus, without wishing to be bound by theory, it is believed that chemokine receptors expressed on CAR expressing cells that recognize chemokines secreted by tumors, e.g. antitumor efficacy of a CAR-expressing cell. The chemokine receptor molecule may comprise a natural or recombinant chemokine receptor or a chemokine-binding fragment thereof. A chemokine receptor molecule suitable for expression in a CAR expressing cell of the present invention includes the CXC chemokine receptor (e.g. CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6 or CXCR7), the CC chemokine receptor (e.g. CCR1, CCR2, , CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, or CCR11), the CX3C chemokine receptor (eg, CX3CR1), the XC chemokine receptor (eg, XCR1), or a chemokine-binding fragment thereof. In one embodiment, the chemokine receptor molecule to be expressed with the CAR of the present invention is selected based on the chemokine(s) secreted by the tumor. In one embodiment, the CAR-expressing cell described herein further comprises, for example, expresses the CCR2b receptor or the CXCR2 receptor. In one embodiment, the CAR of the present invention and the chemokine receptor molecule are on the same vector or are on two different vectors. In embodiments where the CAR of the present invention and the chemokine receptor molecule are on the same vector, the CAR and the chemokine receptor molecule are each under the control of two different promoters or are under the control of the same promoter.

Nucleic acid constructs encoding CAR.

The present invention also relates to nucleic acid molecules encoding one or more of the CAR constructs described herein. In one aspect, the nucleic acid molecule is provided as a messenger RNA transcript. In one aspect, the nucleic acid molecule is provided as a DNA construct.

In one embodiment, the nucleic acid molecule encoding the NKR-CAR of the present invention further comprises a nucleic acid sequence encoding an intracellular signaling domain or adapter molecule.

The present invention also relates to vectors into which the DNA of the present invention is introduced. Vectors derived from retroviruses such as lentivirus are suitable means for obtaining long-term gene transfer, as they allow for long-term, stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have an additional advantage over vectors derived from oncoretroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells such as hepatocytes. They also have the additional advantage of low immunogenicity. The retroviral vector may also be, for example, a retroviral gamma vector. The gamma retroviral vector may contain, for example, a promoter, a packaging signal (Ψ), a primer binding site (PBS), one or more (e.g., two) long terminal repeats (LTRs), and a transgene of interest, e.g., a gene encoding CAR. The gamma retroviral vector may not contain viral structural genes such as gag, pol and env. Exemplary gammaretrovirus vectors include murine leukemia virus (MLV), spleen necrosis virus (SFFV), and myeloproliferative sarcoma virus (MPSV), and vectors derived therefrom. Other gammaretroviral vectors are described, for example, in Tobias Maetzig et al., Gammaretroviral Vectors: Biology, Technology and Application Viruses. Jun 2011; 3(6): 677-713.

In another embodiment, the vector containing the nucleic acid encoding the desired CAR of the invention is an adenoviral vector (A5/35). In another embodiment, expression of CAR-encoding nucleic acids can be performed using transposons such as sleeping beauty, crisper, CAS9, and zinc finger nucleases. See below June et al., 2009 Nature Reviews Immunology, 9.10: 704-716, incorporated herein by reference.

Briefly, expression of natural or synthetic CAR-encoding nucleic acids is generally obtained by operably linking a nucleic acid encoding a CAR polypeptide or region thereof to a promoter and introducing the construct into an expression vector. Vectors may be suitable for replication and integration in eukaryotes. Representative cloning vectors contain transcription and translation terminators, initiation sequences, and promoters suitable for directing the expression of the desired nucleic acid sequence.

The expression constructs of the present invention can also be used for immunization and nucleic acid gene therapy using standard gene delivery protocols. Gene delivery methods are known in the art. See, for example, US Pat. Nos. 5,399,346; 5,580,859; In another embodiment, the invention relates to a gene therapy vector.

The nucleic acid can be cloned into various types of vectors. For example, nucleic

- 136 040145 acid can be cloned into a vector, including, but not limited to, a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe-forming vectors, and sequencing vectors.

In addition, the expression vector can be delivered to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY) and other virology and molecular biology textbooks. Viruses that are suitable as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, suitable restriction endonuclease recognition sites, and one or more selectable markers (e.g., WO 01/96584, WO 01/29058 and US Pat. No. 6,326,193) .

A number of virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a suitable platform for a gene delivery system. The selected gene can be introduced into a vector and packaged into retroviral particles using methods known in the art. The recombinant virus can then be isolated and delivered to the individual's cells in vivo or ex vivo. A number of retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. A number of adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.

Additional promoter elements, such as enhancers, regulate the transcription start frequency. As a rule, they are located in the region of 30-110 bp. upstream of the start site, although a number of promoters have recently been shown to also contain functional elements downstream of the start site. The spacing between promoter elements can often be varied such that promoter function is maintained when the elements are reversed or moved relative to each other. In the thymidine kinase (tk) promoter, the distance between the promoter elements can be increased up to 50 bp. in different directions before the activity begins to decline. Apparently, depending on the promoter, individual elements can function together or independently to activate transcription.

An example of a promoter that is capable of expressing a CAR transgene in a mammalian T cell is the EF1a promoter. The native EF1a promoter directs the expression of the alpha subunit of the elongation factor 1 complex, which ensures the enzymatic delivery of aminoacyl-tRNA to the ribosome. The EF1a promoter has been widely used in mammalian expression plasmids and has been shown to be effective in directing CAR expression from transgenes cloned into a lentiviral vector. See, for example, Milone et al., Mol. Ther., 17(8): 1453-1464 (2009). In one aspect, the EF1a promoter contains the sequence provided as SEQ ID NO: 11.

One example of a promoter is the cytomegalovirus (CMV) immediate early promoter sequence. Such a promoter sequence is a strong constitutive promoter sequence capable of directing high levels of expression of any polynucleotide sequence operably linked to it. However, other constitutive promoter sequences can also be used, including, but not limited to, simian virus 40 early promoter (SV40), mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, promoter MoMuLV, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, elongation factor 1α promoter, and human gene promoters such as, but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter . Moreover, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch capable of turning on expression of a polynucleotide sequence that is operably linked when such expression is desired, or turning off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

In one embodiment, the vector is a lentiviral vector. In one embodiment, the vector further comprises a promoter. In one embodiment, the promoter is the EF-1 promoter.

In one embodiment, the vector is an in vitro transcribable vector, such as a vector that transcribes the RNA of the nucleic acid molecule described in the present invention. In one embodiment, the nucleic acid sequence in the vector further comprises a poly(A) tail, eg a poly(A) tail as described herein, eg containing approximately 150 adenosine bases. In one embodiment, the nucleic acid sequence in the vector further comprises a 3'UTR.

Another example of a promoter is the phosphoglycerate kinase (PGK) promoter. In embodiments, a truncated PGK promoter may be desirable (e.g., a PGK promoter with one or more, e.g., 1, 2, 5, 10, 100, 200, 300, or 400 nucleotide deletions compared to the wild-type PGK promoter sequence ). The nucleotide sequences of exemplary PGK promoters are shown below.

PGK WT promoter.

ACCCCTCTCTCCAGCCACTAAGCCAGTTGCTCCCTCGGCTGACGGCTGCACGCGAGGCC

TCCGAACGTCTTACGCCTTGTGGCGCGCCCGTCCTTGTCCCGGGTGTGATGGCGGGGTGTGGGG

CGGAGGGCGTGGCGGGGAAGGGCCGGCGACGAGAGCCGCGCGGGACGACTCGTCGGCGATAACC

GGTGTCGGGTAGCGCCAGCCGCGCGACGGTAACGAGGGACCGCGACAGGCAGACCGCTCCCATGA

TCACTCTGCACCGCCGAAGGCAAATAGTGCAGGCCGTGCGGCGCTTGGCGTTCCTTGGAAGGGCT

GAATCCCCGCCTCGTCCTTCGCAGCGGCCCCCCGGGTGTTCCCATCGCCGCTTCTAGGCCCACT

GCGACGCTTGCCTGCACTTCTTACACGCTCTGGGTCCCAGCCGCGGCGACGCAAAGGGCCTTGG

TGCGGGTCTCGTCGGCGCAGGGACGCGTTTGGGTCCCGACGGAACCTTTTCCGCGTTGGGGTTG

GGGCACCATAAGCT (SEQ ID NO: 52)

Exemplary truncated PGK promoters.

PGK100:

ACCCCTCTCTCCAGCCACTAAGCCAGTTGCTCCCTCGGCTGACGGCTGCACGCGAGGCC

TCCGAACGTCTTACGCCTTGTGGCGCGCCCGTCCTTGTCCCGGGTGTGATGGCGGGGTG (SEQ ID NO: 53)

PGK200:

ACCCCTCTCTCCAGCCACTAAGCCAGTTGCTCCCTCGGCTGACGGCTGCACGCGAGGCC

TCCGAACGTCTTACGCCTTGTGGCGCGCCCGTCCTTGTCCCGGGTGTGATGGCGGGGTGTGGGG

CGGAGGGCGTGGCGGGGAAGGGCCGGCGACGAGAGCCGCGCGGGACGACTCGTCGGCGATAACC

GGTGTCGGGTAGCGCCAGCCGCGCGACGGTAACG (SEQ ID NO: 54)

PGK300:

ACCCCTCTCTCCAGCCACTAAGCCAGTTGCTCCCTCGGCTGACGGCTGCACGCGAGGCC

TCCGAACGTCTTACGCCTTGTGGCGCGCCCGTCCTTGTCCCGGGTGTGATGGCGGGGTGTGGGG

CGGAGGGCGTGGCGGGGAAGGGCCGGCGACGAGAGCCGCGCGGGACGACTCGTCGGCGATAACC

GGTGTCGGGTAGCGCCAGCCGCGCGACGGTAACGAGGGACCGCGACAGGCAGACCGCTCCCATGA

TCACTCTGCACCGCCGAAGGCAAATAGTGCAGGCCGTGCGGCGCTTGGCGTTCCTTGGAAGGGCT

GAATCCCCG (SEQ ID NO: 55)

PGK400:

ACCCCTCTCTCCAGCCACTAAGCCAGTTGCTCCCTCGGCTGACGGCTGCACGCGAGGCC

TCCGAACGTCTTACGCCTTGTGGCGCGCCCGTCCTTGTCCCGGGTGTGATGGCGGGGTGTGGGG

CGGAGGGCGTGGCGGGGAAGGGCCGGCGACGAGAGCCGCGCGGGACGACTCGTCGGCGATAACC

GGTGTCGGGTAGCGCCAGCCGCGCGACGGTAACGAGGGACCGCGACAGGCAGACCGCTCCCATGA

TCACTCTGCACCGCCGAAGGCAAATAGTGCAGGCCGTGCGGCGCTTGGCGTTCCTTGGAAGGGCT

GAATCCCCGCCTCGTCCTTCGCAGCGGCCCCCCGGGTGTTCCCATCGCCGCTTCTAGGCCCACT

GCGACGCTTGCCTGCACTTCTTACACGCTCTGGGTCCCAGCCG (SEQ ID NO: 56)

The vector may also contain, for example, a signal sequence for facilitating secretion, a polyadenylation signal and a transcription terminator (for example, from the bovine growth hormone (BGH) gene), an element for episomal replication and replication in prokaryotes (for example, origin SV40 and ColE1 or other known in the art), and/or selection elements (eg, ampicillin resistance gene and/or zeocin marker).

In order to evaluate the expression of a CAR polypeptide or regions thereof, the expression vector to be introduced into the cell may also contain a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from the cell population that has been attempted to be transfected or infected with viral vectors. In other aspects, the selectable marker may be carried on a separate stretch of DNA and used in a co-transfection procedure. Selectable markers and reporter genes can be flanked with suitable regulatory sequences to allow expression in host cells. Suitable selectable markers include, for example, antibiotic resistance genes such as neo and the like.

Reporter genes are used to identify likely transfected cells and to evaluate the functionality of regulatory sequences. In general, a reporter gene is a gene that is not contained or expressed by the recipient organism or tissue and which encodes a polypeptide whose expression is manifested by any easily detectable property, such as enzymatic activity. Reporter gene expression is assessed at an appropriate time after the introduction of DNA into recipient cells. Suitable reporter genes may include genes encoding a gene for luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (eg, Ui-Tei et al., 2000 FEBS Letters, 479: 79-82). Suitable expression systems are well known and may be prepared by known methods or obtained commercially. In general, the construct with the smallest 5' flanking region that exhibits the highest level of reporter gene expression is identified as the promoter. Such promoter regions can be linked to a reporter gene and used to evaluate means for the ability to modulate promoter-driven transcription.

In one embodiment, the vector may further comprise a nucleic acid encoding a second CAR, such as a second NKR-CAR, TCAR, or inhibitory CAR. In one embodiment, the second CAR contains an antigen-binding domain that binds to a target antigen, such as a tumor antigen, as described herein. In embodiments, the antigen bound by the first CAR is the same antigen that binds the second CAR. Alternatively, the antigen bound by the first CAR is a different antigen than that bound by the second CAR. For example, the first CAR binds to mesothelin, while the second CAR binds to another tumor antigen or an antigen present on a normal cell.

In one embodiment, the vector contains a nucleic acid encoding an NKRCAR of the present invention and a nucleic acid encoding a TCAR. In one embodiment, the TCAR contains an antigen-binding domain that binds a tumor antigen, eg, a tumor antigen other than that that binds NKR-CAR. In one embodiment, the TCAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the intracellular signaling domain comprises the primary intracellular signaling domain described herein, such as CD3-zeta or provided in Table 2, and optionally one or more costimulatory signaling domains described herein, for example, 4-1BB, CD28, CD27, ICOS or provided in table. 3.

In one embodiment, the vector contains a nucleic acid encoding an NKRCAR of the present invention and a nucleic acid encoding an inhibitory CAR. In one embodiment, the inhibitory CAR contains an antigen-binding domain that binds an antigen found on normal cells but not on malignant cells, eg, normal cells that express an antigen recognized by NKR-CAR. In one embodiment, the inhibitory CAR comprises an antigen-binding domain, a transmembrane domain, and an intracellular domain of an inhibitory molecule. For example, the intracellular domain of an inhibitory CAR may be the intracellular domain of PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine and TGFR beta.

In embodiments, the vector may contain two or more nucleic acid sequences encoding a CAR, e.g., an NKR-CAR as described herein, and a second CAR, e.g., a second NKR-CAR that inhibits a CAR, or a TCAR that specifically binds to an antigen, different from the antigen recognized by the first NKR-CAR. In such embodiments, two or more nucleic acid sequences encoding a CAR are encoded by a single nucleic acid molecule in the same reading frame and as a single polypeptide chain. In this aspect, two or more CARs may, for example, be separated by one or more peptide cleavage sites (eg, a self-cleavage site or an intracellular protease substrate).

In embodiments, a vector containing a nucleic acid sequence encoding the described NKR-CAR may further contain a nucleic acid sequence encoding an adapter molecule. In such embodiments, two or more nucleic acid sequences are encoded by a single nucleic acid molecule in the same reading frame and as a single polypeptide chain. In this embodiment, the NKR-CAR and the adapter molecule can be separated by one or more peptide cleavage sites (e.g., a self-cleavage site or an intracellular protease substrate) where the NKR-CAR, the adapter molecule, and the peptide cleavage site are in the same reading frame and encoded as a single polypeptide chain.

- 139 040145

Examples of peptide cleavage sites include the following, where GSG residues are optional:

T2A: (GSG) EGRGSLLTCGDVEENPGP (SEQ ID NO: 57)

P2A: (GSG) ATNFSLLKQAGDVEENPGP (SEQIDNO: 58)

E2A: (GSG) QCTNYALLKLAGDVESNPGP (SEQIDNO: 59)

F2A: (GSG) VKQTLNFDLLKLAGDVESNPGP (SEQIDNO: 60)

Methods for introducing and expressing genes in a cell are known in the art. With respect to an expression vector, the vector can easily be introduced into a host cell, eg, a mammalian cell, a bacterial cell, a yeast cell, or an insect cell, by any method in the art. For example, the expression vector can be transferred into the host cell by physical, chemical, or biological means.

Polynucleotides can be introduced into target cells by any of a number of different methods, for example, commercially available methods that include, but are not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) ( Harvard Instruments, Boston, Mass.) or Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg Germany), cationic liposome-mediated transfection using lipofection, polymer encapsulation, peptide-mediated transfection, or biolistic-based delivery systems. particles such as gene guns (see, for example, Nishikawa et al., Hum Gene Ther., 12(8):861-70 (2001).

Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing a cell containing vectors and/or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, vol. 1-4, Cold Spring Harbor Press, NY).

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

Chemical means of introducing a polynucleotide into a host cell include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, granules, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a carrier for in vitro and in vivo delivery is a liposome (eg, an artificial membrane vesicle).

In the case where a non-viral delivery system is used, an exemplary delivery vehicle is a liposome. For the introduction of nucleic acids into the host cell (in vitro, ex vivo or in vivo) provide for the use of lipid formulations. In another aspect, the nucleic acid may be linked to a lipid. The nucleic acid associated with the lipid can be encapsulated in the aqueous content of the liposome dispersed in the lipid bilayer of the liposome associated with the liposome through a binding molecule that is associated with the liposome and an oligonucleotide enclosed in the liposome, in a complex formed with the liposome dispersed in solution, containing a lipid, mixed with a lipid, combined with a lipid, contained in a suspension in a lipid, contained in or complexed with a micelle, or otherwise associated with a lipid. Associated with lipid, lipid/DNA or lipid/expression vector compositions are not limited to any particular structure in solution. For example, they can be contained in a bilayer structure in the form of micelles or with a folded structure. They may also simply be dispersed in solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances, which may be natural or synthetic lipids. For example, lipids include fat droplets that occur naturally in the cytoplasm, as well as a class of compounds that contain long chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

Suitable lipids can be obtained from commercial sources. For example, dimyristylphosphatidylcholine (DMPC) can be obtained from Sigma, St. Louis, MO; dicetyl phosphate (DCP) is available from K and K Laboratories (Plainview, NY); cholesterol (Choi) can be obtained from Calbiochem-Behring; Dimyristylphosphatidylglycerol (DMPG) and other lipids are available from Avanti Polar Lipids, Inc. (Birmingham, A.L.). The original solutions of lipids in chloroform or chloroform/methanol can be stored at approximately -20°C. Only chloroform is used as a solvent, as it evaporates much faster than methanol. Liposome is a general term that includes a number of monolayer and multilayer lipid carriers formed by the generation of closed lipid bilayer structures or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an internal aqueous environment.

- 140 040145

Multilayer liposomes contain multiple lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in excess aqueous solution. Lipid components undergo rearrangement before forming closed structures and trapping water and solutes between lipid bilayers (Ghosh et al., 1991 Glycobiology, 5:505-10). However, compositions are also contemplated which contain various structures in solution other than the usual vesicular structure. For example, lipids may allow micellar structure or exist only as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also provided.

Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose the cell to an inhibitor of the present invention, a number of assays can be performed to confirm the presence of a recombinant DNA sequence in a host cell. Such assays include, for example, molecular biological assays well known to those skilled in the art such as Southern and Northern blotting, RT-PCR, and PCR; biochemical assays, such as detecting the presence or absence of a particular peptide, for example, by immunological methods (ELISA types and Western blotting) or by the assays described herein to identify agents within the scope of the invention.

The present invention further relates to a vector containing a CAR-encoding nucleic acid molecule. In one aspect, the CAR vector can be directly transduced into a cell, such as an effector cell of the immune system, such as a T cell or NK cell. In one aspect, the vector is a cloning or expression vector, e.g., a vector including, but not limited to, one or more plasmids (e.g., expression plasmids, cloning vectors, minicircles, minivectors, double microchromosomes), retroviral and lentiviral vector designs. In one aspect, the vector is capable of expressing the CAR construct in effector cells of the mammalian immune system, eg, mammalian T cells or mammalian NK cells. In one aspect, the mammalian T cell is a human T cell.

RNA transfection.

Disclosed herein are methods for producing in vitro transcribed CAR RNA, such as NKR-CAR RNA. The present invention also relates to an RNA construct encoding NKRCAR that can be directly transfected into a cell. In one embodiment, the NKR-CAR encoding RNA construct further comprises a nucleic acid sequence encoding a TCAR of the present invention. The method of obtaining mRNA for use in transfection may include in vitro transcription (IVT) of the template with specially designed primers, followed by the addition of polyA to obtain a construct containing a non-translated 3'- and 5'-sequence (UTR), 5'-cap and/or internal a ribosome binding site (IRES), a nucleic acid to be expressed, and a poly(A) tail, typically 50-2000 bases long (SEQ ID NO: 35). The RNA obtained in this way can be efficiently transfected into various types of cells. In one aspect, the template contains NKR-CAR sequences.

In one aspect, NKR-CAR is encoded by a messenger RNA (mRNA). In one aspect, an mRNA encoding an NKR-CAR is introduced into a T cell to generate an NKR-CAR cell. In one aspect, an mRNA encoding an NKR-CAR is introduced into an NK cell to generate an NKR-CAR cell.

In one embodiment, the in vitro transcribed NKR-CAR RNA can be introduced into the cell in the form of transient transfection. RNA is produced by in vitro transcription using a polymerase chain reaction (PCR) derived template. DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using suitable primers and RNA polymerase. The DNA source may be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence, or any other suitable DNA source. In one embodiment, the desired in vitro transcription template is an NKR-CAR of the present invention. For example, a template for NKR-CAR RNA contains an extracellular region containing a single-chain variable domain of an anti-tumor antibody; hinge region, transmembrane domain (eg, KIR transmembrane domain). In one embodiment, the desired in vitro transcription template contains KIR-CAR and DAP12 on separate templates. In one embodiment, the desired in vitro transcription template contains KIR-CAR and DAP12 on the same template. The template for DAP12 contains a transmembrane domain and an intracellular region.

In one embodiment, the in vitro transcribed NKR-CAR RNA can be introduced into the cell in the form of transient transfection. RNA is produced by in vitro transcription using a polymerase chain reaction (PCR) derived template. DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using suitable primers and RNA polymerase. The DNA source may be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic sequence

- 141 040145 DNA strength or any other suitable source of DNA. In one embodiment, the desired in vitro transcription template is an NKR-CAR of the present invention. For example, a template for NKR-CAR RNA contains an extracellular region containing a single-chain variable domain of an anti-tumor antibody; hinge region, transmembrane domain (eg, KIR transmembrane domain). In one embodiment, the desired in vitro transcription template contains KIR-CAR and DAP12 on separate templates. In one embodiment, the desired in vitro transcription template contains KIR-CAR and DAP12 on the same template. The template for DAP12 contains a transmembrane domain and an intracellular region.

In one embodiment, the DNA to be used for PCR contains an open reading frame. The DNA may be from a naturally occurring DNA sequence from an organism's genome. In one embodiment, the nucleic acid may contain some or all of the 5' and/or 3' untranslated regions (UTRs). A nucleic acid may contain exons and introns. In one embodiment, the DNA to be used for PCR is a human nucleic acid sequence. In another embodiment, the DNA to be used for PCR is a human nucleic acid sequence containing the 5' and 3' UTRs. Alternatively, the DNA may be an artificial DNA sequence that is not normally expressed in a natural organism. An exemplary artificial DNA sequence is a DNA sequence that contains regions of genes that are fused with each other to form an open reading frame encoding a fusion protein. Sections of DNA that are doped with each other may come from a single organism or from more than one organism.

PCR is used to generate an in vitro transcription template for mRNA, which is used for transfection. PCR methods are well known in the art. Primers for use in PCR are designed to contain regions that are substantially complementary to regions of the DNA to be used as a PCR template. Substantially complementary, as used herein, refers to a nucleotide sequence where most or all of the bases in the primer sequence are complementary, or one or more bases are not complementary or mismatched. Substantially complementary sequences are capable of annealing or hybridizing to the intended target DNA under the annealing conditions used for PCR. Primers can be designed to be substantially complementary to any portion of the DNA template. For example, primers can be designed to amplify a portion of a nucleic acid that is normally transcribed in cells (open reading frame), including the 5' and 3' UTRs. Primers can also be designed to amplify a portion of a nucleic acid encoding a particular domain of interest. In one embodiment, the primers are designed to amplify the coding region of the human cDNA, including all or portions of the 5' and 3' UTRs. Primers suitable for PCR can be prepared by synthetic methods that are well known in the art. Forward primers are primers that contain a region of nucleotides that are substantially complementary to nucleotides on the DNA template that are located in the 3'-5' direction of the DNA sequence to be amplified. The 3'-5' direction is used herein to designate region 5 to the DNA sequence to be amplified, relative to the coding strand. Reverse primers are primers that contain a region of nucleotides that are substantially complementary to a double-stranded DNA template that is located 3'-5' from the DNA sequence to be amplified. The 3'-5' direction is used herein to designate the 3' region to the DNA sequence to be amplified relative to the coding strand.

Any DNA polymerase suitable for PCR can be used in the methods described herein. The reagents and polymerase are commercially available from a number of sources.

You can also use chemical structures with the ability to provide stability and/or efficiency of translation. Preferably the RNA contains 5'- and 3'-UTR. In one embodiment, the length of the 5'-UTR is from one to 3000 nucleotides. The length of the 5' and 3' UTR sequences to be added to the coding region can be varied in various ways, including but not limited to designing PCR primers that anneal to different regions of the UTR. Using this approach, one skilled in the art can modify the 5' and 3' UTR lengths necessary to obtain optimal translation efficiency after transfection of non-transcribed RNA.

The 5' and 3' UTRs may be natural, endogenous 5' and 3' UTRs for the nucleic acids of interest. Alternatively, UTR sequences that are not endogenous to the nucleic acid of interest can be added by introducing UTR sequences into the forward and reverse primers, or by any other modification of the template. The use of UTR sequences that are not endogenous to the nucleic acid of interest may be suitable for stability modification.

- 142 040145 and/or the efficiency of RNA translation. For example, it is known that AU-rich elements in 3'-UTR sequences can reduce mRNA stability. Thus, one can choose or design

3'-UTR to enhance the stability of the transcribed RNA based on the properties of the UTR, which are well known in the art.

In one embodiment, the 5'-UTRs may comprise the Kozak sequence of an endogenous nucleic acid. Alternatively, when a 5'-UTR that is not endogenous to the nucleic acid of interest is added to PCR as described above, the Kozak consensus sequence can be reconstructed by adding the 5'-UTR sequence. Kozak sequences can increase the efficiency of translation of some RNA transcripts, but do not appear to be necessary for all RNAs to ensure efficient translation. Kozak sequence requirements for many mRNAs are known in the art. In other embodiments, the 5'UTR may be the 5'UTR of an RNA virus whose RNA genome is stable in cells. In other embodiments, various analogs of nucleotides in the 3' or 5' UTR can be used to block endonuclease degradation of the mRNA.

To enable RNA synthesis from a DNA template without the need for gene cloning, a transcription promoter must be attached to the DNA template upstream of the sequences to be transcribed. When a sequence that functions as a promoter for RNA polymerase is added to the 5' end of the forward primer, the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame to be transcribed. In one preferred embodiment, the promoter is a T7 polymerase promoter, as described elsewhere in this specification. Other suitable promoters include, but are not limited to, the T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for the T7, T3 and SP6 promoters are known in the art.

In a preferred embodiment, the mRNA contains a 5' cap and a 3' poly(A) tail that determines ribosome binding, translation initiation, and cellular stability of the mRNA. On a circulating DNA template, such as plasmid DNA, RNA polymerase produces a large concatamer product that is not suitable for expression in eukaryotic cells. Transcription of plasmid DNA linearized at the 3'-UTR end results in a normal-sized mRNA that is not efficient in eukaryotic transfection even though it is polyadenylated after transcription.

On a linear DNA template, phage T7 RNA polymerase can extend the 3' end of the transcript after the last base of the template (Schenborn and Mierendorf, Nuc. Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J Biochem., 270:1485-65 (2003).

A common method for studying poly-A/T extensions in a DNA template is molecular cloning. However, the poly-A/T sequence integrated into plasmid DNA can cause plasmid instability, which is why plasmid DNA templates derived from bacterial cells are often heavily contaminated with deletions and other aberrations. This results in cloning methods that are not only labor intensive and time consuming, but often unreliable. Therefore, a method that allows the construction of DNA templates without 3' poly-A/T extension without cloning is highly desirable. A segment of a poly-A/T transcription DNA template can be obtained during PCR using a reverse primer containing a poly-T tail such as a 100T tail (SEQ ID NO: 31) (size can be 50-5000T (SEQ ID NO: 32 )), or after PCR by any other method, including, but not limited to, DNA ligation or in vitro recombination. Poly(A) tails also provide RNA stability and reduce its degradation. As a rule, the length of the poly(A) tail positively correlates with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is 100 to 5000 adenosines (SEQ ID NO: 33).

Poly(A) RNA tails can be further extended after in vitro transcription using a poly(A) polymerase such as E. coli poly(A) polymerase (E-PAP). In one embodiment, increasing the length of the poly(A) tail from 100 nucleotides to 300 to 400 nucleotides (SEQ ID NO: 34) results in an approximately twofold increase in RNA translation efficiency. In addition, the attachment of different chemical groups to the 3' end can increase the stability of the mRNA. Such attachment may contain modified/artificial nucleotides, aptamers and other compounds. For example, ATP analogs can be introduced into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase the stability of RNA.

5' caps also provide stability to RNA molecules. In a preferred embodiment, the RNA produced by the methods described herein contains a 5' cap. The 5' cap is prepared by methods known in the art and described herein (Cougot et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski et al., RNA, 7:1468-95 ( 2001); Elango et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

RNA produced by the methods described herein may also contain an internal ribosome binding site (IRES) sequence. The IRES sequence can be any viral, chromosomal, or artificial sequence that initiates cap-independent binding of the ribosome to the mRNA and facilitates the initiation of translation. Any solutes suitable for cell electroporation may be included, which may contain factors that facilitate cell permeability and viability, such as sugars, peptides, lipids, proteins, antioxidants, and surfactants.

RNA can be introduced into target cells by any of a number of different methods, for example, commercially available methods that include, but are not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) ( Harvard Instruments, Boston, Mass.) or Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg Germany), cationic liposome-mediated transfection using lipofection, polymer encapsulation, peptide-modified transfection, or biolistic-based delivery systems. particles such as a gene gun (see, for example, Nishikawa et al., Hum Gene Ther., 12(8):861-70 (2001).

Nucleic acid sequences encoding the desired molecules can be obtained by recombinant methods known in the art, such as, for example, screening libraries from cells expressing the gene, obtaining the gene from a vector known to contain it, or isolating directly from cells and tissues containing it by standard methods. Alternatively, the gene of interest can be obtained by synthesis rather than cloning.

Delivery methods are not based on viruses.

In some aspects, non-viral methods can be used to deliver a nucleic acid encoding a CAR, such as NKR-CAR or TCAR, as described herein, to a cell or tissue, or to an individual.

In some embodiments, the non-viral method includes the use of a transposon (also referred to as a transposable element). In some embodiments, a transposon is a piece of DNA that can insert itself into a site in the genome, such as a piece of DNA that is capable of self-replicating and inserting a copy of itself into the genome, or a piece of DNA that can be cut from a longer nucleic acid and inserted to another location in the genome. For example, a transposon contains a DNA sequence consisting of inverted repeats flanking genes for transposition.

Exemplary nucleic acid delivery methods using transposons include the Sleeping Beauty transposon system (SBTS) and the PiggyBac transposon system (PB). See, for example, Aronovich et al., Hum. Mol. Genet., 20.R1(2011):R14-20; Singh et al., Cancer Res., 15(2008):2961-2971; Huang et al., Mol. Ther., 16(2008):580-589; Grabundzija et al., Mol. Ther., 18(2010):1200-1209; Kebriaei et al., Blood. 122.21(2013):166; Williams, Molecular Therapy, 16.9(2008):151516; Bell et al., Nat. Protoc, 2.12(2007):3153-65 and Ding et al., Cell, 122.3(2005):473-83, which are all incorporated herein by reference.

SBTS includes two components: 1) a transposon containing the transgene, and 2) a source of the transposase enzyme. A transposase can move a transposon from a host plasmid (or other donor DNA) to a target DNA, such as the host cell's chromosome/genome. For example, a transposase binds to a carrier/donor DNA plasmid, cuts the transposon (containing the transgene(s)) from the plasmid, and introduces it into the genome of the host cell. See, for example, Aronovich et al. higher.

Illustrative transposons include the pT2 based transposon. See, for example, Grabundzija et al., Nucleic Acids Res., 41.3(2013):1829-47 and Singh et al., Cancer Res. 68.8(2008): 2961-2971, which are all incorporated herein by reference. Exemplary transposases include a Tc1/mariner type transposase, eg, SB10 transposase or SB11 transposase (a highly active transposase that can be expressed, for example, from the cytomegalovirus promoter). See, for example, Aronovich et al.; Kebriaei et al. and Grabundzija et al., which are all incorporated herein by reference.

The use of SBTS allows for efficient integration and expression of a transgene, for example a nucleic acid encoding a CAR of the present invention. Disclosed herein are methods for obtaining a cell, eg a T cell or NK cell, that stably expresses a CAR of the present invention, eg using a transposon system such as SBTS.

According to the methods described herein, in some embodiments, one or more nucleic acids, eg, plasmids, containing SBTS components are delivered to a cell (eg, a T or NK cell). For example, the nucleic acid(s) are delivered by standard nucleic acid delivery methods (eg, plasmid DNA), eg, by the methods described herein, eg, electroporation, transfection, or lipofection. In some embodiments, the implementation of the nucleic acid contains a transposon containing a transgene, for example, a nucleic acid encoding a CAR described in the present invention. In some embodiments, the nucleic acid comprises a transposon containing a transgene (eg, a nucleic acid encoding a CAR as described herein) as well as a nucleic acid sequence encoding a transposase enzyme. Other embodiments relate to a system with two nucleic acids, for example, a system based on double

- 144 040145 plasmids, for example, where the first plasmid contains a transposon containing a transgene, and the second plasmid contains a nucleic acid sequence encoding a transposase enzyme. For example, the first and second nucleic acids are co-delivered to the host cell.

In some embodiments, cells, e.g., T or NK cells, that express a CAR of the present invention are obtained using a combination of gene insertion using SBTS and gene editing using a nuclease (e.g., zinc finger nucleases (ZFN), effector transcription activator-like nucleases (TALEN), the CRISPR/Cas system, or reshaped homing endonuclease engineered meganucleases).

In some embodiments, the use of a non-viral delivery method allows cells, such as T or NK cells, to be reprogrammed and directly administered to an individual. The advantages of non-viral vectors include, but are not limited to, ease and relatively low cost of obtaining sufficient quantities to fit a patient population, stability during storage, and lack of immunogenicity.

In the case where a non-viral delivery system is used, an exemplary delivery vehicle is a liposome. For the introduction of nucleic acids into the host cell (in vitro, ex vivo or in vivo) provide for the use of lipid formulations. In another aspect, the nucleic acid may be linked to a lipid. The nucleic acid associated with the lipid can be encapsulated in the aqueous content of the liposome dispersed in the lipid bilayer of the liposome associated with the liposome through a binding molecule that is associated with the liposome and an oligonucleotide enclosed in the liposome, in a complex formed with the liposome dispersed in solution, containing a lipid, mixed with a lipid, combined with a lipid, contained in a suspension in a lipid, contained in or complexed with a micelle, or otherwise associated with a lipid. Associated with lipid, lipid/DNA or lipid/expression vector compositions are not limited to any particular structure in solution. For example, they can be contained in a bilayer structure in the form of micelles or with a folded structure. They may also simply be dispersed in solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances, which may be natural or synthetic lipids. For example, lipids include fat droplets that occur naturally in the cytoplasm, as well as a class of compounds that contain long chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

Suitable lipids can be obtained from commercial sources. For example, dimyristylphosphatidylcholine (DMPC) can be obtained from Sigma, St. Louis, MO; dicetyl phosphate (DCP) is available from K and K Laboratories (Plainview, NY); cholesterol (Choi) can be obtained from Calbiochem-Behring; Dimyristylphosphatidylglycerol (DMPG) and other lipids are available from Avanti Polar Lipids, Inc. (Birmingham, A.L.). The original solutions of lipids in chloroform or chloroform/methanol can be stored at approximately -20°C. Only chloroform is used as a solvent, as it evaporates much faster than methanol. Liposome is a general term that includes a number of monolayer and multilayer lipid carriers formed by the generation of closed lipid bilayer structures or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an internal aqueous environment. Multilayer liposomes contain multiple lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in excess aqueous solution. Lipid components undergo rearrangement before forming closed structures and trapping water and solutes between lipid bilayers (Ghosh et al., 1991 Glycobiology, 5:505-10). However, compositions are also contemplated which contain various structures in solution other than the usual vesicular structure. For example, lipids can be micellar or exist only as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also provided.

Cell sources.

Prior to propagation and genetic modification, a source of cells (eg, immune system effector cells, eg, T cells or NK cells) is obtained from the individual. The term subject is intended to include living organisms in which an immune response can be elicited (eg, mammals). Examples of individuals include humans, dogs, cats, mice, rats and their transgenic species. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors.

In certain embodiments of the present invention, any number of immune system effector cell lines (eg, T cells or NK cells) available in the art can be used. In certain embodiments of the present invention, T cells can be obtained from a unit of blood collected from an individual using any number of techniques known to one of skill in the art, such as Ficoll™ separation. In one preferred embodiment, cells are obtained from the individual's circulating blood by apheresis. Product

- 145 040145 apheresis typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes and platelets. In one embodiment, the cells harvested by apheresis can be washed to remove the plasma fraction and placed in a suitable buffer or medium for subsequent processing steps. In one aspect of the invention, the cells are washed with phosphate buffered saline (PBS). In an alternative embodiment, the wash solution does not contain calcium and may not contain magnesium, or may not contain many if not all of the divalent cations. The initial stages of activation in the absence of calcium lead to increased activation. As will be readily understood by those skilled in the art, the washing step can be carried out by methods known to those skilled in the art, such as using a semi-automatic flow through centrifuge (e.g., Cobe 2991 cell processor, Baxter CytoMate, or Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, cells can be resuspended in various biocompatible buffers such as, for example, Ca-free, Mg-free PBS, PlasmaLyte A, or other saline with or without buffer. Alternatively, unwanted components of the apheresis sample can be removed and the cells directly resuspended in culture media.

It has been found that the methods of use can use culture media conditions providing 5% or less, e.g. 2%, human AB serum and use known culture media conditions and formulations, e.g., such as those described in Smith et al., Ex vivo expansion of human T cells for adoptive immunotherapy using the novel Xeno-free CTS Immune Cell Serum Replacement, Clinical and Translational Immunology (2015) 4, e31; doi:10.1038/cti.2014.31.

In another embodiment, T cells are isolated from peripheral blood lymphocytes by erythrocyte lysis and monocyte depletion, eg, PERCOLL™ gradient centrifugation or flow elution centrifugation. You can further isolate a specific subset of T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+ and CD45RO+ T cells, by positive or negative selection methods. For example, in one embodiment, T cells are isolated by incubation with anti-CD3/anti-CD28 (e.g., 3x28) conjugated beads, such as DYNABEADS® M-450 CD3/CD28T, for a period of time sufficient to positively select for the desired T cells. In one embodiment, the time period is approximately 30 minutes. In a further embodiment, the time period is in the range of 30 minutes to 36 hours or more and all integer values in this range. In a further embodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred embodiment, the time period is 10 to 24 hours. In one aspect, the incubation time period is 24 hours. For the isolation of T cells from patients with leukemia, the use of longer incubation periods, such as 24 h, may increase cell yield. A longer incubation time can be used to isolate T cells in any situation where there are multiple T cells compared to other cell types, such as in the isolation of tumor infiltrating lymphocytes (TIL) from tumor tissue or in immunocompromised individuals. In addition, using longer incubation times may increase the efficiency of CD8+ T cell uptake. Thus, by simply reducing or increasing the time, T cell binding to CD3/CD28 beads can be achieved and/or increasing or decreasing the bead to T cell ratio (as described further herein) T cell subpopulations can be advantageously selected for or against in the moment of cultivation start or at any other time points during the process. Furthermore, by increasing or decreasing the ratio of anti-CD3 antibody and/or anti-CD28 antibody on a bead or other surface, a subset of T cells can be preferably selected for or against at the start of culture or at other desired time points. The person skilled in the art will appreciate that many selection steps can also be used in the context of the present invention. In certain embodiments, it may be desirable to carry out a selection method and use unselected cells in the activation and propagation method. Unselected cells can also be subjected to additional selection steps.

Enrichment of a population of T cells for negative selection can be carried out using a combination of antibodies directed to surface markers unique to the negatively selected cells. One method is cell sorting and/or selection by negative magnetic immune adhesion or flow cytometry, which uses a mixture of monoclonal antibodies directed to cell surface markers present on the negatively selected cells. For example, for enrichment in CD4 ⁺ cells by negative selection, the mixture of monoclonal antibodies typically contains antibodies against CD14, CD20, CD11b, CD16, HLA-DR and CD8. In certain embodiments, it may be desirable to enrich or positively select for regulatory T cells that typically express CD4+, CD25+, CD62L ^hl , GITR+ and FoxP3 ⁺ . Alternatively, in certain embodiments, regulatory T cells are depleted by anti-C25 antibody-conjugated beads or other similar selection method.

- 146 040145

The methods described herein may include, for example, selecting a specific subset of immune system effector cells, e.g. described in the present description. Preferably, the population of depleted regulatory T cells contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% CD25+ cells.

In one embodiment, regulatory T cells, eg, CD25+ T cells, are removed from the population using an anti-CD25 antibody or fragment thereof, or a CD25 binding ligand, IL-2. In one embodiment, the CD25 antibody, or fragment thereof, or CD25 binding ligand, is conjugated to a substrate, eg a bead, or otherwise applied to a substrate, eg a bead. In one embodiment, the CD25 antibody, or fragment thereof, is substrate-conjugated as described herein.

In one embodiment, regulatory T cells, eg, CD25+ T cells, are removed from the population using Miltenyi™ CD25 Depletion Reagent. In one embodiment, the ratio of cells to CD25 depletion reagent is 1e7 cells to 20 µl, or 1e7 cells to 15 µl, or 1e7 cells to 10 µl, or 1e7 cells to 5 µl, or 1e7 cells to 2 .5 µl, or from 1e7 cells to 1.25 µl. In one embodiment, for example, for regulatory T cells, for example, a CD25+ depletion of more than 500 million cells/ml is used. In a further aspect, a cell concentration of 600, 700, 800, or 900 million cells/mL is used.

In one embodiment, the immune system effector cell population to be depleted contains approximately 6x109 CD25+ T cells. In other aspects, the immune system effector cell population to be depleted contains approximately 1x109 to 1x10 ¹⁰ CD25+ T cells and any integer value within that range. In one embodiment, the resulting population of depleted regulatory T cells contains 2x109 regulatory T cells, eg, CD25+ cells or less (eg, 1x109, 5x108, 1x10 ⁸ , 5x10 ⁷ , 1x10 ⁷ or less CD25+ cells).

In one embodiment, regulatory T cells, eg, CD25+ cells, are removed from the population using a CliniMAC system with a depletion tube set, such as, for example, a 162-01 tube. In one embodiment, the CliniMAC system operates with a set depletion parameter such as, for example, DEPLETION2.1.

Without wishing to be bound by a particular theory, reducing the level of negative immune cell regulators (e.g., reducing the number of unwanted immune cells, e.g., T _REG cells) in an individual prior to apheresis or during production of a CAR expressing cell product may reduce the risk of relapse in the individual. For example, methods for depleting T _REG cells are known in the art. Methods for reducing TREQ cells include, but are not limited to, cyclophosphamide, anti-GITR antibody (an anti-GITR antibody described herein), CD25 depletion, and combinations thereof.

In some embodiments, the production methods include reducing (eg, depleting) T _REG cells to produce a CAR-expressing cell. For example, preparation methods include bringing a sample, such as an apheresis sample, into contact with an anti-GITR antibody and/or an anti-CD25 antibody (or fragment thereof, or a CD25 binding ligand), for example, to deplete T _REG cells to produce a CAR expressing cell product. (eg, T cells, NK cells).

In one embodiment, the subject is pre-treated with one or more therapies that lower T _REG cells prior to harvesting cells to produce a CAR expressing cell product, thereby reducing the individual's risk of relapse when treated with CAR expressing cells. In one embodiment, methods for reducing T _REG cells include, but are not limited to, administering to the individual one or more of cyclophosphamide, an anti-GITR antibody, CD25 depletion, or a combination thereof. Administration of one or more of cyclophosphamide, anti-GITR antibody, CD25 depletion, or a combination thereof may occur during or after infusion of the CAR-expressing cell product.

In one embodiment, the individual receives pre-treatment with cyclophosphamide prior to harvesting cells to obtain a CAR expressing cell product, thereby reducing the individual's risk of relapse when treated with CAR expressing cells. In one embodiment, the subject is pre-treated with an anti-GITR antibody prior to harvesting cells to obtain a CAR expressing cell product, thereby reducing the individual's risk of relapse when treated with CAR expressing cells.

In one embodiment, the cell population to be removed is not regulatory T cells or tumor cells, but cells that otherwise interfere with proliferation and/or function of CART cells, such as cells expressing CD14, CD11b, CD33 , CD15, or other markers expressed by possible immunosuppressive cells. In one embodiment, such cells are contemplated to be removed simultaneously with regulatory T cells and/or tumor cells, or after said depletion, or in another order.

The methods described in the present invention may include more than one selection step, such as more than one depletion step. Enrichment of a population of T cells by negative selection can be carried out, for example, with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method is cell sorting and/or selection by negative magnetic immune adhesion or flow cytometry, which uses a mixture of monoclonal antibodies directed to cell surface markers present on the negatively selected cells. For example, to enrich CD4+ cells through negative selection, a mixture of monoclonal antibodies may contain antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

The methods described herein may further include removing cells from a population that expresses a tumor antigen, e.g., a tumor antigen, that does not contain CD25, e.g., CD19, CD30, CD38, CD123, CD20, CD14, or CD11b, thereby providing a population of depleted regulatory T cells, eg CD25+ depleted and tumor antigen depleted cells, which are suitable for expression of a CAR, eg the CAR described herein. In one embodiment, tumor antigen-expressing cells are removed at the same time as regulatory T cells, such as CD25+ cells. For example, an anti-CD25 antibody or fragment thereof and an antibody against a tumor antigen or fragment thereof can be attached to the same substrate, for example, a bead that can be used to remove cells, or an anti-CD25 antibody or fragment thereof, or an antibody against a tumor antigen, or a fragment thereof can be attached to separate beads, the mixture of which can be used to remove cells. In other embodiments, the removal of regulatory T cells, eg, CD25+ cells, and the removal of tumor antigen-expressing cells are sequential and may occur, for example, in any order.

Also disclosed are methods that include removing cells from a population that express a checkpoint inhibitor, e.g., a checkpoint inhibitor described herein, e.g., one or more PD1+ cells, LAG3+ cells, and TIM3+ cells, to thereby provide a population of depleted regulatory T .beta.-cells, eg, CD25+ depleted cells and checkpoint inhibitor-depleted cells, eg, PD1+, LAG3+ and/or TIM3+ depleted cells. Exemplary checkpoint inhibitors include B7-H1, B7-1, CD160, P1H, 2B4, PD1, TIM3, CEACAM (e.g. CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, TIGIT, CTLA-4, BTLA and LAIR1. In one embodiment, checkpoint inhibitor-expressing cells are removed at the same time as regulatory T cells, such as CD25+ cells. For example, an anti-CD25 antibody or fragment thereof and an anti-checkpoint inhibitor antibody or fragment thereof can be bound to the same bead that can be used to remove cells, or an anti-CD25 antibody or fragment thereof and an anti-checkpoint inhibitor antibody or fragment thereof. can be associated with individual beads, a mixture of which can be used to remove cells. In other embodiments, the removal of regulatory T cells, eg, CD25+ cells, and the removal of inhibitor-expressing checkpoint cells is sequential and may occur, for example, in any order.

In one embodiment, a T cell population can be selected that expresses one or more of IFN-γ, TNFa, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10, IL- 13, granzyme B and perforin or other suitable molecules such as other cytokines. It is possible to determine methods for screening cell expression, for example, the methods described in PCT Publication No. WO 2013/126712.

To highlight the desired population of cells by positive or negative selection, the concentration of cells and the surface (for example, particles, such as granules) can be changed. In certain embodiments, it may be desirable to significantly reduce the volume in which the beads and cells are mixed with each other (eg, increase the concentration of cells) to ensure maximum contact between cells and beads. For example, in one embodiment, a concentration of 2 billion cells/mL is used. In one embodiment, a concentration of 1 billion cells/mL is used. In a further embodiment, more than 100 million cells/ml are used. In a further embodiment, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/mL is used. In yet another embodiment, a cell concentration of 75, 80, 85, 90, 95, or 100 million cells/mL is used. In further embodiments, concentrations of 125 or 150 million cells/mL may be used. The use of high concentrations can result in increased cell yield, cell activation and cell proliferation. In addition, the use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells, or from samples that contain many tumor cells (i.e., leukemic blood, tumor tissue, etc.). Such populations of cells may be of therapeutic value and their production is desirable. For example, the use of a high concentration of cells provides a more efficient selection of CD8+ T cells, which are usually characterized by a weaker expression of CD28.

In a related embodiment, it may be desirable to use lower cell concentrations. By significantly diluting the mixture of T cells and surfaces (eg, particles such as beads), interactions between particles and cells can be minimized. This results in the selection of cells that express high amounts of the desired antigens to bind to the particles. For example, CD4+ T cells express higher levels of CD28 and are more efficiently taken up compared to CD8+ T cells at dilute concentrations. In one embodiment, the cell concentration used is 5x10 ⁶ /ml. In other embodiments, the concentration used may be from about 1x10 ⁵ /ml to 1x10 ⁶ /ml and any integer value in this range.

In other embodiments, the cells can be incubated on a shaker for various periods of time at various speeds at 2-10°C or at room temperature.

T cells for stimulation can also be frozen after the washing step. Without wishing to be bound by theory, the step of freezing followed by thawing provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After a washing step that removes plasma and platelets, the cells can be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and useful in this context, one method involves using PBS containing 20% DMSO and 8% human serum albumin or culture media containing 10% dextran 40 and 5 % dextrose, 20% human serum albumin and 7.5% DMSO or 31.25% Plasmalyte A, 31.25% dextrose 5%, 0.45% NaCl, 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO or other suitable cell culture freezing media containing, for example, Hespan and PlasmaLyte A, the cells are then frozen to -80° C. at 1° per minute and stored in the gas phase in a liquid nitrogen storage tank. Other methods of controlled freezing can be used, as well as uncontrolled flash freezing at -20°C or in liquid nitrogen.

In certain embodiments, cryopreserved cells are thawed and washed as described herein and left to equilibrate for 1 hour at room temperature prior to activation by the methods of the present invention.

It is also within the context of the invention to collect blood samples or apheresis from an individual at a point in time before expanded cells as described herein may be needed. Thus, a source of cells to be expanded can be harvested at any desired time and desired cells, such as T cells, isolated and frozen for further use in T cell therapy for any possible disease or condition for which T cell therapy will be effective, such as the T cell therapy described herein. In one embodiment, a blood sample is taken or apheresis is performed from a primarily healthy individual. In certain embodiments, a blood sample is taken or apheresis is performed from a substantially healthy individual who is at risk of developing a disease but who has not yet developed a disease, and the cells of interest are isolated and frozen for further use. In certain embodiments, T cells can be expanded, frozen, and used later. In certain embodiments, samples are collected from the patient immediately after the diagnosis of a concurrent disease, as described herein, but prior to any treatment. In a further embodiment, the cells are isolated from a blood sample or by apheresis in an individual prior to any possible appropriate treatments, including, but not limited to, treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents such as such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunodestructive agents such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228 and radiation. These drugs inhibit calcium-dependent calcineurin phosphatase (cyclosporine and FK506) or inhibit p70S6 kinase, which is important for growth factor-induced signaling (rapamycin). (Liu et al., Cell, 66:807-815, 1991; Henderson et al., Immun., 73:316-321, 1991; Bierer et al., Curr. Opin. Immun., 5:763-773, 1993). In a further embodiment, cells are isolated from a patient and frozen for use at subsequent time points in combination with (e.g., before, concurrently, or after) bone marrow or stem cell transplantation, T-cell ablative therapy using chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In another embodiment, cells are isolated prior to use and may be frozen for use at subsequent time points for treatment following B-cell ablative therapy, such as agents

- 149 040145 which interact with CD20, such as Rituxan.

In a further embodiment of the present invention, T cells are obtained from a patient immediately following treatment, which leaves the subject with functional T cells. In this regard, it has been observed that after certain cancer treatments, in particular treatments with drugs that damage the immune system, immediately after treatment during the period when patients generally recover from treatment, the quality of the T cells obtained can be optimal or improved in terms of their ability to propagate ex vivo. Likewise, following ex vivo manipulation by the methods described herein, these cells may be in a preferred state for increased engraftment and expansion in vivo. Thus, in the context of the present invention, the collection of blood cells, including T cells, dendritic cells, or other hematopoietic stem cells, is contemplated during the recovery phase. In addition, in certain embodiments, a mobilization (e.g., GM-CSF mobilization) and conditioning regimen can be used to create conditions in an individual that favor repopulation, recycling, regeneration, and/or expansion of specific cell types, especially during a specific time window. after therapy. Exemplary cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

In one embodiment, immune system effector cells expressing a CAR molecule, such as the CAR molecule described herein, are obtained from an individual who has received a low immunostimulatory dose of an mTOR inhibitor. In one embodiment, a population of immune system effector cells, e.g., T cells to be engineered to express CAR, is harvested after a sufficient period of time, or after sufficient dosing with a low immunostimulatory dose of an mTOR inhibitor, such that the level of PD1-negative immune effector cells system, eg, T cells, or the ratio of PD1-negative immune system effector cells, eg, T-cells/PD1-positive immune system effector cells, eg, T cells, in or harvested from the individual was at least transiently increased.

In other embodiments, a population of immune system effector cells, e.g., T cells that have been or will be engineered to express CAR, can be treated ex vivo by contacting an amount of an mTOR inhibitor that increases the number of PD1-negative immune system effector cells, for example, T-cells or increases the ratio of PD1-negative effector cells of the immune system, for example, T-cells/PD1-positive effector cells of the immune system, for example, T-cells.

In one embodiment, the T cell population is diaglycerol kinase (DGK) deficient. DGK-deficient cells include cells that do not express DGK RNA or DGK protein or have reduced or inhibited DGK activity. DGK-deficient cells can be obtained through genetic approaches, for example, the introduction of RNA interference agents, such as siRNA, shRNA, miRNA to reduce or prevent the expression of DGK. Alternatively, DGK-deficient cells can be obtained by treatment with the DGK inhibitors described herein.

In one embodiment, the T cell population is Ikaros deficient. Ikaros-deficient cells include cells that do not express Ikaros RNA RNA or Ikaros protein or have reduced or inhibited Ikaros activity. preventing expression of Ikaros. Alternatively, Ikaros-deficient cells can be obtained by treatment with Ikaros inhibitors, such as lenalidomide.

In embodiments, the T cell population is DGK-deficient and Ikaros-deficient, eg, does not express DGK and Ikaros, or has reduced or inhibited DGK and Ikaros activity. Such DGK- and Ikaros-deficient cells can be obtained by any method described in the present invention.

In one embodiment, NK cells are obtained from an individual. In another embodiment, the NK cells are an NK cell line, for example, the NK-92 cell line (Conkwest).

Allogeneic effector cells of the immune system with CAR.

In the embodiments described herein, the immune system effector cell may be an allogeneic immune system effector cell, such as a T cell or NK cell. For example, the cell may be an allogeneic T cell, e.g., an allogeneic T cell that lacks expression of a functional T cell receptor (TCR) and/or human leukocyte antigen (HLA), e.g., HLA class I and/or HLA II class.

A T cell lacking a functional TCR can, for example, be engineered such that it does not express any functional TCR on its surface, engineered such that it does not express one or more subunits that contain a functional

- 150 040145 native TCR (e.g., engineered to not express (or have reduced expression of) TCR alpha, TCR beta, TCR gamma, TCR delta, TCR epsilon, and/or TCR zeta), or be designed in such a way that it produces very little functional TCR on its surface. Alternatively, the T cell may express a substantially damaged TCR, for example, by expressing mutated or truncated forms of one or more of the TCR subunits. The term substantially damaged TCR means that such a TCR does not elicit an adverse immune response in the host.

The T cell described herein can, for example, be engineered to not express functional HLA on its surface. For example, a T cell as described herein can be engineered such that cell surface expression of HLA, eg HLA class I and/or HLA class II, is reduced. In some aspects, the suppression of HLA can be carried out by reducing or eliminating the expression of beta-2 microglobulin (B2M).

In some embodiments, the implementation of the T cell may not contain a functional TCR and functional HLA, for example, HLA class I and/or HLA class II.

Modified T cells that lack functional TCR and/or HLA expression can be generated by any suitable means, including knocking out or knocking down one or more TCR or HLA subunits. For example, a T cell may involve TCR and/or HLA knockdown using siRNA, shRNA, evenly spaced short palindromic repeats (CRISPR), transcriptional activator-like effector nuclease (TALEN), or zinc finger endonuclease (ZFN). ).

In some embodiments, the allogeneic cell may be a cell that does not express or expresses the inhibitory molecule at low levels, for example, by any method described in the present invention. For example, the cell may be a cell that does not express or expresses an inhibitory molecule at low levels, for example, which can reduce the ability of a CAR-expressing cell to increase an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g. CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 , CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine and TGFR beta. Inhibition of the inhibitory molecule, for example by inhibiting the level of DNA, RNA, or protein, can optimize the performance of a CAR-expressing cell. In embodiments, an inhibitory nucleic acid can be used, e.g., an inhibitory nucleic acid, e.g., dsRNA, e.g., siRNA or shRNA, uniformly spaced short palindromic repeats (CRISPR), effector nuclease like transcription activators (TALEN) or zinc finger endonuclease (ZFN), for example, as described herein. siRNA and shRNA to inhibit TCR or HLA.

In some embodiments, TCR expression and/or HLA expression can be inhibited using an siRNA or shRNA that targets a TCR and/or HLA encoding nucleic acid and/or an inhibitory molecule of the present invention (e.g., PD1, PD- L1, PD-L2, CTLA4, TIM3, CEACAM (e.g. CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine and TGFR beta) in a cell.

Expression of siRNA and shRNA in T cells can be obtained using any conventional expression system, such as, for example, a lentivirus-based expression system.

Exemplary shRNAs that repress the expression of one or more TCR components are described, for example, in US Publication No. 2012/0321667. Exemplary siRNAs and shRNAs that repress the expression of HLA class I and/or HLA class II genes are described, for example, in US Publication No. 2007/0036773.

CRISPR to inhibit TCR or HLA.

CRISPR or CRISPR to TCR and/or HLA or CRISPR to inhibit TCR and/or HLA, as used herein, refers to a group of short palindromic repeats arranged in groups evenly spaced from each other, or a system containing such a group of repeats. As used herein, Cas refers to a CRISPR-associated protein. The CRISPR/Cas system refers to a system derived from CRISPR and Cas that can be used to silence or mutate a TCR and/or HLA gene and/or an inhibitory molecule of the present invention (e.g., PD1, PD-L1, PD- L2, CTLA4, TIM3, CEACAM (e.g. CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGFR beta).

Natural CRISPR/Cas systems are found in approximately 40% of sequenced eubacterial genomes and 90% of sequenced archaebacteria. Grissa et al. (2007) BMC Bioinformatics, 8:172. This system is a type of prokaryotic immune system that provides resistance to foreign genetic elements such as plasmids and phages and provides a form of acquired immunity. Barrangou et al. (2007) Science, 315: 1709-1712; Marragini et al. (2008) Science, 322: 18431845.

The CRISPR/Cas system has been modified for use in genome editing (silencing, amplification or alteration of specific genes) in eukaryotes such as mice or primates. Wiedenheft et al. (2012) Nature, 482: 331-8. This is done by introducing into a eukaryotic cell a plasmid containing the specifically designed CRISPR and one or more appropriate Cas.

The CRISPR sequence, sometimes referred to as the CRISPR locus, contains alternating repeats and spacers. In natural CRISPR, spacers typically contain sequences foreign to bacteria, such as a plasmid or phage sequence; in TCR and/or the HLA CRISPR/Cas system, spacers are derived from the TCR or HLA gene sequence.

RNA from the CRISPR locus is constitutively expressed and processed by Cas proteins into small RNAs. They contain a spacer flanked by a repeat sequence. RNA directs other Cas proteins to silence exogenous genetic elements at the RNA or DNA level. Horvath et al. (2010) Science, 327: 167-170; Makarova et al. (2006) Biology Direct, 1:7. Thus, spacers serve as templates for RNA molecules, similar to miRNAs. Pennisi, (2013) Science, 341: 833-836.

Because they occur naturally in many different types of bacteria, the precise locations of CRISPR and the structure, function, and number of Cas genes and their product vary slightly between species. Haft et al. (2005) PLoS Comput. Biol., 1: e60; Kunin et al. (2007) Genome Biol., 8: R61; Mojica et al. (2005) J. Mol. Evol., 60: 174-182; Bolotin et al., (2005) Microbiol., 151: 2551-2561; Pourcel et al. (2005) Microbiol., 151: 653-663 and Stern et al. (2010) Trends. Genet., 28: 335-340. For example, Cse proteins (a subtype of Cas, E. coli) (eg, CasA) form a functional complex, Cascade, that processes CRISPR RNA transcripts in the spacer-repeat units that Cascade contains. Browns et al. (2008) Science, 321: 960-964. In other prokaryotes, Cas6 processes the CRISPR transcript. For CRISPR-based phage inactivation in E. coli, Cascade and Cas3 are required, but not Cas1 or Cas2. Cmr proteins (Cas RAMP module) in Pyrococcus furiosus and other prokaryotes form a functional complex with small CRISPR RNAs that recognizes and cleaves complementary target RNAs. The simplest CRISPR system is based on the Cas9 protein, which is a nuclease with two active restriction sites, one for each strand of the double helix. The combination of Cas9 and modified CRISPR locus RNA can be used in a genome editing system. Pennisi, (2013) Science, 341: 833-836.

Thus, the CRISPR/Cas system can be used to edit the TCR and/or HLA gene (add or remove a base pair) or introduce a premature stop codon, thus reducing TCR and/or HLA expression. The CRISPR/Cas system can alternatively be used as RNAi, TCR and/or HLA gene knockout in a reversible manner. In a mammalian cell, for example, RNA can direct the Cas protein to the TCR and/or HLA promoter, sterically blocking the RNA polymerase.

It is possible to generate artificial CRISPR/Cas systems that inhibit TCR and/or HLA using technology known in the art, such as that described in US Publication No. 20140068797 and Cong (2013), Science, 339: 819-823. Other artificial CRISPR/Cas systems that are known in the art that inhibit TCR and/or HLA can also be made, such as those described in Tsai (2014), Nature Biotechnol., 32:6569-576, US Pat. No. 8,871,445, 8865406, 8795965, 8771945 and 8697359.

TALEN to inhibit TCR and/or HLA.

TALEN or TALEN to HLA and/or TCR or TALEN to inhibit HLA and/or TCR refers to a transcription activator-like effector nuclease, an artificial nuclease that can be used to edit the HLA and/or TCR gene, and/or an inhibitory molecule described in of the present invention (e.g. PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g. CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine and TGFR beta).

TALEN is produced artificially by fusing the TAL effector DNA binding domain with the DNA cleaving domain. Transcription activator-like effectors (TALE) can be designed to bind to any desired DNA sequence, including the HLA or TCR gene region. Combining the engineered TALE with a DNA cleaving domain can generate a restriction enzyme that is specific for any desired DNA sequence, including an HLA or TCR sequence. They can then be introduced into a cell where they can be used to edit the genome. Boch (2011), Nature Biotech., 29: 135-6 and Boch et al. (2009) Science, 326: 1509-12; Moscow et al. (2009) Science, 326: 3501.

TALE are proteins secreted by Xanthomonas bacteria. The DNA-binding domain contains a repetitive, highly conserved sequence of 33-34 amino acids in length, except for the 12th and 13th amino acids. These two positions are highly variable, showing a strong correlation with particular nucleotide recognition. Thus, they can be designed to bind to a desired DNA sequence.

To obtain TALEN, the TALE protein is fused with a nuclease (N), which is a wild type endonuclease or a FokI mutant endonuclease. Several mutations have been made in FokI for use in TALEN; for example, they improve the specificity or activity of the cleavage. Cermak et al. (2011) Nucl. Acids Res., 39: e82; Miller et al. (2011) Nature Biotech., 29: 143-8; Hockemeyer et al. (2011) Nature Biotech., 29: 731-734; Wood et al. (2011) Science, 333: 307; Doyon et al. (2010) Nature Methods, 8: 7479; Szczepek et al. (2007) Nature Biotech.? 25:786-793? and Guo et al. (2010) J. Mol. Biol., 200: 96.

The FokI domain functions as a dimer, requiring two constructs with unique DNA-binding domains to sites in the target genome with the correct orientation and spacing. The number of amino acid residues between the TALE DNA-binding domain and the FokI cleavage domain and the number of bases between the two separate TALEN binding sites appear to be important parameters for obtaining high levels of activity. Miller et al. (2011) Nature Biotech., 29: 143-8.

TALEN HLA or TCR can be used intracellularly to produce a double strand break (DSB). Mutation can occur at the site of a break if repair mechanisms mistakenly repair the break by attaching non-homologous ends. For example, a misrepair can introduce a frameshift mutation. Alternatively, foreign DNA can be introduced into the cell along with TALEN; depending on the foreign DNA sequences and chromosomal sequence, this process can be used to create a defect in the HLA or TCR gene, or introduce such a defect in the wild-type HLA or TCR gene, thereby downregulating HLA or TCR expression.

You can design TALEN specific to sequences in HLA or TCR by any method known in this field, including various schemes using modular components. Zhang et al. (2011) Nature Biotech. 29: 149-53; Geibler et al. (2011) PLoS ONE 6: e19509.

Nuclease zinc fingers for HLA and/or TCR inhibition.

ZFN or zinc finger nuclease or ZFN to HLA and/or TCR or ZFN to inhibit HLA and/or TCR refers to zinc finger nuclease, an artificial nuclease that can be used to edit the HLA and/or TCR gene and/or the inhibitory molecule described in of the present invention (e.g. PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g. CEACAM1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine and TGFR beta).

Like TALEN, ZFN contains a FokI nuclease domain (or a derivative thereof) fused to a DNA-binding domain. In the case of ZFN, the DNA binding domain contains one or more zinc fingers. Carroll et al. (2011) Genetics Society of America, 188: 773-782, and Kim et al. (1996) Proc. Natl. Acad. sci. USA, 93: 1156-1160.

The zinc finger is a small protein structural motif stabilized by one or more zinc ions. A zinc finger may contain, for example, Cys2His2 and may recognize an approximately 3 bp sequence. Various zinc fingers of known specificity can be combined to produce multi-finger polypeptides that recognize sequences of approximately 6, 9, 12, 15, or 18 bp in length. Various methods are available for selecting and assembling modules to produce zinc fingers (and combinations thereof) recognizing specific sequences, including phage display, yeast single-hybrid systems, bacterial single-hybrid and two-hybrid systems, and mammalian cells.

Like TALEN, ZFN must dimerize to cleave DNA. Thus, it is necessary for the ZFN pair to target nonpalindromic DNA regions. The two individual ZFNs must bind opposite strands of DNA with their nucleases at the correct distance from each other. Bitinaite et al. (1998) Proc. Natl. Acad. sci. USA 95: 10570-5.

Also similar to TALEN, ZFN can create a double-strand break in DNA, which can create a frameshift mutation when misrepaired, resulting in a decrease in the expression and amount of HLA and/or TCR in the cell. ZFN can also be used with homologous recombination to mutate in the HLA or TCR gene.

ZFNs specific for sequences in HLA and/or TCR can be constructed by any method known in the art. Cathomen et al. (2008) Mol. Ther., 16:1200-7; Guo et al., (2010) J. Mol. Biol., 400: 96; U.S. patent publication 2011/0158957 and U.S. Patent Publication. 2012/0060230.

Expression of telomerase.

Without wishing to be bound by any particular theory, in some embodiments, the therapeutic T cell has a short lifespan in the patient due to shortened telomerases in the T cell; thus, transfection with the telomerase genome can lengthen T cell telomeres and improve T cell maintenance in the patient. See Carl June, Adoptive T cell therapy for cancer in

- 153 040145 the clinic, Journal of Clinical Investigation, 117:1466-1476 (2007). Thus, in one embodiment, an effector cell of the immune system, eg, a T cell, ectopically expresses a telomerase subunit, eg, a telomerase catalytic subunit, eg TERT, eg hTERT. In some aspects, the present invention provides a method for producing a CAR-expressing cell, comprising contacting the cell with a nucleic acid encoding a telomerase subunit, eg a telomerase catalytic subunit, eg TERT, eg hTERT. The cell can be brought into contact with the nucleic acid before, simultaneously with, or after contact with the CAR-encoding construct.

In one aspect, the invention relates to a method for obtaining a population of immune system effector cells (eg, T cells, NK cells). In one embodiment, the method includes: providing a population of immune system effector cells (e.g., T cells or NK cells), bringing the population of immune system effector cells into contact with a nucleic acid encoding a CAR, and bringing the population of immune system effector cells into contact with a nucleic acid encoding a telomerase subunit, such as hTERT, under conditions that allow expression of CAR and telomerase.

In one embodiment, the nucleic acid encoding the telomerase subunit is DNA. In one embodiment, the nucleic acid encoding the telomerase subunit contains a promoter capable of directing the expression of the telomerase subunit.

In one embodiment, hTERT comprises the amino acid sequence of GenBank ID protein AAC51724.1 (Meyerson et al., hEST2, the Putative Human Telomerase Catalytic Subunit Gene, Is Up-Regulated in Tumor Cells and during Immortalization, Cell Volume 90, Issue 4, 22 August 1997, Pages 785-795) as listed below:

MPRAPRCRAVRSLLRSHYREVLPLATFVRRLGPQGWRLVQRGDPAAFRALVAQCLVCVP WDARPPPAAPSFRQVSCLKELVARVLQRLCERGAKNVLAFGFALLDGARGGPPEAFTTSVRSYL PNTVTDALRGSGAWGLLLRRVGDDVLVHLLARCALFVLVAPSCAYQVCGPPLYQLGAATQARPP PHASGPRRRLGCERAWNHSVREAGVPLGLPAPGARRRGGSASRSLPLPKRPRRGAAPEPERTPV GQGSWAHPGRTRGPSDRGFCWSPARPAEEATSLEGALSGTRHSHPSVGRQHHAGPPSTSRPPR PWDTPCPPVYAETKHFLYSSGDKEQLRPSFLLSSLRPSLTGARRLVETIFLGSRPWMPGTPRRL PRLPQRYWQMRPLFLELLGNHAQCPYGVLLKTHCPLRAAVTPAAGVCAREKPQGSVAAPEEEDT DPRRLVQLLRQHSSPWQVYGFVRACLRRLVPPGLWGSRHNERRFLRNTKKFISLGKHAKLSLQE LTWKMSVRGCAWLRRSPGVGCVPAAEHRLREEILAKFLHWLMSVYWELLRSFFYVTETTFQKN RLFFYRKSVWSKLQSIGIRQHLKRVQLRELSEAEVRQHREARPALLTSRLRFIPKPDGLRPIVN MDYWGARTFRREKRAERLTSRVKALFSVLNYERARRPGLLGASVLGLDDIHRAWRTFVLRVRA QDPPPELYFVKVDVTGAYDTIPQDRLTEVIAS IIKPQNTYCVRRYAWQKAAHGHVRKAFKSHV STLTDLQPYMRQFVAHLQETSPLRDAWIEQSSSLNEASSGLFDVFLRFMCHHAVRIRGKSYVQ CQGIPQGSILSTLLCSLCYGDMENKLFAGIRRDGLLLRLVDDFLLVTPHLTHAKTFLRTLVRGV PEYGCWNLRKTWNFPVEDEALGGTAFVQMPAHGLFPWCGLLLDTRTLEVQSDYSSYARTSIR ASLTFNRGFKAGRNMRRKLFGVLRLKCHSLFLDLQV NSLQTVCTNIYKILLLQAYRFHACVLQL PFHQQVWKNPTFFLRVISDTASLCYSILKAKNAGMSLGAKGAAGPLPSEAVQWLCHQAFLLKLT RHRVTYVPLLGSLRTAQTQLSRKLPGTTLTALEAAANPALPSDFKTILD (SEQ ID NO: 61)

In one embodiment, hTERT contains a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 61. In one embodiment, hTERT contains the sequence of SEQ ID NO: 61. In one embodiment, hTERT contains a deletion (eg, no more than 5, 10, 15, 20, or 30 amino acids) at the N-terminus, C-terminus, or both. In one embodiment, hTERT contains a transgenic amino acid sequence (eg, no more than 5, 10, 15, 20, or 30 amino acids) at the N-terminus, C-terminus, or both.

In one embodiment, hTERT is encoded by a nucleic acid sequence with GeneBank accession number AF018167 (Meyerson et al., hEST2, the Putative Human Telomerase Catalytic Subunit Gene, Is Up-Regulated in Tumor Cells and during Immortalization, Cell Volume 90, Issue 4, 22 August 1997, Pages 785-795):

- 154 040145 caggcagcgt ggtcctgctg cgcacgtggg aagccctggc cccggccacc cccgcgatgc

61 cgcgcgctcc ( ccgctgccga gccgtgcgct ccctgctgcg cagccactac cgcgaggtgc 121 tgccgctggc cacgttcgtg cggcgcctgg ggccccaggg ctggcggctg gtgcagcgcg 181 gggacccggc ggctttccgc gcgctggtgg cccagtgcct ggtgtgcgtg ccctgggacg 241 cacggccgcc ccccgccgcc ccctccttcc gccaggtgtc ctgcctgaag gagctggtgg 301 cccgagtgct gcagaggctg tgcgagcgcg gcgcgaagaa cgtgctggcc ttcggcttcg 361 cgctgctgga cggggcccgc gggggcccc ccgaggcctt caccaccagc gtgcgcagct 421 acctgcccaa cacggtgacc gacgcactgc gggggagcgg ggcgtggggg ctgctgttgc 481 gccgcgtgggg cgacgacgtg ctggttcacc tgctggcacg ctgcgcgctc tttgtgctgg 541 tggctcccag ctgcgcctac caggtgtgcg ggccgccgct gtaccagctc ggcgctgcca 601 ctcaggcccg gccccgcca cacgctagtg gaccccgaag gcgtctggga tgcgaacggg 661 cctggaacca tagcgtcagg gaggccgggg tccccctggg cctgccagcc ccgggtgcga 721 ggaggcgcgg gggcagtgcc agccgaagtc tgccgttgcc caagaggccc aggcgtggcg 781 ctgcccctga gccggagcgg acgcccgttg ggcaggggtc ctgggcccac ccggggcagga 841 cgcgtggacc gagtgaccgt ggttctgtg tggtgtcacc tgccagaccc gccgaagaag 901 cccctcttt ggagggtgcg ctctctggca cgcgccactc ccacccatcc gtgggccgcc 961 agcaccacgc gggcccccca tccacatcgc ggccaccacg tccctgggac acgccttgtc 1021 ccccggtgta cgccgagacc aagcacttcc tctactcctc aggcgacaag gagcagctgc 1081 ggccctcctt cctactcagc tctctgaggc ccagcctgac tggcgctcgg aggctcgtgg 1141 agcatctt tctgggttcc aggccctgga tgccagggac tccccgcagg ttgccccgcc 1201 tgccccagcg ctactggcaa atgcggcccc tgtttctgga gctgcttggg aaccacgcgc 1261 agtgccccta cggggtgctc ctcaagacgc actgcccgct gcgagctgcg gtcaccccag 1321 cagccggtgt ctgtgcccgg gagaagcccc agggctctgt ggcggcccc gaggaggagg 1381 acacagaccc ccgtcgcctg gtgcagctgc tccgccagca cagcagcccc

tggcaggtgt

- 155 040145

1441 acggcttcgt gcgggcctgc ctgcgccggc tggtgcccc aggcctctgg ggctccaggc 1501 acaacgaacg ccgcttcctc aggaacacca agaagttcat ctccctgggg aagcatgcca 1561 agctctcgct gcaggagctg acgtggaaga tgagcgtgcg gggctgcgct tggctgcgca 1621 ggagcccagg ggttggctgt gttccggccg cagagcaccg tctgcgtgag gagatcctgg 1681 ccaagttcct gcactggctg atgagtgtgt acgtcgtcga gctgctcagg tctttctttt 1741 atgtcacgga gaccacgttt caaaagaaca ggctcttttt ctacgggaag agtgtctgga 1801 gcaagttgca aagcattgga atcagacagc acttgaagag ggtgcagctg cgggagctgt 1861 cggaagcaga ggtcaggcag catcgggaag ccaggcccgc cctgctgacg tccagactcc 1921 gcttcatccc caagcctgac gggctgcggc cgattgtgaa catggactac gtcgtgggag 1981 ccagaacgtt ccgcagagaa aagaggggccg agcgtctcac ctcgaggggtg aaggcactgt 2041 tcagcgtgct caactacgag cgggcgcggc gccccggcct cctgggcgcc tctgtgctgg 2101 gcctggacga tatccacagg gcctggcgca ccttcgtgct gcgtgtgcgg gccggacc 2161 cgccgcctga gctgtacttt gtcaaggtgg atgtgacggg cgcgtacgac accatccccc 2221 aggacaggct cacgggaggtc atcgccagca tcatcaaacc ccagaacacg tactgcgtgc 2281 gtcggtatgc cgtggtccag aaggccgccc atgggcacgt ccgcaaggcc ttcaagagcc 2341 acgtctctac cttgacagac ctccagccgt acatgcgaca gttcgtggct cacctgcagg 2401 agaccagccc gctgagggat gccgtcgtca tcgagcagag ctcctccctg aatgaggcca 2461 gcagtggcct cttcgacgtc ttcctacgct tcatgtgcca ccacgccgtg

cgcatcaggg

- 156 040145

2521 gcaagtccta cgtccagtgc caggggatcc cgcagggctc catcctctcc acgctgctct 2581 gcagcctgtg ctacggcgac atggagaaca agctgtttgc ggggattcgg ggggacggggc 2641 tgctcctgcg tttggtggat gatttcttgt tggtgacacc tcacctcacc cacgcgaaaa 2701 ccttcctcag gaccctggtc cgaggtgtcc ctgagtatgg ctgcgtggtg aacttgcgga 2761 agacagtggt gaacttccct gtagaagacg aggccctggg tggcacggct tttgttcaga 2821 tgccggccca cggcctattc ccctggtgcg gcctgctgct ggatacccgg accctggagg 2881 tgcagagcga ctactccagc tatgccggga cctccatcag agccagtctc accttcaacc 2941 gcggcttcaa ggctgggagg aacatgcgtc gcaaactctt tggggtcttg cggctgaagt 3001 gtcacagcct gtttctggat ttgcaggtga acagcctcca gacggtgtgc accaacatct 3061 acaagatcct cctgctgcag gcgtacaggt ttcacgcatg tgtgctgcag ctcccatttc 3121 atcagcaagt ttggaagaac cccacatttt tcctgcgcgt catctctgac acggcctccc 3181 tctgctactc catcctgaaa gccaagaacg cagggatgtc gctgggggcc aagggcgccg 3241 ccggccctct gccctccgag gccgtgcagt ggctgtgcca ccaagcattc ctgctcaagc 3301 tgactcgaca ccgtgtcacc tacgtgccac tcctggggtc actcaggaca gccgacgc 3361 agctgagtcg gaagctcccg gggacgacgc tgactgccct ggaggccgca gccaacccgg 3421 cactgccctc agacttcaag accatcctgg actgatggcc acccgcccac agccaggccg 3481 agagcagaca ccagcagccc tgtcacgccg ggctctacgt cccagggagg gaggggcggc 3541 ccacaccag gcccgcaccg ctgggagtct gaggcctgag tgagtgtttg gccgaggcct 3601 gcatgtccgg ctgaaggctg agtgtccggc tgaggcctga gcgagtgtcc agccaagggc 3661 tgagtgtcca gcacacctgc cgtcttcact tccccacagg ctggcgctcg gctccacccc 3721 agggccagct tttcctcacc aggagcccgg cttccactcc ccacatagga atagtccatc 3781 cccagattcg ccattgttca cccctcgccc tgccctcctt tgccttccac ccccaccatc 3841 caggtggaga ccctgagaag gaccctggga gctctgggaa tttggagtga ccaaaggtgt 3901 gccctgtaca caggcgagga ccctgcacct ggatgggggt ccctgtgggt caaattgggg 3961 ggaggtgctg tgggagtaaa atactgaata tatgagtttt tcagttttga

aaaaaaaaa

4021 aaaaaaa (SEQ ID NO: 62)

In one embodiment, hTERT is encoded by a nucleic acid with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:

- 157 040145

62. In one embodiment, hTERT is encoded by the nucleic acid of SEQ ID NO: 62.

Cell activation and reproduction.

Клетки можно активировать и размножать, как правило, способами, как описано, например, в патентах США 6352694, 6534055, 6905680, 6692964, 5858358, 6887466, 6905681, 7144575, 7067318, 7172869, 7232566, 7175843, 5883223, 6905874, 6797514, 6867041 and in US Patent Application Publication No. 20060121005.

Typically, T cells of the invention are propagated by bringing into contact with a surface-attached agent that stimulates the CD3/TCR complex-associated signal and a ligand that stimulates a co-stimulatory molecule on the surface of the T cells. In particular, T cell populations can be stimulated as described herein, such as by contacting an anti-CD3 antibody or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contacting a protein kinase C activator (e.g. , bryostatin) in combination with a calcium ionophore. To costimulate a helper molecule on the surface of T cells, a ligand is used that binds to the helper molecule. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions suitable to promote T cell proliferation. To stimulate the proliferation of CD4+ T cells or CD8+ T cells, an anti-CD3 antibody and an anti-CD28 antibody can be used. Examples of anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone, Besangon, France), which can be used as well as other methods well known in the art (Berg et al., Transplant Proc, 30(8):3975 -3977, 1998; Haanen et al., J. Exp. Med., 190(9):13191328, 1999; Garland et al., J. Immunol Meth., 227(1-2):53-63, 1999) .

In certain embodiments, the primary stimulatory signal and the costimulatory signal for the T cell may be provided by different protocols. For example, the means providing each signal may be in solution or associated with the surface. In the case of surface binding, the agents may be bound to the same surface (ie, in the cis configuration) or to separate surfaces (ie, in the trans conformation). Alternatively, one agent may be bound to the surface and another agent in solution. In one embodiment, the agent providing the co-stimulatory signal is cell surface bound and the primary activation signal providing agent is in solution or surface bound. In certain aspects, both agents may be in solution. In another embodiment, the agents may be in soluble form and then crosslinked to a surface, such as a cell expressing Fc receptors, or an antibody or other binding agent that will bind to the agents. In this regard, see, for example, US Patent Application Publication Nos. 20040101519 and 20060034810 for artificial antigen presenting cells (aAPC) which are contemplated for use in T cell activation and expansion in the present invention.

In one embodiment, the two agents are immobilized on the beads, on the same bead, ie. cis or on separate granules, ie. trance. As an example, the agent providing the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof, and the agent providing a costimulatory signal is an anti-CD28 antibody or an antigen-binding fragment thereof; and both agents are co-immobilized on the same bead in equimolar amounts. In one embodiment, a 1:1 ratio of each bead-bound antibody is used for expansion of CD4+ T cells and growth of T cells. In certain embodiments, the implementation of the present invention uses the ratio of antibodies against CD3:CD28 associated with the beads, so that you see an increase in expansion of T cells compared to reproduction observed using the ratio of 1:1. In one particular embodiment, an increase of about 1 to about 3 times is observed compared to reproduction observed using a 1:1 ratio. In one embodiment, the ratio of CD3:CD28 antibody bound to the beads is in the range of 100:1 to 1:100 and all integer values in this range. In one embodiment of the present invention, more of the anti-CD28 antibody is bound to the particles compared to the anti-CD3 antibody, i. e. the CD3:CD28 ratio is less than one. In certain embodiments, the ratio of anti-CD28 antibody to bead-bound anti-CD3 antibody is greater than 2:1. In one specific embodiment, a 1:100 ratio of CD3:CD28 of the bead-bound antibody is used. In another embodiment, a CD3:CD28 ratio of 1:75 of the bead-bound antibody is used. In a further embodiment, a CD3:CD28 ratio of 1:50 of the bead-bound antibody is used. In another embodiment, a CD3:CD28 ratio of 1:30 of the bead-bound antibody is used. In one preferred embodiment, a 1:10 CD3:CD28 ratio of the bead-bound antibody is used. In another embodiment, a CD3:CD28 ratio of 1:3 of the bead-bound antibody is used. In another embodiment, a CD3:CD28 ratio of 3:1 of the bead-bound antibody is used.

- 158 040145

To stimulate T cells or other target cells, particle to cell ratios of 1:500 to 500:1 and any integer values in this range can be used. As those skilled in the art can easily understand, the ratio of particles to cells may depend on the size of the particles relative to the target cell. For example, small granules may bind only a few cells, while larger granules may bind many cells. In certain embodiments, a ratio of cells to particles ranging from 1:100 to 100:1 and any integer values in this range can also be used to stimulate T cells, and in additional embodiments, the ratio is from 1:9 to 9:1 and any integer values in that range. The ratio of anti-CD3 antibody and anti-CD2 antibody bound particles to T cells that results in T cell stimulation can be varied as above, however certain preferred values include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2: 1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1 and 15:1, where one of the preferred ratios is at least 1:1 particles per T cell. In one embodiment, a particle to cell ratio of 1:1 or less is used. In one particular embodiment, the preferred particle:cell ratio is 1:5. In additional embodiments, the implementation of the ratio of particles to cells may vary depending on the day of stimulation. For example, in one embodiment, the ratio of particles to cells is from 1:1 to 10:1 on the first day, and additional particles are added to the cells every day or every other day thereafter for a period of up to 10 days in final ratios from 1:1 up to 1:10 (based on cell counts per day of addition). In one specific embodiment, the ratio of particles to cells is 1:1 on the first day of stimulation, and is adjusted to 1:5 on the third and fifth days of stimulation. In another embodiment, the particles are added every day or every other day to a final ratio of 1:1 on the first day and 1:5 on the third and fifth days of stimulation. In another embodiment, the ratio of particles to cells is 2:1 on the first day of stimulation and is adjusted to 1:10 on the third and fifth days of stimulation. In another embodiment, the particles are added every day or every other day to a final ratio of 1:1 on the first day, and 1:10 on the third and fifth days of stimulation. One skilled in the art will appreciate that a number of other relationships may be suitable for use in the present invention. In particular, the ratios vary with particle size and cell size and type. In one embodiment, the most typical ratios for use are approximately 1:1, 2:1, and 3:1 on the first day.

In further embodiments of the present invention, cells, such as T cells, are combined with agent-coated beads, then the beads and cells are separated, and then the cells are cultured. In an alternative embodiment, the agent-coated beads and cells are not separated but cultured together prior to culturing. In a further embodiment, the beads and cells are first concentrated by applying a force, such as the force of a magnetic field, which results in increased ligation of cell surface markers, thus inducing stimulation of the cells.

As an example, cell surface proteins can be ligated by bringing paramagnetic beads to which anti-CD3 and anti-CD28 antibodies (3x28 beads) are attached into contact with T cells. In one embodiment, cells (e.g., 104 to 109 T cells) and beads (e.g., DYNABEADS® M-450 CD3/CD28 T paramagnetic beads in a 1:1 ratio) are pooled in a buffer, such as PBS (no divalent cations). such as calcium and magnesium). Again, those skilled in the art will readily appreciate that any concentration of cells can be used. For example, the target cell may be very rare in the sample and may represent only 0.01% of the sample, or the entire sample (ie, 100%) may be the target cell of interest. Thus, any number of cells is contemplated within the context of the present invention. In certain embodiments, it may be desirable to significantly reduce the volume in which the particles and cells are mixed with each other (ie, increase the concentration of cells) to ensure maximum cell-particle contact. For example, in one aspect, a concentration of approximately 2 billion cells/mL is used. In another embodiment, more than 100 million cells/ml are used. In additional embodiments, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/mL is used. In yet another aspect, a cell concentration of 75, 80, 85, 90, 95, or 100 million cells/mL is used. In additional aspects, concentrations of 125 or 150 million cells/ml can be used. The use of high concentrations can lead to increased cell yield, cell activation and cell proliferation. In addition, the use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells. Such cell populations may be of therapeutic value, and in certain embodiments, their production is desirable. For example, the use of a high concentration of cells provides a more efficient selection of CD8+ T cells, which are usually characterized by a weaker expression of CD28.

In one embodiment, the nucleic acid transduced cells encode

- 159 040145 CAR, for example, NKR-CAR, described in the present invention, propagate, for example, by the method described in the present invention. In one embodiment, the cells are expanded in culture over a period of several hours (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18, 21 hours) to about 14 days (e.g., , 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days). In one embodiment, cells are propagated over a period of 4 to 9 days. In one embodiment, cells are propagated over a period of 8 days or less, such as 7, 6, or 5 days. In one embodiment, cells, for example, a NKRCAR cell as described herein, is propagated in culture for 5 days and the resulting cells are more active than the same cells propagated in culture for 9 days under the same culture conditions. . Activity can be determined, for example, by various functions of T cells, for example, proliferation, cytolysis of the target cell, production of cytokines, activation, migration, or combinations thereof. In one embodiment, for cells, e.g., NKR-CAR, cells of the present invention that are expanded for 5 days show at least a one, two, three, or four-fold increase in cell doubling when challenged with antigen compared to those the same cells that were propagated in culture for 9 days under the same cultivation conditions. In one embodiment, cells, e.g., cells expressing the NKR-CAR of the present invention, are expanded in culture for 5 days, and the resulting cells are characterized by higher production of pro-inflammatory cytokines, e.g., levels of IFN-γ and/or GM- CSF compared to the same cells that were propagated in culture for 9 days under the same culture conditions. In one embodiment, for cells, e.g., NKR-CAR cells of the present invention that have been propagated for 5 days, show an increase of at least one, two, three, four, five, ten times or more pg/ml the production of pro-inflammatory cytokines, eg IFN-γ and/or GM-CSF levels compared to the same cells that were expanded in culture for 9 days under the same culture conditions.

In one of the embodiments of the present invention, the mixture can be cultured for several hours (approximately 3 h) up to approximately 14 days, or any integer value of time in this interval. In another embodiment, the mixture can be cultured for 21 days. In another embodiment, the beads and T cells are co-cultured for about eight days. In another embodiment, the beads and T cells are co-cultured for 2-3 days. Multiple cycles of stimulation may also be desirable, such that the T cell culture time may be 60 days or more. Conditions suitable for culturing T cells include suitable media (eg, minimal culture media or RPMI 1640 or X-vivo 15, (Lonza) medium) which may contain factors necessary for proliferation and viability, including serum (eg, fetal bovine serum or human serum), interleukin 2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFe and TNF-α or any other cell growth supplements known to the person skilled in the art. Other cell growth additives include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetylcysteine and 2-mercaptoethanol. Media may include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15 and XVivo 20, Optimizer with added amino acids, sodium pyruvate and vitamins, or serum-free or supplemented with an appropriate amount of serum (or plasma), or a certain set of hormones and / or the amount of cytokine (s) sufficient for the growth and reproduction of T cells. Antibiotics, such as penicillin and streptomycin, are only administered to the experimental cultures, not to the cell cultures to be infused into the individual. The target cells are maintained under conditions necessary to support growth, eg at a specific temperature (eg 37° C.) and atmosphere (eg air plus 5% CO ₂ ).

In one embodiment, the cells are expanded in a suitable medium (e.g., the medium described in the present invention) that contains one or more interleukins, resulting in at least a 200-fold increase (e.g., 200, 250, 300, 350-fold) cells during a breeding period of 14 days, for example, as measured by the method described in the present invention, such as flow cytometry. In one embodiment, cells are propagated in the presence of IL-15 and/or IL-7 (eg, IL-15 and IL-7).

In embodiments, the methods described herein, e.g., methods for producing a CAR-expressing cell, comprise removing regulatory T cells, e.g., CD25+ T cells, from a cell population, e.g., using an anti-CD25 antibody or fragment thereof, or a CD25 binding ligand, IL-2. Methods for removing regulatory T cells, eg CD25+ T cells, from a population of cells are described herein. In embodiments, the methods, e.g., methods of preparation, further comprise bringing a cell population (e.g., a cell population in which regulatory T cells, such as CD25+ T cells, have been depleted, or a cell population that has previously been contacted with an anti-CD25 antibody , its fragment or CD25-160 040145 binding ligand) in contact with IL-15 and/or IL-7. For example, a cell population (eg, which has previously been contacted with an anti-CD25 antibody, a fragment thereof, or a CD25 binding ligand) is expanded in the presence of IL-15 and/or IL-7.

In some embodiments, a CAR-expressing cell described herein is contacted with a composition comprising an interleukin 15 (IL-15) polypeptide, an interleukin 15 receptor alpha (IL-15Ra) polypeptide, or a combination of an IL-15 polypeptide and an IL-15Ra polypeptide. eg hetIL-15 during production of a CAR expressing cell, eg ex vivo. In embodiments, a CAR-expressing cell as described herein is contacted with a composition comprising an IL-15 polypeptide during production of a CAR-expressing cell, eg, ex vivo. In embodiments, a CAR expressing cell as described herein is contacted with a composition comprising a combination of an IL-15 polypeptide and an IL-15 Ra polypeptide during production of a CAR expressing cell, eg ex vivo. In embodiments, a CAR-expressing cell as described herein is contacted with a composition containing hetIL-15 during production of a CAR-expressing cell, eg, ex vivo.

In one embodiment, a CAR-expressing cell as described herein is contacted with a composition containing hetIL-15 during ex vivo propagation. In one embodiment, a CAR expressing cell as described herein is contacted with a composition containing an IL-15 polypeptide during ex vivo propagation. In one embodiment, a CAR expressing cell as described herein is contacted with a composition comprising an IL-15 polypeptide and an IL-15Ra polypeptide during ex vivo propagation. In one embodiment, the implementation of bringing into contact leads to the survival and proliferation of the population of lymphocytes, such as CD8+ T cells.

T cells that have been stimulated for different times may have different characteristics. For example, typical blood mononuclear or peripheral blood products after apheresis contain a population of helper T cells (TH, CD4+), which is a population of more cytotoxic or suppressor T cells (TC, CD8+). Ex vivo expansion of T cells by stimulation of the CD3 and CD28 receptors results in a T cell population that is predominantly TH cells up to about days 8-9, while after about 8-9 days, the T cell population contains an increasing population of TC cells. Thus, depending on the goal of treatment, it may be advantageous to infuse the individual with a population of T cells containing predominantly TH cells. Similarly, if a subpopulation of TC cells specific for an antigen is isolated, it may be preferable to expand this subpopulation to a greater extent.

Furthermore, in addition to the CD4 and CD8 markers, other phenotypic markers are significantly altered, but largely reproducibly during the cell expansion process. Thus, this reproducibility allows for tailoring the activated T cell product for specific purposes.

Alternatively, or in combination with the methods described herein, methods and compositions are provided for one or more of: detecting and/or quantifying CAR-expressing cells (eg, in vitro or in vivo (eg, clinical monitoring)); expansion and/or activation of immune cells and/or CAR-directed selection, which include the use of a CAR ligand. In one exemplary embodiment, the CAR ligand is an antibody that binds to a CAR molecule, e.g., binds to the extracellular antigen-binding domain of CAR (e.g., an antibody that binds to an antigen-binding domain, e.g., an anti-idiotypic antibody, or an antibody that binds to an extracellular constant region). linking domain). In other embodiments, the CAR ligand is a CAR antigenic molecule (eg, a CAR antigenic molecule as described herein).

In one aspect, a method for detecting and/or quantifying CAR-expressing cells is described. For example, a CAR ligand can be used to detect and/or quantify CAR-expressing cells in vitro or in vivo (eg, clinical monitoring of CAR-expressing cells in a patient or a patient dosing regimen).

The method includes providing a CAR ligand (optionally a labeled CAR ligand, such as a CAR ligand that contains a label, bead, radioactive or fluorescent label);

acquisition of a CAR-expressing cell (eg, acquisition of a sample containing CAR-expressing cells, such as an industrial design or a clinical sample);

bringing the CAR-expressing cell into contact with the CAR ligand under conditions where binding occurs, thereby detecting the level (eg, number) of CAR-expressing cells present. Binding of a CAR expressing cell to a CAR ligand can be detected by standard methods such as FACS, ELISA, and the like.

- 161 040145

In another aspect, a method for expanding and/or activating cells (eg, effector cells of the immune system) is described.

The method includes providing a CAR-expressing cell (eg, a first CAR-expressing cell or a transiently CAR-expressing cell);

bringing said CAR expressing cell into contact with a CAR ligand, e.g., a CAR ligand as described herein) under conditions where expansion and/or proliferation of immune cells occurs, thereby obtaining an activated and/or expanded population of cells.

In certain embodiments, the CAR ligand is present (eg, is immobilized or attached to a substrate, eg, a non-natural substrate). In some embodiments, the substrate is a non-cellular substrate. The non-cellular substrate may be a solid support selected from, for example, a plate (eg, microtiter plate), membrane (eg, nitrocellulose membrane), matrix, chip, or bead. In embodiments, the CAR ligand is contained in the substrate (eg, on the surface of the substrate). The CAR ligand can be immobilized, attached, or linked covalently or non-covalently (eg, cross-linked) to a substrate. In one embodiment, the CAR ligand is attached (eg, covalently) to the bead. In the above embodiments, the immune cell population can be expanded in vitro or ex vivo. The method may further include culturing a population of immune cells in the presence of a CAR molecule ligand, for example, by any of the methods described herein.

In other embodiments, the method for expanding and/or activating the cell further comprises adding a second stimulatory molecule, such as CD28. For example, the CAR ligand and the second stimulatory molecule can be immobilized on a substrate, such as one or more beads, thus allowing for increased cell proliferation and/or activation.

In yet another aspect, a method is provided for selecting or enriching a CAR-expressing cell. The method includes contacting a CAR expressing cell with a CAR ligand as described herein and selecting a cell based on CAR ligand binding.

Other embodiments relate to a method for depleting, reducing and/or cytolyzing a CAR expressing cell. The method includes contacting a CAR expressing cell with a CAR ligand as described herein and targeting the cell based on binding of the CAR ligand, thereby reducing the number and/or killing the CAR expressing cell. In one embodiment, the CAR ligand is bound to a toxic agent (eg, a toxin or cell-damaging drug). In another embodiment, the anti-idiotypic antibody can elicit an effector cell activity, such as ADCC or ADC activities.

Exemplary anti-CAR antibodies that can be used in the methods described herein are described, for example, in WO 2014/190273 and Jena et al., Chimeric Antigen Receptor (CAR)-Specific Monoclonal Antibody to Detect CD19-Specific T cells in Clinical Trials, PLOS March 2013 8:3 e57838, the contents of which are incorporated by reference. In one embodiment, the anti-idiotypic antibody molecule recognizes an anti-CD19 antibody molecule, for example an anti-CD19 scFv. For example, an anti-idiotypic antibody molecule can compete for binding to clone #136.20.1 of the CD19-specific CAR mAb described in Jena et al., PLOS March 2013 8:3 e57838; may contain the same CDRs (e.g., one or more, e.g., all of CDR1 VH, CDR2 VH, CDR3 CH, CDR1 VL, CDR2 VL, and CDR3 VL using the Kabat definition, the Chothia definition, or a combination of the Kabat and Chothia definitions) as clone no. 136.20.1 mAb CAR specific to CD19; may contain one or more (eg, 2) variable regions as clone #13 6.20.1 mAb CAR specific to CD19, or may contain clone # 136.20.1 mAb CAR specific to CD19. In some embodiments, an anti-idiotypic antibody is generated by the method described in Jena et al. In another embodiment, the anti-idiotypic antibody molecule is an anti-idiotypic antibody molecule as described in WO 2014/190273. In some embodiments, the anti-idiotypic antibody molecule contains the same CDRs (e.g., one or more, e.g., all of CDR1 VH, CDR2 VH, CDR3 CH, CDR1 VL, CDR2 VL, and CDR3 VL) as an antibody molecule from WO 2014/190273, such as 13 6.20.1; may contain one or more (eg 2) variable regions of an antibody molecule from WO 2014/190273, or may contain an antibody molecule from WO 2014/190273 such as 136.20.1. In other embodiments, the anti-CAR antibody binds to the constant region of the extracellular binding domain of the CAR molecule, for example, as described in WO 2014/190273. In some embodiments, an anti-CAR antibody binds to a constant region of the extracellular binding domain of a CAR molecule, such as a heavy chain constant region (eg, a CH2-CH3 hinge region) or a light chain constant region. For example, in some embodiments, an anti-CAR antibody competes for binding with a 2D3 monoclonal antibody described in WO 2014/190273, contains the same CDRs (e.g., one or

- 162 040145 more, for example, all of CDR1 VH, CDR2 VH, CDR3 CH, CDR1 VL, CDR2 VL and CDR3 VL) as 2D3, or contains one or more (for example, 2) 2D3 variable regions, or contains 2D3, as described V

WO 2014/190273.

In some aspects and embodiments, the compositions and methods herein are optimized for a particular subset of T cells, for example, as described in US Serial No. 62/031699, registered July 31, 2014, the contents of which are herein fully incorporated by reference. In some embodiments, the optimized T cell subsets are characterized by increased resistance compared to a control T cell, eg, a different type of T cell (eg, CD8+ or CD4+) expressing the same construct.

In some embodiments, the CD4+ T cell comprises a CAR as described herein, wherein the CAR comprises an intracellular signaling domain suitable for (e.g., optimized for, e.g., resulting in increased resistance to) CD4+ T cells, e.g., an ICOS domain. In some embodiments, the CD8+ T cell comprises a CAR as described herein, wherein the CAR comprises an intracellular signaling domain suitable for (e.g., optimized for, e.g., resulting in increased resistance to) CD8+ T cells, e.g., 4-1BB domain, a CD28 domain or other costimulatory domain other than the ICOS domain. In some embodiments, the implementation of the CAR described in the present invention contains antigennegative domain described in the present invention, for example, CAR containing antigennegative domain that specifically binds to the tumor antigen described in the present description.

In one aspect, the present description describes a method of treating an individual, for example, an individual suffering from a malignant tumor. The method includes administering to said individual an effective amount of:

1) CD4+ T-cell containing CAR (CAR ^cd4+ ) containing:

an antigen-binding domain, eg an antigen-binding domain of the present invention, eg an antigen-binding domain that specifically binds to the tumor antigen of the present invention;

a transmembrane domain, and an intracellular signaling domain, such as a first costimulatory domain, such as an ICOS domain; And

2) CD8+ T-cell containing CAR (CAR ^cd8+ ) containing:

an antigen-binding domain, eg an antigen-binding domain of the present invention, eg an antigen-binding domain that specifically binds to the tumor antigen of the present invention;

a transmembrane domain, and an intracellular signaling domain, such as a second costimulatory domain, such as a 4-1BB domain, a CD28 domain, or another costimulatory domain other than the ICOS domain;

where CAR ^cd4+ and CAR ^cd8+ differ from each other.

Optionally, the method further comprises administering:

3) a second CD8+ T cell containing a CAR (second CAR ^cd8+ ) containing:

an antigen-binding domain, eg an antigen-binding domain of the present invention, eg an antigen-binding domain that specifically binds to a tumor antigen of the present invention;

a transmembrane domain; and an intracellular signaling domain, wherein the second ^cd8+ CAR contains an intracellular signaling domain, eg, a costimulatory signaling domain not contained in the ^cd8+ CAR, and optionally does not contain an ICOS signaling domain.

CAR activity assays.

Once the CARs, such as the NKR-CARs of the present invention, are constructed, various assays can be used to assess the activity of the molecule, such as, but not limited to, the ability of T cells to proliferate following antigen challenge, long-term expansion of T cells in the absence of restimulation and anti-cancer activity in suitable in vitro and animal models. Assays for evaluating the effects of NKR-CAR are described in more detail below.

Western blot analysis of NKR-CAR expression in primary T cells can be used to detect the presence of monomers and dimers. See, for example, Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). Very briefly, T cells (a 1:1 mixture of CD4+ and CD8+ T cells) expressing CAR are expanded in vitro for more than 10 days, followed by lysis and SDS-PAGE in reductive

- 163 040145 conditions. CARs are detected by Western blotting using an antibody specific for CAR components, eg, NKR component, eg, KIR component. The same T cell subsets are used for SDS-PAGE analysis under non-reductive conditions to provide an assessment of covalent dimer formation.

In vitro expansion of CAR+ T cells following antigen challenge can be measured by flow cytometry. For example, a mixture of CD4+ and CD8+ T cells are stimulated with aAPC aCD3/aCD28 followed by transduction with lentiviral vectors expressing GFP under the control of promoters to be analyzed. Illustrative promoters include the CMV IE, EF-1a, ubiquitin C, or phosphoglycerokinase (PGK) gene promoters. GFP fluorescence was assessed on day 6 of culture in subpopulations of CD4+ and/or CD8+ T cells by flow cytometry. See, for example, Milone et al., Molecular Therapy, 17(8): 1453-1464 (2009). Alternatively, a mixture of CD4 ⁺ and CD8 ⁺ T cells are stimulated with aCD3/aCD28 coated magnetic beads on day 0 and CAR is transduced on day 1 using a bicistronic lentiviral vector expressing CAR along with eGFP using the ribosome gapping sequence, 2A. Cultures are restimulated with antigen-expressing K562 cells, wild-type K562 cells (K562 wild-type), or negative control cells that do not express tumor antigen. A day later, exogenous IL-2 is added to the cultures at a concentration of 100 IU/ml. GFP+ T cells are counted by flow cytometry using bead-based counting. See, for example, Milone et al., Molecular Therapy, 17(8): 1453-1464 (2009).

It is also possible to measure sustained proliferation of CAR+ T cells in the absence of restimulation. See, for example, Milone et al., Molecular Therapy, 17(8): 1453-1464 (2009). Briefly, mean T cell volume (fl) is measured on day 8 of culture using a Coulter Multisizer III particle counter, Nexcelom Cellometer Vision, or Millipore Scepter after stimulation with aCD3/aCD28 coated magnetic beads on day 0, and transduction with the indicated CAR on day 1 .

Animal models can also be used to measure the activity of a CAR-expressing cell. For example, a xenograft model using human mesothelin-specific KIR-CAR+ T cells can be used to treat a primary human malignancy that expresses mesothelin, such as mesothelioma or ovarian cancer, in immunodeficient mice. See, for example, Milone et al., Molecular Therapy, 17(8): 1453-1464 (2009). Very briefly, once the tumor is established, mice are randomly assigned to treatment groups. Varying amounts of CAR-expressing T cells are administered. At various time points, CAR expression and proliferation/resistance of CAR-expressing cells can be monitored. Animals are evaluated for tumor progression at weekly intervals. Survival curves for groups are compared using a log-rank test. A dose-dependent response to CAR treatment can be assessed. See, for example, Milone et al., Molecular Therapy 17(8): 1453-1464 (2009).

Assessment of cell proliferation and cytokine production of CAR expressing cells has been previously described, for example, in Milone et al., Molecular Therapy, 17(8): 1453-1464 (2009). Briefly, the evaluation of CAR mediated proliferation is performed on microtiter plates by mixing washed T cells with K562 cells expressing CD19 (K19) or CD32 and CD137 (KT32-BBL) to a final T cell:K562 ratio of 2:1. Prior to use, K562 cells are irradiated with gamma radiation.

Anti-CD3 (clone OKT3) and anti-CD28 (clone 9.3) monoclonal antibodies are added to cultures with KT32-BBL cells in order to serve as a positive control for the stimulation of T cell proliferation, since these signals support long-term expansion of CD8+ T cells ex vivo. T cells are counted in cultures using CountBright™ fluorescent beads (Invitrogen, Carlsbad, CA) and flow cytometry as described by the manufacturer. CAR+ T cells are identified by GFP expression using T cells that are constructed using lentiviral vectors expressing CAR associated with eGFP-2A. For CAR+ T cells not expressing GFP, CAR+ T cells are detected using a biotinylated recombinant tumor antigen protein and avidin-PE secondary conjugate. It is also possible to simultaneously detect the expression of CD4+ and CD8+ on T cells with specific monoclonal antibodies (BD Biosciences). Cytokine measurements are performed in supernatants collected 24 h after restimulation using the human TH1/TH2 cytokine cytometric bead array (BD Biosciences, San Diego, CA) according to the manufacturer's instructions or using the Luminex 30-plex kit (Invitrogen). Fluorescence was assessed using a BD Fortessa flow cytometer and analyzed according to the manufacturer's instructions. Similar experiments can be done with CD123S CART.

Cytotoxicity can be assessed by a standard 51Cr release assay. See, for example, Milone et al., Molecular Therapy., 17(8): 1453-1464 (2009). Briefly, target cells (lines K562 expressing tumor antigen and primary tumor cells) are loaded with 51Cr (as NaCrO ₄ , New England Nuclear, Boston, MA) at 37° C. for 2 hours with frequent agitation, washing 164 040145 were plated twice in complete RPMI and plated on microtiter plates. Effector T cells are mixed with target cells in complete RPMI wells in various effector cell:target cell (E:T) ratios. Also prepare additional wells containing only medium (spontaneous release, SR) or 1% solution of Triton-X 100 detergent (full release, TR). After 4 hours of incubation at 37°C, collect the supernatant from each well. The released 51Cr is then measured using a gamma counter (Packard Instrument Co., Waltham, MA). Each condition is run for at least those repetitions and percentage lysis is calculated using the formula: % lysis=(ER-SR)/(TR-SR) where ER is the average 51Cr released under each experimental condition.

Imaging techniques can be used to evaluate the specific migration and proliferation of CARs in a tumor-bearing animal model. Such assays have been described, for example, in Barrett et al., Human Gene Therapy, 22:1575-1586 (2011). Briefly, NOD/SCID/yc'/' (NSG) mice are injected iv with Nalm-6 cells followed by T cell injection 7 days later 4 hours after electroporation with CAR constructs. T cells are stably transfected with a lentiviral construct to express firefly luciferase and bioluminescence imaging is performed in mice. Alternatively, the therapeutic efficacy and specificity of a single injection of CAR+ T cells can be measured in a Nalm-6 xenograft model as follows: NSG mice are injected with Nalm-6 transduced to stably express firefly luciferase, followed by a single tail vein injection of T cells, electroporated CAR, after 7 days. Animals were visualized at various time points after injection. For example, photon density heat maps of firefly luciferase positive leukemia can be obtained from representative mice on day 5 (2 days before treatment) and day 8 (24 hours after CAR ⁺ PBL).

Other assays can also be used to evaluate the CAR design of the invention, including assays described in the Examples section of this specification as well as assays known in the art.

Therapeutic application.

The present invention relates to a cell (e.g., T cell or NK cell) modified to express an NKR-CAR, e.g. KIR-CAR, or a set of NKR-CAR types, e.g. eg KIR-CAR combines the antigen recognition domain of a specific antibody with an NKR component, eg KIR.

In one embodiment, the KIR-CARs of the invention comprise an activating KIR that delivers its signal through interaction with an immunoreceptor tyrosine activation motif (ITAM) containing the DAP12 membrane protein, which is mediated by residues in the transmembrane domains of these proteins.

In one embodiment, the KIR-CARs of the invention comprise an inhibitory KIR that delivers its signal through one or more immunoreceptor tyrosine inhibitory motifs (ITIMs) that interact directly or indirectly with cytoplasmic signaling proteins such as SHP-1, SHP-2 and the Vav protein family. KIRs carrying cytoplasmic domains that contain (ITIM) abolish the activating signal, resulting in inhibition of cytolytic activity and NK cytokine production activity. In some instances, a modified T cell expressing the KIR-CAR of the invention can elicit a KIRCAR-mediated T cell response. In one embodiment, the dependence of binding to more than one type of antigen allows the modified T cell to have increased specificity to elicit a response when it binds to a tumor cell rather than when it binds to a normal uninvolved cell.

The invention relates to the use of a set of KIR-CAR types to target the specificity of a primary T cell for a tumor antigen. Thus, the present invention also relates to a method of stimulating a T-cell mediated immune response to a population of target cells or tissue in a mammal, comprising the step of administering to the mammal a T cell that expresses a constellation of KIR-CAR types, where each KIR-CAR type contains a binding molecule, which specifically interacts with a predetermined target. In one embodiment, the cell contains a first KIR-CAR containing an activating KIR (actKIR-CAR) and a second KIR-CAR containing an inhibitory KIR (inhKIR-CAR).

Without wishing to be bound by any particular theory, the antitumor immune response elicited by modified KIR-CAR T cells may be an active or passive immune response. In addition, the KIR-CAR mediated immune response may be part of an adoptive immunotherapy approach in which KIR-CAR modified T cells induce an immune response specific for the antigen-binding domain in KIR-CAR.

Cancers that can be treated include tumors that are not or are not yet substantially vascularized, as well as vascularized tumors. Malignant tumors may include non-solid tumors (such as hematologic tumors such as leukemias and lymphomas) or may include solid tumors. Types of malignancy

- 165 040145 venous tumors that are treated with the CARs of the invention include, but are not limited to, carcinoma, blastoma and sarcoma and certain types of leukemia or lymphoid malignancies, benign and malignant tumors and malignancies such as sarcomas, carcinomas and melanomas. Also included are tumors/malignancies in adults and tumors/malignancies in children. In one embodiment, the cancer to be treated is a solid tumor, such as the solid tumor described herein.

Hematologic malignancies are malignant tumors of the blood or bone marrow. Examples of hematologic (or hematogenous) malignancies include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia, and myeloblastic, promyelocytic, myelomonocytic, monocytic leukemia, and erythroleukemia), chronic leukemias (such as chronic myelocytic leukemia (granulocytic ) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high-grade forms), multiple myeloma, Waldenström's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

Solid tumors are abnormal masses of tissue that usually do not contain cysts or fluid areas. Solid tumors may be benign or malignant. Different types of solid tumors are named after the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer , lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas, sebaceous carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors (such as glioma (such as glioma of the brainstem and mixed gliomas), glioblastoma (also known as glioblastoma multiforme), astrocytoma, CNS lymphoma, germinoma, medulloblastoma, schwannoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, and brain metastases).

In one embodiment, the invention relates to methods for treating cancer by providing an individual in need thereof with immune system effector cells (e.g., T cells, NK cells) that are engineered to express the CD19 CAR, e.g., with a binding an anti-CD19 domain of the present invention, wherein the cancer cells express CD19. In one embodiment, the cancer being treated is ALL (acute lymphoblastic leukemia), CLL (chronic lymphocytic leukemia), DLBCL (diffuse large B-cell lymphoma), MCL (mantle cell lymphoma), or MM (multiple myeloma).

In one embodiment, the invention relates to methods for treating cancer by providing an individual in need thereof with immune system effector cells (e.g., T cells, NK cells) that are engineered to express EGFRvIIICAR, e.g., with a binding domain against EGRFvIII described in the present invention, where malignant cells express EGFRvIII. In one embodiment, the cancer being treated is a glioblastoma.

In one embodiment, the invention relates to methods for treating cancer by providing an individual in need thereof with immune system effector cells (e.g., T cells, NK cells) that are engineered to express mesothelin CAR, e.g. binding domain against mesothelin described in the present invention, where malignant cells express mesothelin. In one embodiment, the cancer to be treated is a solid tumor, such as the solid tumor described herein, such as mesothelioma, pancreatic cancer, lung cancer, or ovarian cancer.

In one embodiment, the invention relates to methods for treating cancer by providing an individual in need thereof with immune system effector cells (e.g., T cells, NK cells) that are engineered to express CD123CAR, e.g., with a binding domain against CD123 described in the present invention, where malignant cells express CD123. In one embodiment, the cancer being treated is AML.

- 166 040145

In one embodiment, the invention relates to methods for treating cancer by providing an individual in need thereof with immune system effector cells (e.g., T cells, NK cells) that are engineered to express CLL-1CAR, e.g., an anti-CLL-1 binding domain of the present invention, wherein the cancer cells express CLL-1. In one embodiment, the cancer being treated is AML.

In one embodiment, the invention relates to methods for treating cancer by providing an individual in need thereof with immune system effector cells (e.g., T cells, NK cells) that are engineered to express CD33CAR, e.g., with a binding domain against CD33 described in the present invention, where malignant cells express CD33. In one embodiment, the cancer being treated is AML.

In one embodiment, the invention relates to methods for treating cancer by providing an individual in need thereof with immune system effector cells (e.g., T cells, NK cells) that are engineered to express GD2CAR, e.g., with a binding domain against GD2 described in the present invention, where malignant cells express GD2. In one embodiment, the cancer being treated is a neuroblastoma.

In one embodiment, the invention relates to methods for treating cancer by providing an individual in need thereof with immune system effector cells (e.g., T cells, NK cells) that are engineered to express oacetyl-GD2CAR, wherein the cancer cells express o-acetyl-602. In one embodiment, the cancer being treated is a neuroblastoma or melanoma.

In one embodiment, the invention relates to methods for treating cancer by providing an individual in need thereof with immune system effector cells (e.g., T cells, NK cells) that are engineered to express BCMACAR, e.g., with a binding domain against BCMA described in the present invention, where malignant cells express BCMA. In one embodiment, the cancer being treated is multiple myeloma.

In one embodiment, the invention relates to methods for treating cancer by providing an individual in need thereof with immune system effector cells (e.g., T cells, NK cells) that are engineered to express CLDN6CAR, e.g., with a binding domain against CLDN6 described in the present invention, where malignant cells express CLDN6. In one embodiment, the cancer to be treated is a solid tumor, such as the solid tumor described herein, such as ovarian cancer, lung cancer, or breast cancer.

In one embodiment, the invention relates to methods for treating cancer by providing an individual in need thereof with immune system effector cells (e.g., T cells, NK cells) that are engineered to express WT1CAR, e.g., with a binding domain against WT1 described in the present invention, where malignant cells express WT1.

In one embodiment, the antigen-binding domain of KIR-CAR cells, eg, T cells or NK cells, of the invention is for the treatment of a particular cancer. In one embodiment, KIR-CAR cells, e.g., T cells or NK cells of the invention, are modified to express a first actKIR-CAR targeting a first antigen and a second inhKIR-CAR targeting a second antigen, wherein the first the antigen is expressed on the particular tumor or cancer, and the second antigen is not expressed on the particular tumor or cancer. Thus, conditional T-cell activation is obtained by the participation of actKIR-CAR (or standard TCR-zeta CAR carrying scFv to an antigen on the cancer cell of interest) and inhKIR-CAR carrying, for example, scFv directed against an antigen that is present. on normal, but not malignant tissue, which ensures that the activation signal from actKIR-CAR is inhibited when a KIR-CAR T cell encounters normal cells. Examples of antigens that serve as useful targets for inhibitory CARs include ephrin receptors (Pasquale, 2010, Nat. Rev. Cancer, 10(3):165-80) and claudins (Singh et al., 2010, J. Oncol. , 2010:541957), which are expressed by epithelial cells from normal tissues but are often selectively lost by malignant tumors (eg, EPHA7).

Therapeutic applications for diseases and disorders in which mesothelin is expressed.

The present invention relates to compositions and methods for the treatment of diseases and disorders associated with the expression of mesothelin. Examples of a Disease or Disorder Associated with Expression

- 167 040145 mesothelin include mesothelioma (eg pleural mesothelioma, peritoneal mesothelioma, pericardial mesothelioma), pancreatic cancer or ovarian cancer. The disease or disorder associated with the expression of mesothelin is a solid tumor described in the present description.

Malignant mesothelioma is a type of cancer that occurs in the thin layer of cells lining the internal organs of the body, known as the mesothelium. There are three established types of mesothelioma. Pleural mesothelioma (eg, malignant pleural mesothelioma or MPM) is the most common form of the disease, accounting for approximately 70% of cases, and occurs in the lung lining known as the pleura. Peritoneal mesothelioma occurs in the lining of the abdomen, known as the peritoneum.

Pericardial mesothelioma occurs in the pericardium, which lines the heart.

An individual may be at risk of developing mesothelioma if the individual has been exposed to asbestos. Asbestos exposure and inhalation of asbestos particles can cause mesothelioma. In most cases, symptoms of mesothelioma do not appear in an asbestos-exposed individual for many years after exposure to asbestos.

Symptoms of pleural mesothelioma include, for example, lower back pain or lateral chest pain and difficulty breathing. Other symptoms include difficulty swallowing, persistent cough, fever, weight loss or fatigue. Additional symptoms that some patients experience are muscle weakness, loss of sensory ability, coughing up blood, swelling of the face and hands, and hoarseness. In the early stages of the disease, such as stage 1 mesothelioma, symptoms may be mild. Patients typically complain of pain in one area of the chest that never goes away, weight loss, and fever.

Peritoneal mesothelioma occurs in the abdomen, and as a result, symptoms often include abdominal pain, weight loss, nausea, and vomiting. There may be accumulation of fluid in the abdominal cavity, and also as a result of a malignant tumor. Peritoneal mesothelioma begins in the abdomen and often spreads to other organs in the area, including the liver, spleen, or intestines. Severe abdominal pain is the most common complaint that patients first present with. An uncomfortable level may also be present with accumulation of fluid in the abdomen. Other symptoms of peritoneal mesothelioma may include difficulty passing a bowel movement, nausea and vomiting, fever, and swollen legs.

Pericardial mesothelioma is the least common form of mesothelioma. Pericardial mesothelioma, as the name implies, affects the heart. This rare type of mesothelioma-type cancer affects the pericardium, the membrane that surrounds the heart. As the cancer progresses, the heart becomes unable to deliver oxygen as efficiently to the body, causing a further decline in health at an increasing rate. The symptoms most commonly associated with pericardial mesothelioma mimic those of a myocardial infarction: nausea, chest pain, and shortness of breath.

Individuals for whom the treatment of the invention is effective include individuals with mesothelioma or individuals suspected of having mesothelioma, for example as evidenced by the presence of one or more of the symptoms described herein and/or exposure to asbestos. In specific embodiments, the mesothelioma is pleural mesothelioma (eg, malignant pleural mesothelioma). In other aspects, an individual may be treated who suffers from a precancerous condition such as, for example, pleural plaques, benign mesothelioma, or mesothelial hyperplasia.

Another example of a disease or disorder associated with mesothelin is pancreatic cancer. Pancreatic cancers that can be treated with the methods described herein include, but are not limited to, exocrine pancreatic cancers and endocrine pancreatic cancers. Exocrine pancreatic cancers include, but are not limited to, adenocarcinomas, acinar cell carcinomas, adenosquamous carcinomas, colloid carcinomas, undifferentiated osteoclast-like giant cell carcinomas, hepatoid carcinomas, intraductal papillary mucinous neoplasias, mucinous cystic neoplasias, peristenocystoblastomas carcinomas, solid and pseudopapillary tumors, pancreatic carcinomas and undifferentiated carcinomas. In some embodiments, the exocrine pancreatic cancer is a pancreatic carcinoma. Endocrine pancreatic cancers include, but are not limited to, insulinomas and glucagonomas.

In some embodiments, the pancreatic cancer is any early stage pancreatic cancer, non-metastatic pancreatic cancer, primary pancreatic cancer, resected pancreatic cancer, late stage pancreatic cancer, locally advanced pancreatic cancer, metastatic pancreatic cancer , unresectable pancreatic cancer, pancreatic cancer in remission, recurrent pancreatic cancer, adjuvant pancreatic cancer

- 168 040145 therapy or pancreatic cancer in non-adjuvant therapy. In some embodiments, the pancreatic cancer is locally advanced pancreatic cancer, unresectable pancreatic cancer, or metastatic pancreatic carcinoma. In some embodiments, the pancreatic cancer is refractory to gemcitabine therapy. In some embodiments, the pancreatic cancer is refractory to gemcitabine-based therapy.

In other aspects, the mesothelin expression disorder is ovarian cancer. Ovarian cancer is classified according to the histology of the tumor. Superficial epithelial stromal tumor, also known as ovarian epithelial carcinoma, is the most common type of ovarian cancer. It includes serous tumor (including serous papillary cystadenocarcinoma), endometrioid tumor, and mucus-forming cystadenocarcinoma.

The methods described in the present invention can be used to treat various stages of ovarian cancer, such as stage I, stage II, stage III or stage IV. Staging can be performed, for example, when ovarian cancer is removed. The stage of ovarian cancer is determined as follows:

A stage I malignant tumor is limited to one or both ovaries. A malignant tumor is stage II if one or both ovaries are involved and it has spread to the uterus and/or fallopian tubes or other areas of the pelvis. A malignant tumor is a stage III malignant tumor if one or both ovaries are involved and it has spread to the lymph nodes or other sites outside the pelvis, but is still in the abdominal cavity, such as the intestinal surface or the liver. A malignant tumor is a stage IV malignant tumor if one or both ovaries are involved and the malignant tumor has spread outside the abdomen or into the liver.

In some embodiments, the ovarian cancer is resistant to one or more chemotherapeutic agents. In some embodiments, the ovarian cancer is refractory to one or more chemotherapeutic agents.

Other cancers that can be treated with the CAR compositions described herein include, for example, brain cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, liver cancer, kidney cancer, lymphoma, leukemia, cancer lung (eg, lung adenocarcinoma), melanoma, metastatic melanoma, mesothelioma, neuroblastoma, ovarian cancer, prostate cancer, pancreatic cancer, kidney cancer, skin cancer, thioma, sarcoma, non-Hodgkin's lymphoma, Hodgkin's lymphoma, uterine cancer, and any of these combination.

The present invention relates to methods for inhibiting the proliferation or reducing the population of mesothelin expressing cells, wherein the methods include contacting a cell population containing a mesothelin expressing cell with a mesothelin CAR expressing cell of the invention that binds to the mesothelin expressing cell. In a specific embodiment, the invention relates to methods for inhibiting the proliferation or reducing the population of mesothelin-expressing malignant cells, wherein the methods include bringing the cell population containing a mesothelin-expressing cell into contact with a mesothelin-expressing CAR cell of the invention that binds to the mesothelin-expressing cell. In another embodiment, the invention relates to methods for inhibiting the proliferation or reducing the population of mesothelin-expressing malignant cells, wherein the methods comprise bringing the population of mesothelin-expressing malignant cells with a CAR-expressing cell to a mesothelin-expressing cell of the invention that binds to a mesothelin-expressing cell. In certain embodiments, the mesothelin CAR-expressing cell of the invention reduces the content, number, number, or percentage of cells and/or malignant cells by at least 25%, at least 30%, at least 40%, at least 50%, at least 65%, at least 75%, at least 85%, at least 95%, or at least 99% in an individual with mesothelioma or other mesothelin-expressing malignancy cells, or in an animal model of mesothelioma or other malignancy associated with mesothelin-expressing cells, relative to a negative control. In one aspect, the individual is a human.

The invention also relates to methods for the prevention, treatment and/or management of a disorder associated with mesothelin expressing cells (e.g. mesothelioma), wherein the methods include administering to an individual in need thereof a mesothelioma CAR expressing cell of the invention that binds to a mesothelin expressing cell. In one aspect, the individual is a human.

The invention relates to methods for preventing recurrence of a cancer associated with mesothelin expressing cells, wherein the methods comprise administering to an individual in need thereof a mesothelin CAR expressing cell of the invention that binds to a mesothelin expressing cell. In another embodiment, the methods comprise administering to an individual in need an effective amount of an anti-mesothelin CAR-expressing cell of the invention that binds to a mesothelin-expressing cell, in combination with an effective amount of another therapy.

In one aspect, the invention relates to a vector containing a sequence encoding a mesothelin CAR operably linked to a promoter for expression in effector cells of the mammalian immune system. In one aspect, the invention relates to a recombinant immune system effector cell expressing an anti-mesothelin CAR for use in the treatment of mesothelin-expressing tumors. In one aspect, a mesothelin CAR expressing cell of the invention is capable of contacting a tumor cell with at least one mesothelin CAR of the invention expressed on its surface such that the mesothelin CAR expressing cell is activated in response to the antigen, and the CAR expressing cell targets the malignant cell, and the growth of the malignant tumor is inhibited.

In one aspect, the invention relates to a method for inhibiting the growth of a mesothelin-expressing cancer cell, comprising bringing the tumor cell into contact with a mesothelin-expressing CAR cell such that the CAR-expressing cell is activated in response to the antigen and targets the cancer cell, where the growth of the cancer tumors are inhibited. In one aspect, activated CART targets and causes death of the cancer cell.

In one aspect, the invention relates to a method for treating cancer in an individual. The method includes administering to an individual a mesothelin CAR-expressing cell, thereby treating the individual's cancer. An example of a cancer that can be treated with a mesothelin-expressing cell of the invention is a mesothelin-expressing cancer. In one aspect, the mesothelin expression cancer is selected from mesothelioma, pancreatic cancer, ovarian cancer, and lung cancer.

The invention relates to a type of cell therapy, where effector cells of the immune system, for example, T cells or NK cells, are genetically modified so that they express a chimeric antigen receptor (CAR), and the CAR expressing cell is infused into a recipient in need of it. The infused cell is capable of inducing cytolysis of tumor cells in the recipient. Unlike antibody therapies, CAR-modified effector cells of the immune system are able to replicate in vivo, resulting in long-term persistence that can lead to permanent tumor suppression. In various aspects, cells administered to a patient or progeny thereof are retained in the patient for at least four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, thirteen months, fourteen months , fifteen months, sixteen months, seventeen months, eighteen months, nineteen months, twenty months, twenty-one months, twenty-two months, twenty-three months, two years, three years, four years, or five years after the introduction of the cell to the patient.

The invention also relates to a type of cellular therapy where effector cells of the immune system are modified, for example, with in vitro transcribed RNA for transient expression of a chimeric antigen receptor (CAR), and the CAR expressing cell is infused into a recipient in need thereof. The infused cell is capable of inducing cytolysis of malignant cells in the recipient. Thus, in various aspects, cells administered to a patient are present for less than one month, eg, three weeks, two weeks, one week after administration of the cell to the patient.

Without wishing to be bound by any particular theory, the anti-cancer immune response elicited by CAR-modified effector cells of the immune system may be an active or passive immune response, or alternatively may be due to direct and indirect immune responses. In one aspect, CAR transduced T cells are characterized by the secretion of specific pro-inflammatory cytokines and high cytolytic activity in response to human malignant cells expressing mesothelin and mediate non-specific cytolysis and mediate regression of an established human tumor. For example, antigen-deficient tumor cells in a heterogeneous field of mesothelin-expressing tumors may be susceptible to indirect destruction by mesothelin-redirected T cells that previously acted against adjacent antigen-positive cancer cells.

In one aspect, the fully human scFv-bearing, CAR-modified immune system effector cells of the invention may be a type of vaccine for ex vivo immunization and/or in vivo therapy in a mammal. In one aspect, the mammal is a human.

With respect to ex vivo immunization, at least one of the following steps is carried out in vitro prior to cell administration to a mammal: i) cell expansion, ii) introduction of a nucleic acid encoding KIR-CAR into cells, and/or iii) cell cryopreservation.

Ex vivo methods are well known in the art and are described in more detail below. In brief

- 170 040145 statement, the cells are isolated from a mammal (preferably human) and genetically modified (ie, transduced or transfected in vitro) with a vector expressing the KIR-CAR described in the present invention. The modified KIR-CAR cell may be administered to a mammalian recipient to provide a therapeutic effect. The mammalian recipient may be a human, and the modified KIR-CAR cell may be autologous to the recipient. Alternatively, the cells may be allogeneic, isogenic, or xenogeneic with respect to the recipient.

The ex vivo propagation method for hematopoietic stem and progenitor cells described in US Pat. No. 5,199,942, incorporated herein by reference, can be applied to the cells of the present invention. Other suitable methods are known in the art, thus the present invention is not limited to any particular method for expanding cells ex vivo. Briefly, ex vivo culture and expansion of T cells includes: (1) collecting CD34+ hematopoietic stem and progenitor cells from a mammal from a peripheral blood sample or bone marrow explants; and (2) expansion of such cells ex vivo. In addition to the cell growth factors described in US Pat. No. 5,199,942, other factors such as flt3-L, IL-1, IL-3 and c-kit ligand can be used to culture and expand cells.

In addition to the use of a cell-based vaccine in relation to ex vivo immunization, the present invention also relates to in vivo immunization compositions and methods for eliciting an immune response directed against an antigen in a patient.

In general, cells activated and propagated as described herein can be used in the treatment and prevention of diseases that occur in individuals who have a weakened immune system. In particular, the modified KIR-CAR T cells of the invention are used in the treatment of cancer. In certain embodiments, cells of the invention are used to treat patients at risk of developing cancer. Thus, the present invention provides methods for treating or preventing cancer, comprising administering to an individual in need thereof a therapeutically effective amount of the modified KIR-CAR T cells of the invention.

The modified KIR-CAR T cells of the present invention can be administered alone or as a pharmaceutical composition in combination with diluents and/or other components such as IL-2 or other cytokines or cell populations. Briefly, the pharmaceutical compositions of the present invention may contain a population of target cells, as described in the present description, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may contain buffers such as neutral buffered saline, phosphate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (eg aluminum hydroxide); and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

The present invention also relates to methods for inhibiting the proliferation or reducing the population of mesothelin-expressing cells, wherein the methods comprise bringing the cell population containing the mesothelin-expressing cell into contact with a mesothelin-expressing CAR cell (e.g., an NKR-CAR of the invention that binds to a mesothelin-expressing cell). ). In a specific aspect, the invention relates to methods for inhibiting the proliferation or reducing the population of mesothelin-expressing malignant cells, wherein the methods comprise bringing the population of mesothelin-expressing malignant cells into contact with a mesothelin-expressing CAR cell of the invention that binds to the mesothelin-expressing cell. In one aspect, the present invention relates to methods for inhibiting the proliferation or reducing the population of mesothelin-expressing malignant cells, wherein the methods comprise contacting the mesothelin-expressing malignant cell population with a mesothelin-expressing CAR cell of the invention that binds to the mesothelin-expressing cell. In certain aspects, the mesothelin CAR-expressing cell of the invention reduces the content, number, number, or percentage of cells and/or malignant cells by at least 25%, at least 30%, at least 40%, at least 50 %, at least 65%, at least 75%, at least 85%, at least 95%, or at least 99% in an individual with mesothelioma or other malignant tumor associated with mesothelin-expressing cells , or in an animal model of mesothelioma or other malignancy associated with mesothelin-expressing cells, relative to a negative control. In one aspect, the individual is a human.

The present invention also provides methods for the prevention, treatment and/or management of a disease associated with mesothelin expressing cells (e.g., mesothelioma), wherein the methods comprise administering to an individual in need thereof a mesothelin-expressing cell according to

- 171 040145 of the invention, which binds to a mesothelin-expressing cell. In one aspect, the individual is a human.

The present invention relates to methods for preventing recurrence of a cancer associated with mesothelin expressing cells, wherein the methods comprise administering to an individual in need thereof a mesothelin CAR expressing cell of the invention that binds to a mesothelin expressing cell. In one aspect, the methods comprise administering to an individual in need thereof an effective amount of an anti-mesothelin CAR-expressing cell of the invention that binds to a mesothelin-expressing cell, in combination with an effective amount of another therapy.

Methods of combined treatment.

The CAR-expressing cell described herein can be used in combination with other known agents and therapies. Administered in combination, as used herein, means that two (or more) different drugs are delivered to an individual during the course of the individual's disease, e.g., two or more drugs are delivered after the individual has been diagnosed with a disorder and before how the violation went or was eliminated, or treatment was stopped for other reasons. In some embodiments, delivery of one drug is still maintained when delivery of the second drug is started, so there is overlap in administration. In some cases, this is referred to in the present description as a one-time or concurrent delivery. In other embodiments, delivery of one drug ends before delivery of another drug begins. In some embodiments of this case, the drug is more effective due to the combined administration. For example, the second drug is more effective, e.g., an equivalent effect is seen with less of the second drug, or the second drug reduces symptoms more than when the second drug is administered in the absence of the first drug, or the like. the situation is observed for the first drug. In some embodiments, delivery is such that the reduction in a symptom or other parameter associated with the disorder is greater than would be observed for one drug delivered in the absence of the other. The effect of the two drugs may be partially additive, fully additive, or more than additive. The delivery may be such that the effect of the first drug delivered is still detectable when the second drug is delivered.

Expressing CAR cell described in the present description, and at least one additional therapeutic agent can be administered simultaneously in the same or separate compositions or sequentially. For sequential administration, the CAR expressing cell described herein may be administered first and the additional agent may be administered second, or the order of administration may be reversed.

CAR therapy and/or other therapies, procedures, or methods may be administered during a period of active disorder, or during a period of remission or less active disease. CAR therapy can be given before other treatment, concurrently with treatment, after treatment, or during remission of the disorder.

When administered in combination, the CAR therapy and an additional agent (eg, second or third agent) or all may be administered in an amount or dose that is higher, lower, or the same as the amount or dose of each agent used alone, eg, as monotherapy. In certain embodiments, the administered amount or dose of CAR therapy, additional agent (e.g., second or third agent), or all is lower (e.g., at least 20%, at least 30%, at least 40%, or by at least 50%) compared to the amount or dose of each agent used alone, for example, as monotherapy. In other embodiments, the amount or dose of CAR therapy, additional agent (e.g., second or third agent), or all that results in the desired effect (e.g., treatment of cancer) is lower (e.g., at least 20% lower, at least by at least 30%, at least 40% or at least 50%) compared to the amount or dose of each agent used alone, for example, as monotherapy, required to obtain the same therapeutic effect.

In additional aspects, a CAR expressing cell as described herein may be used in a treatment regimen in combination with surgery, cytokines, radiation, or chemotherapy such as cytoxan, fludarabine, histone deacetylase inhibitors, demethylating agents, or a peptide vaccine such as the vaccine described in Izumoto et al., 2008 J. Neurosurg., 108:963-971.

In some instances, the compounds of the present invention are combined with other therapeutic agents such as other anti-cancer agents, anti-allergy agents, anti-nausea (or anti-emetics), analgesics, cytoprotective agents, and combinations thereof.

In one embodiment, the CAR-expressing cell described herein can be used in combination with a chemotherapeutic agent. Exemplary chemotherapeutic agents include an anthracycline (eg, doxorubicin (eg, liposomal doxorubicin)), vinca alkaloid (eg, vinblastine, vincristine, vindesine, vinorelbine), an alkylating agent (eg, cyclophosphamide, decarbazine, melphalan, ifosfamide, temozolomide), anti-immune cells (eg, alemtuzamab, gemtuzumab, rituximab, ofatumumab, tositumomab, brentuximab), antimetabolite (including, e.g., folic acid antagonists, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors (eg, fludarabine)), mTOR inhibitor, agonist of TNFR-inducible glucocorticoid related TNFR protein (GITR), proteasome inhibitor (eg aclacinomycin A, gliotoxin or bortezomib), immunomodulator such as thalidomide or a thalidomide derivative (eg lenalidomide).

Major chemotherapy agents contemplated for use in combination treatments include anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®) , N4pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposomal injection (DepoCyt®), dacarbazine (DTIC-Dome®), dactinomycin (Actinomycin D, Cosmegen), daunorubicin hydrochloride (Cerubidine®), daunorubicin citrate liposomal injection (DaunoXome®), dexamethasone, docetaxel (Taxotere®), doxorubicin hydrochloride (Adriamycin®, Rubex®), etoposide (Vepesid®), fludarabine phosphate (Fludara®), 5-fluorouracil (Adrucil®, Efudex®), flutamide (Eulexin®), tezacitibine, gemcitabine (difluorodeoxycytidine), hydroxyurea (Hydrea® ), idarubicin (Idamycin®), ifosfamide (IFEX®), irinotecan (Camptosar®), L-asparaginase (ELSPAR®), calcium leucovorin, melphalan (Alkeran®), 6-mercaptopurine (Purinethol®), methotrexate (Folex®) , mitoxantrone (Novantrone®), mylotarg, paclitaxel (Taxol®), phoenix (Yttrium90/MX-DTPA), pentostatin, polyfeprosan 20 with carmustine implant (Gliadel®), tamoxifen citrate (Nolvadex®), teniposide (Vumon®), 6 α-thioguanine, thiotepa, tirapazamine (Tirazone®), topotecan hydrochloride injection (Hycamptin®), vinblastine (Velban®), vincristine (Oncovin®), and vinorelbine (Navelbine®).

Anti-cancer agents of particular interest in combination with the compounds of the present invention include: anthracyclines; alkylating agents; antimetabolites; drugs that inhibit calcium-dependent calcineurin phosphatase or p70S6 kinase (FK506) or inhibit p70S6 kinase; mTOR inhibitors; immunomodulators; anthracyclines; vinca alkaloids; proteasome inhibitors; GITR agonists; protein tyrosine phosphatase inhibitors; a CDK4 kinase inhibitor; BTK inhibitor; an MKN kinase inhibitor; a DGK kinase inhibitor; or an oncolytic virus.

Illustrative antimetabolites include, but are not limited to, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors: methotrexate (Rheumatrex®, Trexall®), 5-fluorouracil (Adrucil®, Efudex®, Fluoroplex®), floxuridine (FUDF®), cytarabine (Cytosar -U®, Tarabine PFS), 6-mercaptopurine (Puri-Nethol®)), 6-thioguanine (Thioguanine Tabloid®), fludarabine phosphate (Fludara®), pentostatin (Nipent®), pemetrexed (Alimta®), raltitrexed (Tomudex®) , cladribine (Leustatin®), clofarabine (Clofarex®, Clolar®), azacitidine (Vidaza®), decitabine, and gemcitabine (Gemzar®). Preferred antimetabolites include cytarabine, clofarabine and fludarabine.

Exemplary alkylating agents include, but are not limited to, nitrogen mustards, ethyleneimine derivatives, alkylsulfonates, nitrosoureas, and triazenes): uramustine (Aminouracil Mustard®, Chlorethaminacil®, Demethyldopan®, Desmethyldopan®, Haemanthamine®, Nordopan®, Uracil nitrogen mustard®, Uracillost ®, Uracilmostaza®, Uramustin®, Uramustine®), chlormethine (Mustargen®), cyclophosphamide (Cytoxan®, Neosar®, Clafen®, Endoxan®, Procytox®, Revimmune™), ifosfamide (Mitoxana®), melphalan (Alkeran®) , chlorambucil (Leukeran®), pipobromane (Amedel®, Vercyte®), triethylenemelamine (Hemel®, Hexalen®, Hexastat®), triethylenethiophosphoramine, temozolomide (Temodar®), thiotepa (Thioplex®), busulfan (Busilvex®, Myleran®) , carmustine (BiCNU®), lomustine (CeeNU®), streptozocin (Zanosar®), and dacarbazine (DTIC-Dome®). Additional exemplary alkylating agents include, but are not limited to, oxaliplatin (Eloxatin®); temozolomide (Temodar® and Temodal®); dactinomycin (also known as Actinomycin-D, Cosmegen®); melphalan (also known as L-PAM, L-sarcolysin and phenylalanine mustine, Alkeran®); altretamine (also known as hexamethylmelamine (HMM), Hexalen®); carmustine (BiCNU®); bendamustine (Treanda®); busulfan (Busulfex® and Myleran®); carboplatin (Paraplatin®); lomustine (also known as CCNU, CeeNU®); cisplatin (also known as CDDP, Platinol® and Platinol®-AQ); chlorambucil (Leukeran®); cyclophosphamide

- 173 040145 (Cytoxan® and Neosar®); dacarbazine (also known as DTIC, DIC and imidazole carboxamide, DTICDome®); altretamine (also known as hexamethylmelamine (HMM), Hexalen®); ifosfamide (Ifex®); prenumustine; procarbazine (Matulane®); mechlorethamine (also known as nitrogen mustard, mustine, and mechlorethamine hydrochloride, Mustargen®); streptozocin (Zanosar®); thiotepa (also known as thiophosphoamide, TESPA and TSPA, Thioplex®); cyclophosphamide (Endoxan®, Cytoxan®, Neosar®, Procytox®, Revimmune®) and bendamustine HCl (Treanda®).

In embodiments, a CAR-expressing cell as described herein is administered to an individual in combination with fludarabine, cyclophosphamide, and/or rituximab. In embodiments, a CAR-expressing cell as described herein is administered to an individual in combination with fludarabine, cyclophosphamide, and rituximab (FCR). In embodiments, the subject is suffering from CLL. For example, an individual has a deletion in the short arm of chromosome 17 (del(17p), for example, in a leukemic cell). In other examples, the individual does not have del(17p). In embodiments, the individual contains a leukemia cell containing a mutation in the immunoglobulin heavy chain variable region (IgV _H ) gene. In other embodiments, the individual does not contain a leukemia cell containing a mutation in the immunoglobulin heavy chain variable region (IgVH) gene. In embodiments, fludarabine is administered at a dose of about 10-50 mg/ ^m mg / m ² ), for example, intravenously. In embodiments, cyclophosphamide is administered at a dose of about 200-300 mg/m ² (eg, about 200-225, 225-250, 250-275, or 275-300 mg/m ² ), eg, intravenously. In embodiments, rituximab is administered at a dose of about 400-600 mg/m ² (eg, 400-450, 450-500, 500-550, or 550-600 mg/m ² ), eg, intravenously.

In embodiments, a CAR expressing cell as described herein is administered to an individual in combination with bendamustine and rituximab. In embodiments, the subject is suffering from CLL. For example, an individual has a deletion in the short arm of chromosome 17 (del(17p), for example, in a leukemic cell). In other examples, the individual does not have a del(17p) deletion. In embodiments, the individual contains a leukemia cell containing a mutation in the immunoglobulin heavy chain variable region (IgV _H ) gene. In other embodiments, the individual does not contain a leukemic cell containing a mutation in the immunoglobulin heavy chain variable region (IgV _H ) gene. In embodiments, bendamustine is administered at a dose of about 70-110 mg/m ² (eg, 70-80, 80-90, 90-100, or 100-110 mg/m ² ), eg, intravenously. In embodiments, rituximab is administered at a dose of about 400-600 mg/m ² (eg, 400-450, 450-500, 500-550, or 550600 mg/m ² ), eg, intravenously.

In embodiments, a CAR-expressing cell as described herein is administered to an individual in combination with rituximab, cyclophosphamide, doxorubicin, vincristine, and/or a corticosteroid (eg, prednisone). In embodiments, a CAR expressing cell as described herein is administered to an individual in combination with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). In embodiments, the individual suffers from diffuse large B-cell lymphoma (DLBCL). In embodiments, the subject suffers from localized non-generalized DLBCL (eg, has a tumor less than 7 cm in size/diameter). In embodiments, the individual receives radiation treatment in combination with R-CHOP. For example, an individual is administered R-CHOP (eg, 1-6 cycles, eg, 1, 2, 3, 4, 5, or 6 cycles of R-CHOP) followed by irradiation. In some cases, the subject is administered R-CHOP (eg 1-6 cycles, eg 1, 2, 3, 4, 5 or 6 cycles of R-CHOP) after exposure.

In embodiments, a CAR expressing cell as described herein is administered to an individual in combination with etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and/or rituximab. In embodiments, a CAR expressing cell as described herein is administered to an individual in combination with etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab (EPOCH-R). In embodiments, a CAR-expressing cell described herein is administered to an individual in combination with a dose-adjusted EPOCH-R (DA-EPOCH-R). In embodiments, the individual suffers from a B-cell lymphoma, eg, aggressive B-cell lymphoma with Myc rearrangement.

In embodiments, a CAR-expressing cell as described herein is administered to an individual in combination with rituximab and/or lenalidomide. Lenalidomide ((RS)-3-(4amino-1-oxo-1,3-dihydro-2H-isoindol-2-yl)piperidine-2,6-dione) is an immunomodulator. In embodiments, a CAR expressing cell as described herein is administered to an individual in combination with rituximab and lenalidomide. In embodiments, the individual suffers from follicular lymphoma (FL) or mantle cell lymphoma (MCL). In embodiments, the individual suffers from FL and has not previously received treatment with cancer therapy. In embodiments, lenalidomide is administered at a dose of about 10-20 mg (eg, 10-15 or 15-20 mg), eg, every day. In embodiments, rituximab is administered at a dose

- 174 040145 approximately 350-550 mg/m ² (for example, 350-375, 375-400, 400-425, 425-450, 450-475 or 475-500 mg/m ² ), for example, intravenously.

Exemplary mTOR inhibitors include, for example, temsirolimus; ridaforolimus (formerly known as deferolimus, (1R,2R,4S)-4-[(2R)-2-[(1R,9S,12S,15R,16E,18R,19R,21R, 23S,24E,26E,28Z, 30S,32S,35R)-1,18-dihydroxu-19,30-gymethoxy-15,17,21,23,29,35-hexamethyl2,3,10,14,20-pentaoxo-11,36-dioxa-4 -azatricyclo[30.3.1.0 ^4,9 ]hexatriaconta-16,24,26,28-tetraen-12yl]propyl]-2-methoxycyclohexyldimethylphosphinate, also known as AP23573 and MK8669 and described in PCT Publication No. WO 03/064383); everolimus (Afinitor® or RAD001); rapamycin (AY22989, Sirolimus®); simapimod (CAS 164301-51-3); emsirolimus, (5-{2,4-bis[(3S)-3-methylmorpholin-4yl]pyrido[2,3-d]pyrimidin-7-yl}-2-methoxyphenyl)methanol (AZD8055); 2-amino-8-[trans-4-(2hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methylpyrido[2,3-d]pyrimidin-7(8H)-one (PF04691502, CAS 1013101-36-4) and N ² -[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2yl)morpholinium-4-yl]methoxy]butyl ]-L-arginylglycyl-L-α-aspartyl-L-serine- (SEQ ID NO: 64), internal salt (SF1126, CAS 936487-67-1) and XL765.

Illustrative immunomodulators include, for example, afutuzumab (available from Roche®); pegfilgrastim (Neulasta®); lenalidomide (CC-5013, Revlimid®); thalidomide (Thalomid®), actimide (CC4047) and IRX-2 (a mixture of human cytokines including interleukin 1, interleukin 2 and interferon y, CAS 951209-71-5, available from IRX Therapeutics). Illustrative anthracyclines include, for example, doxorubicin (Adriamycin® and Rubex®); bleomycin (Lenoxane®); daunorubicin (daunorubicin hydrochloride, daunomycin and rubidomycin hydrochloride, Cerubidine®); liposomal daunorubicin (liposomal daunorubicin citrate, DaunoXome®); mitoxantrone (DHAD, Novantrone®); epirubicin (Ellence™); idarubicin (Idamycin®, Idamycin PFS®); mitomycin C (Mutamycin®); geldanamycin; herbimycin; ravidomycin and deacetylravidomycin.

Exemplary vinca alkaloids include, for example, vinorelbine tartrate (Navelbine®), vincristine (Oncovin®) and vindesine (Eldisine®)); vinblastine (also known as vinblastine sulfate, vincaleukoblastin and VLB, Alkaban-AQ® and Velban®) and vinorelbine (Navelbine®).

Exemplary proteasome inhibitors include bortezomib (Velcade®); carfilzomib (PX171 -007, (S)-4-methyl-N-((S)-1 -(((S)-4-methyl-1 -((R)-2-methyloxiran-2-yl)-1 -oxopentan-2-yl)amino)-1 oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide); marizomib (NPI-0052); ixazomib citrate (MLN-9708); delanzomib (CEP-18770) and O-methyl-N-[(2-methyl-5thiazolyl)carbonyl]-L-seryl-O-methyl-N-[(1S)-2-[(2R)-2-methyl- 2-oxiranyl]-2-oxo-1-(phenylmethyl)ethyl]L-serinamide (ONX-0912).

In embodiments, a CAR expressing cell as described herein is administered to an individual in combination with brentuximab. Brentuximab is an antibody-drug conjugate of an anti-CD30 antibody and monomethylauristatin E. In embodiments, the subject has Hodgkin's lymphoma (HL), eg, relapsed or refractory HL. In embodiments, the subject is suffering from CD30+ HL. In embodiments, the subject has undergone an autologous stem cell transplant (ASCT). In embodiments, the individual has not undergone ASCT. In embodiments, brentuximab is administered at a dose of about 1-3 mg/kg (e.g., about 1-1.5, 1.5-2, 2-2.5, or 2.5-3 mg/kg), such as intravenously, e.g. every 3 weeks.

In embodiments, a CAR expressing cell as described herein is administered to an individual in combination with brentuximab and dacarbazine, or in combination with brentuximab and bendamustine. Dacarbazine is an alkylating agent with the chemical name 5-(3,3-dimethyl-1-triazenyl)imidazole-4-carboxamide. Bendamustine is an alkylating agent with the chemical name 4-[5-[bis(2-chloroethyl)amino]-1-methylbenzimidazol-2-yl]butanoic acid. In embodiments, the subject is suffering from Hodgkin's lymphoma (HL). In embodiments, the individual has not previously received treatment with cancer therapy. In embodiments, the age of the individual is at least 60 years old, such as 60, 65, 70, 75, 80, 85, or more. In embodiments, dacarbazine is administered at a dose of about 300-450 mg/m ² (e.g., about 300-325, 325-350, 350-375, 375-400, 400-425, or 425-450 mg/m ² ), e.g., intravenously. In embodiments, bendamustine is administered at a dose of about 75-125 mg/m ² (eg, 75-100 or 100-125 mg/m ² , eg, about 90 mg/m ² ), eg, intravenously. In embodiments, brentuximab is administered at a dose of about 1-3 mg/kg (e.g., about 1-1.5, 1.5-2, 2-2.5, or 2.5-3 mg/kg), such as intravenously, e.g. every 3 weeks.

In some embodiments, a CAR-expressing cell as described herein is administered to an individual in combination with a CD20 inhibitor, such as an anti-CD20 antibody (eg, a mono- or bispecific anti-CD20 antibody) or fragment thereof. Exemplary anti-CD20 antibodies include, but are not limited to, rituximab, ofatumumab, ocrelizumab, veltuzumab, obinutuzumab, TRU-015 (Trubion Pharmaceuticals), ocaratuzumab, and Pro131921 (Genentech). See, for example, Lim et al., Haematologica., 95, 1 (2010):135-43.

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In some embodiments, the anti-CD20 antibody comprises rituximab. Rituximab is an IgG1 kappa chimeric mouse/human monoclonal antibody that binds to CD20 and induces cytolysis of a CD20 expressing cell, for example, as described in www.accessdata.fda.gov/drugsatfda_docs/label/2010/103705s5311lbl.pdf. In embodiments, a CAR-expressing cell as described herein is administered to an individual in combination with rituximab. In embodiments, the subject is suffering from CLL or SLL.

In some embodiments, rituximab is administered intravenously, for example, as an intravenous infusion. For example, each infusion provides approximately 500-2000 mg (e.g., approximately 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 950-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400, 1400-1500, 1500-1600, 1600-1700, 1700-1800, 1800-1900 or 1900-2000 mg) rituximab. In some embodiments, rituximab is administered at a dose of 150 to 750 mg/m ² , such as about 150-175, 175-200, 200-225, 225-250, 250-300, 300-325, 325-350, 350375, 375 -400, 400-425, 425-450, 450-475, 475-500, 500-525, 525-550, 550-575, 575-600, 600-625, 625-650, 650-675 or 675-700 mg/m ² where m ² means the surface area of the body of the individual. In some embodiments, rituximab is administered at a dosing interval of at least 4 days, such as 4, 7, 14, 21, 28, 35 days or more. For example, rituximab is administered at a dosing interval of at least 0.5 weeks, such as 0.5, 1, 2, 3, 4, 5, 6, 7, 8 weeks or more. In some embodiments, rituximab is administered at a dose and dosing interval as described herein over a period of time, such as at least 2 weeks, such as at least 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 weeks or more. For example, rituximab is administered at a dose and dosing interval as described herein, for a total of at least 4 doses per treatment cycle (e.g., at least 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16 or more doses per treatment cycle).

In some embodiments, the CD20 antibody comprises ofatumumab. Ofatumumab is an anti-CD20 human IgG1K monoclonal antibody with a molecular weight of approximately 149 kDa. For example, ofatumumab is produced using transgenic mouse and hybridoma technology and expressed and isolated from a recombinant mouse cell line (NS0). See, for example, www.accessdata.fda.gov/drugsatfda_docs/label/2009/125326lbl.pdf and clinical trial ID NCT01363128, NCT01515176, NCT01626352 and NCT01397591. In embodiments, a CAR-expressing cell as described herein is administered to an individual in combination with ofatumumab. In embodiments, the subject is suffering from CLL or SLL.

In some embodiments, ofatumumab is administered as an intravenous infusion. For example, each infusion provides approximately 150-3000 mg (eg, approximately 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 600-650 650-700 700-750 750800 800-850 850-900 900-950 950-1000 1000-1200 1200-1400 1400-1600 1600-1800 20002200, 2200-2400, 2400-2600, 2600-2800 or 2800-3000 mg) ofatumumab. In embodiments, ofatumumab is administered at an initial dose of about 300 mg followed by 2000 mg, eg, for about 11 doses, eg, over 24 weeks. In some embodiments, ofatumumab is administered at a dosing interval of at least 4 days, such as 4, 7, 14, 21, 28, 35 days or more. For example, ofatumumab is administered at a dosing interval of at least 1 week, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 26, 28, 20, 26, 28, 30 weeks or more. In some embodiments, ofatumumab is administered at a dose and dosing interval as described herein over a period of time, e.g., at least 1 week, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 40, 50, 60 weeks or more, or 1, 2, 3, 4, 5 , 6, 7, 8, 9, 10, 11, 12 months or more, or 1, 2, 3, 4, 5 years or more. For example, ofatumumab is administered at a dose and at a dosing interval as described herein, for a total of at least 2 doses per treatment cycle (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13, 14, 15, 16, 18, 20 or more doses per treatment cycle).

In some cases, the CD20 antibody includes ocrelizumab. Ocrelizumab is a humanized anti-CD20 monoclonal antibody, for example, as described in clinical trials with identification numbers NCT00077870, NCT01412333, NCT00779220, NCT00673920, NCT01194570 and Kappos et al., Lancet., 19.378(2011):1779-87.

In some cases, the anti-CD20 antibody includes veltuzumab. Veltuzumab is a humanized anti-CD20 monoclonal antibody. See, for example, clinical trial identification no. NCT00547066, NCTO0546793, NCT01101581 and Goldenberg et al., Leuk. Lymphoma., 51 (5) (2010): 747-55.

In some instances, the anti-CD20 antibody includes GA101. GA101 (also called obinutuzumab or RO5072759) is a humanized anti-CD20 monoclonal antibody with a modified glycosylation pattern. See, for example, Robak., Curr. Opin. Investig. Drugs., 10.6(2009):588-96; clinical trials with identification number: NCT01995669, NCT01889797, NCT02229422 and NCT01414205; and www.accessdata.fda.gov/drugsatfda_docs/label/2013/125486s000lbl. pdf.

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In some instances, the CD20 antibody includes AME-133v. AME-133v (also called

LY2469298 or ocaratuzumab) is a humanized anti-CD20 IgG1 monoclonal antibody with increased affinity for the FcYRIIIa receptor and increased antibody dependent cellular cytotoxicity (ADCC) activity compared to rituximab. See, for example, Robak et al.,

BioDrugs, 25.1(2011):13-25 and Forero-Torres et al., Clin. Cancer Res., 18, 5 (2012):1395-403.

In some instances, the anti-CD20 antibody includes PRO131921. PRO131921 is a humanized anti-CD20 monoclonal antibody designed to better bind to FcyRIIIa and have increased ADCC compared to rituximab. See, for example, Robak et al., BioDrugs, 25.1(2011): 13-25 and Casulo et al., Clin Immunol., 154.1(2014): 37-46; and clinical trial ID NCT00452127.

In some instances, the anti-CD20 antibody includes TRU-015. TRU-015 is an anti-CD20 fusion protein derived from the domains of an anti-CD20 antibody. TRU-015 is smaller than monoclonal antibodies but retains Fc-mediated effector functions. See, for example, Robak et al., BioDrugs, 25.1(2011):13-25. TRU-015 contains a single chain variable fragment (scFv) of the anti-CD20 antibody associated with the hinge, CH2 and CH3 domains of human IgG1, but lacking the CH1 and CL domains.

In some embodiments, an anti-CD20 antibody described herein is conjugated or otherwise linked to a therapeutic agent, e.g., a chemotherapeutic agent (e.g., cytoxan, fludarabine, histone deacetylase inhibitor, demethylating agent, peptide vaccine, antitumor antibiotic, tyrosine kinase inhibitor, alkylating agent , a microtubule-acting agent, or an antimitotic agent), an antiallergic agent, an antinausea agent (or an antiemetic), an analgesic agent, or a cytoprotective agent of the present invention.

In embodiments, a CAR-expressing cell as described herein is administered to an individual in combination with a B-cell lymphoma 2 (BCL-2) inhibitor (eg, venetoclax, also referred to as ABT-199 or GDC-0199) and/or rituximab. In embodiments, a CAR-expressing cell as described herein is administered to an individual in combination with venetoclax and rituximab. Venetoclax is a small molecule compound that inhibits the anti-apoptotic protein BCL-2. Structure of venetoclax (4-(4-{[2-(4-chlorophenyl)4,4-dimethylcyclohekC-1-en-1-yl]methyl}π and [(tetrahydro-2H-pyran-4ylmethyl)amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]pyridin-5-ylκoxy)benzamide) is shown below.

In embodiments, the subject is suffering from CLL. In embodiments, the subject is suffering from recurrent CLL, eg, the subject has previously received therapy for cancer. In embodiments, venetoclax is administered at a dose of about 15-600 mg (e.g., 15-20, 20-50, 50-75, 75-100, 100-200, 200-300, 300-400, 400-500, or 500-600 mg), for example, every day. In embodiments, rituximab is administered at a dose of about 350-550 mg/m ² (eg, 350-375, 375-400, 400-425, 425-450, 450-475, or 475-500 mg/m ² ), such as intravenously , for example, every month.

In some embodiments, a CAR-expressing cell as described herein is administered in combination with an oncolytic virus. In embodiments, oncolytic viruses are capable of selectively replicating and activating the death or growth inhibition of a malignant cell. In some cases, oncolytic viruses have no or minimal effect on non-cancerous cells. Oncolytic virus includes, but is not limited to, oncolytic adenovirus, oncolytic herpes simplex viruses, oncolytic retrovirus, oncolytic parvovirus, oncolytic vaccinia virus, oncolytic Sindbis virus, oncolytic influenza virus, or oncolytic RNA virus (e.g., oncolytic reovirus, oncolytic Newcastle disease virus ( NDV), measles oncolytic virus, or oncolytic vesicular stomatitis virus (VSV)).

In some embodiments, the oncolytic virus is a virus, such as the recombinant oncolytic virus described in US2010/0178684 A1, incorporated herein by reference in its entirety. In some embodiments, the recombinant oncolytic virus contains a nucleic acid sequence (eg, a heterologous nucleic acid sequence) encoding an inhibitor of an immune or inflammatory response, for example, as described in US2010/0178684 A1, incorporated herein by reference in its entirety. In embodiments, the recombinant oncolytic virus, e.g., oncolytic NDV, contains a proapoptotic protein (e.g., apoptin), a cytokine (e.g., GM-CSF, interferon-gamma, interleukin 2 (IL-2), tumor necrosis factor alpha), an immunoglobulin (e.g., anti-ED-B fibronectin antibody), a tumor-associated antigen, a bispecific adapter protein (e.g., a bispecific antibody or antibody fragment directed against the NDV HN protein and a T-cell costimulatory receptor such as CD3 or CD28; or a fusion protein between human IL-2 and single chain antibody directed against the NDV HN protein). See, for example, Zamarin et al., Future Microbiol., 7.3(2012):347-67, incorporated herein by reference in its entirety. In some embodiments, the oncolytic virus is a chimeric oncolytic NDV as described in US 8591881 B2, US 2012/0122185 A1, or US 2014/0271677 A1, each of which is incorporated herein by reference in its entirety.

In some embodiments, the oncolytic virus includes a conditionally replicating adenovirus (CRAd) that is engineered to replicate only in malignant cells. See, for example, Alemany et al., Nature Biotechnol., 18(2000): 723-27. In some embodiments, the implementation of the oncolytic adenovirus includes the virus described in table. 1 on page 725 of Alemany et al., incorporated herein by reference in its entirety.

Illustrative oncolytic viruses include, but are not limited to, the following: oncolytic adenovirus group B (ColoAd1) (PsiOxus Therapeutics Ltd.) (see, for example, clinical trial ID: NCT02053220);

ONCOS-102 (formerly called CGTG-102), which is an adenovirus containing granulocyte-monocyte colony stimulating factor (GM-CSF) (Oncos Therapeutics) (see, for example, clinical trial ID: NCT01598129);

VCN-01, which is a genetically modified human oncolytic adenovirus encoding human hyaluronidase PH20 (VCN Biosciences, S.L.) (see, for example, clinical trial identifiers: NCT02045602 and NCT02045589);

the conditionally replicating adenovirus ICOVIR-5, which is a virus derived from wild-type human adenovirus serotype 5 (Had5) that has been modified to selectively replicate in malignant cells with a deregulated retinoblastoma/E2F pathway (Institut Catala d'Oncologia) (see, e.g., clinical trial ID: NCT01864759);

selivir (celyvir), which contains bone marrow-derived autologous mesenchymal stem cells (MSC) infected with ICOVIR5, an oncolytic adenovirus (Hospital Infantil Universitario Nino Jesύs, Madrid, Spain/Ramon Alemany) (see, for example, clinical trial ID: NCT01844661 );

CG0070, which is a conditionally replicating oncolytic adenovirus serotype 5 (Ad5) in which the human E2F-1 promoter directs the expression of essential genes of the E1a virus, thus limiting viral replication and cytotoxicity against tumor cells with a defective Rb pathway (Cold Genesys, Inc. ) (see, for example, clinical trial ID: NCT02143804); or

DNX-2401 (formerly Delta-24-RGD), which is an adenovirus engineered to be selectively expressed in pathway-deficient (Rb) retinoblastoma cells and infect cells that express certain RGD-binding integrins more efficiently (Clinica Universidad de Navarra, Universidad de Navarra/DNAtrix, Inc.) (see, for example, clinical trial ID: NCT01956734).

In some embodiments, the oncolytic virus of the present invention is administered by injection, such as subcutaneous, intra-arterial, intravenous, intramuscular, intrathecal, or intraperitoneal injection. In embodiments, the oncolytic virus of the present invention is administered intratumorally, transdermally, transmucosally, orally, intranasally, or via pulmonary administration.

In one embodiment, cells expressing the CARs of the present invention are administered to an individual in combination with a molecule that reduces the Treg cell population. Methods that lead to a decrease in the number (eg, depletion) of Treg cells are known in the art and include, for example, CD25 depletion, administration of cyclophosphamide, modulating GITR function. Without wishing to be bound by theory, it is believed that reducing the number of Treg cells in an individual prior to apheresis or prior to administration of a CAR expressing cell of the present invention reduces the number of unwanted immune cells (e.g., Treg) in the tumor microenvironment and reduces the individual's risk of recurrence. .

In one embodiment, a CAR expressing cell as described herein is administered to an individual in combination with a GITR-targeting and/or GITR-modulating molecule, such as a GITR agonist and/or an anti-GITR antibody that depletes regulatory T-178 040145 cells. (treg). In embodiments, cells expressing the CAR of the present invention are administered to an individual in combination with cyclophosphamide. In one embodiment, GITR binding molecules and/or molecules that modulate GITR functions (e.g., an agonist

GITR and/or Treg-depleting anti-GITR antibodies) are administered prior to administration of the CAR-expressing cell.

For example, in one embodiment, the GITR agonist may be administered prior to cell apheresis. In embodiments, cyclophosphamide is administered to the individual prior to administration (eg, infusion or reinfusion) of the CAR-expressing cell or prior to cell apheresis. In embodiments, the cyclophosphamide and the anti-GITR antibody are administered to the individual prior to administration (eg, infusion or reinfusion) of the CAR-expressing cell or prior to apheresis of the cells. In one embodiment, the subject is suffering from a cancer (eg, a solid cancer or a hematologic cancer such as ALL or CLL). In one embodiment, the individual suffers from CLL. In embodiments, the individual suffers from ALL. In embodiments, the individual suffers from a solid cancer, such as a solid cancer described in the present invention. Exemplary GITR agonists include, for example, GITR fusion proteins and anti-GITR antibodies (e.g., bivalent anti-GITR antibodies) such as, for example, the GITR fusion protein described in US Pat. PCT Nos. WO 2010/003118 and 2011/090754, or an anti-GITR antibody as described in, for example, US Pat. No. 7,025,962, EP No. 1,947,183B1, US Pat. No. 7,812,135, US Pat. 1866339, PCT Publication No. WO 2011/028683, PCT Publication No. WO 2013/039954, PCT Publication No. WO 2005/007190, PCT Publication No. WO 2007/133822, PCT Publication No. WO 2005/055808, PCT Publication No. WO 99/40196, PCT Publication No. WO 2001/03720; PCT Publication No. WO 99/20758; PCT Publication No. WO 2006/083289; PCT Publication No. WO 2005/115451;

In one embodiment, a CAR expressing cell as described herein is administered to an individual in combination with a GITR agonist, eg, a GITR agonist as described herein. In one embodiment, the GITR agonist is administered prior to the CAR expressing cell.

For example, in one embodiment, the GITR agonist may be administered prior to cell apheresis.

In one embodiment, a CAR expressing cell as described herein is administered to an individual in combination with an mTOR inhibitor, eg an mTOR inhibitor as described herein, eg rapalogue such as everolimus. In one embodiment, the mTOR inhibitor is administered prior to the CAR expressing cell. For example, in one embodiment, the mTOR inhibitor may be administered prior to cell apheresis.

In one embodiment, a CAR expressing cell as described herein is administered to an individual in combination with a protein tyrosine phosphatase inhibitor, eg, a protein tyrosine phosphatase inhibitor as described herein. In one embodiment, the protein tyrosine phosphatase inhibitor is an SHP-1 inhibitor, such as an SHP-1 inhibitor as described herein, such as, for example, sodium stibogluconate. In one embodiment, the protein tyrosine phosphatase inhibitor is an SHP-2 inhibitor.

In one embodiment, a CAR expressing cell as described herein can be used in combination with a kinase inhibitor. In one embodiment, the kinase inhibitor is a CDK4 inhibitor, such as a CDK4 inhibitor as described herein, such as a CD4/6 inhibitor such as, for example, 6-acetyl-8-cyclopentyl-5-methyl-2-(5- piperazin-1-yl-pyridin-2-ylamino)-8H-pyrido[2,3-d]pyrimidin-7-one (also referred to as palbociclib or PD0332991). In one embodiment, the kinase inhibitor is a BTK inhibitor, such as a BTK inhibitor as described herein, such as, for example, ibrutinib. In one embodiment, the kinase inhibitor is an mTOR inhibitor, such as an mTOR inhibitor as described herein, such as, for example, rapamycin, a rapamycin analog, OSI-027. An mTOR inhibitor may be, for example, an mTORC1 inhibitor and/or an mTORC2 inhibitor, such as an mTORC1 inhibitor and/or an mTORC2 inhibitor as described herein. In one embodiment, the kinase inhibitor is an MNK inhibitor, such as an MNK inhibitor as described herein, such as, for example, 4-amino-5-(4-fluoroanilino)pyrazolo-[3,4-d]pyrimidine. The MNK inhibitor may be, for example, an inhibitor of MNK1a, MNK1b, MNK2a and/or MNK2b. In one embodiment, the kinase inhibitor is a DGK inhibitor, such as a DGK inhibitor as described herein, such as, for example, DGKinhl (D5919) or DGKinh2 (D5794). In one embodiment, the kinase inhibitor is a CDK4 inhibitor selected from aloisin A; flavopyridol or HMR-1275, 2-(2-chlorophenyl)-5,7dihydroxy-8-[(3S,4R)-3-hydroxy-1-methyl-4-piperidinyl]-4-chromenone; crizotinib (PF-02341066;

- 179 040145 2-(2-chlorophenyl)-5,7-dihydroxy-8-[(2R,3S)-2-(hydroxymethyl)-1-methyl-3-pyrrolidinyl]4H-1-benzopyran-4-one hydrochloride (P276-00); 1-methyl-5-[[2-[5-(trifluoromethyl)-1 H-imidazol-2-yl]-4pyridinyl]oxy]-N-[4-(trifluoromethyl)phenyl]-1H-benzimidazol-2-amine (RAF265); indisulama (E7070); roscovitine (CYC202); palbociclib (PD0332991); dinacyclib (SCH727965); N[5-[[(5-tert-butyloxazol-2-yl)methyl]thio]thiazol-2-yl]piperidine-4-carboxamide (BMS 387032); 4-[[9-chloro-7-(2,6difluorophenyl)-5H-pyrimido[5,4-d][2]benzazepin-2-yl]amino]benzoic acid (MLN8054); 5-[3-(4,6difluoro-1H-benzimidazol-2-yl)-1H-indazol-5-yl]-N-ethyl-4-methyl-3-pyridinemethanamine (AG-024322); 4-(2,6-dichlorobenzoylamino)-1H-pyrazole-3-carboxylic acid N-(piperidin-4-yl)amide (AT7519); 4[2-methyl-1-(1-methylethyl)-1H-imidazol-5-yl]-N-[4-(methylsulfonyl)phenyl]-2-pyrimidinamine (AZD5438) and XL281 (BMS908662).

In one embodiment, the kinase inhibitor is a CDK4 inhibitor such as palbociclib (PD0332991) and palbociclib is administered at a dose of about 50, 60, 70, 75, 80, 90, 100, 105, 110, 115, 120, 125, 130 , 135 mg (eg, 75 mg, 100 mg, or 125 mg) every day for a specified period of time, such as every day for 14-21 days in a cycle of 28 days or every day for 7-12 days in a cycle of 28 days 21 days. In one embodiment, palbociclib is administered for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles.

In embodiments, a CAR-expressing cell as described herein is administered to an individual in combination with a cyclin-dependent kinase (CDK) 4 or 6 inhibitor, such as a CDK4 inhibitor or a CDK6 inhibitor as described herein. In embodiments, a CAR expressing cell as described herein is administered to an individual in combination with a CDK4/6 inhibitor (e.g., an inhibitor that targets CDK4 and CDK6), e.g., a CDK4/6 inhibitor as described herein. In one embodiment, the subject is suffering from MCL. MCL is an aggressive malignant tumor that responds poorly to currently available therapies, i.e. essentially incurable. In many cases, MCL cyclin D1 (CDK4/6 regulator) is expressed (eg, due to a chromosomal translocation involving the immunoglobulin and cyclin D1 genes) in MCL cells. Thus, without being bound by theory, it is believed that MCL cells are highly sensitive to CDK4/6 inhibition with high specificity (ie, minimal effect on normal cells of the immune system). CDK4/6 inhibitors alone had some efficacy in the treatment of MCL, but achieved only partial remission with a high relapse rate. An exemplary CDK4/6 inhibitor is LEE011 (also referred to as ribociclib), the structure of which is shown below.

Without wishing to be bound by theory, it is believed that by administering a CAR expressing cell of the present invention with a CDK4/6 inhibitor (e.g., LEE011 or another CDK4/6 inhibitor of the present invention), a higher response to treatment can be obtained, for example, with a higher remission rate and/or a lower relapse rate, for example, compared to CDK4/6 inhibitor alone.

In one embodiment, the kinase inhibitor is a BTK inhibitor selected from ibrutinib (PCI-32765); GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO4059; CNX-774 and LFM-A13. In a preferred embodiment, the BTK inhibitor does not reduce or inhibit the kinase activity of interleukin 2 inducible kinase (ITK) and is selected from GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059; CNX-774 and LFM-A13.

In one embodiment, the kinase inhibitor is a BTK inhibitor, such as ibrutinib (PCI-32765). In embodiments, a CAR expressing cell as described herein is administered to an individual in combination with a BTK inhibitor (eg, ibrutinib). In embodiments, a CAR expressing cell as described herein is administered to an individual in combination with ibrutinib (also referred to as PCI-32765). Ibrutinib structure -en-1-she) is presented below.

In embodiments, the individual suffers from CLL, mantle cell lymphoma (MCL), or small cell lymphoma (SLL). For example, an individual has a deletion in the short arm of the chromosome

- 180 040145 (del(17p), for example, in a leukemic cell). In other examples, the individual does not have del(17p). In embodiments, the individual suffers from recurrent CLL or SLL, eg, the individual has previously received cancer therapy (eg, previously received one, two, three, or four cancer therapies). In embodiments, the subject is suffering from refractory CLL or SLL. In other embodiments, the individual suffers from follicular lymphoma, such as relapsed or refractory follicular lymphoma. In some embodiments, ibrutinib is administered at a dose of about 300-600 mg/day (e.g., about 300-350, 350-400, 400-450, 450-500, 500-550, or 550-600 mg/day, for example, about 420 mg/day or approximately 560 mg/day), for example, orally. In embodiments, ibrutinib is administered at a dose of about 250, 300, 350, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600 mg (e.g., 250, 420, or 560 mg) every day for period of time, for example, every day for a cycle of 21 days or every day for a cycle of 28 days. In one embodiment, ibrutinib is administered for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles.

In some embodiments, ibrutinib is administered in combination with rituximab. See, for example, Burger et al. (2013) Ibrutinib In Combination With Rituximab (iR) Is Well Tolerated and Induces a High Rate Of Durable Remissions In Patients With High-Risk Chronic Lymphocytic Leukemia (CLL): New, Updated Results Of a Phase II Trial In 40 Patients, Abstract 675 presented at ^55th ASH Annual Meeting and Exposition, New Orleans, LA 7-10 Dec. Without being bound by theory, it is believed that the addition of ibrutinib enhances the T cell proliferative response and may shift T cells from the T helper 2 (Th2) to T helper 1 (Th1) phenotype. Th1 and Th2 are helper T cell phenotypes where Th1 directs different immune response pathways compared to Th2. The Th1 phenotype is associated with pro-inflammatory responses, for example to kill cells such as intracellular pathogens/viruses or malignant cells, or to perpetuate autoimmune responses. The Th2 phenotype is associated with eosinophil accumulation and anti-inflammatory responses.

In some embodiments of the methods, uses, and compositions herein, the BTK inhibitor is the BTK inhibitor described in WO 2015/079417, which is incorporated in its entirety. For example, in some embodiments, the BTK inhibitor is a compound of formula (I) or a pharmaceutically acceptable salt thereof:

R7

(I) where R1 is hydrogen, C1- _C6 alkyl optionally substituted with hydroxy;

R2 is hydrogen or halogen;

R3 is hydrogen or halogen;

R4 is hydrogen;

R5 is hydrogen or halogen;

or R4 and R5 are connected to each other and represent the bond -CH2-, -CH2-CH2-, -CH=CH-, -CH=CHCH2-; -CH2-CH=CH- or -CH2-CH2-CH2-;

R6 and R7 are independently H, C ₁ -C ₆ -alkyl optionally substituted hydroxyl, C ₃ -C ₆ -cycloalkyl optionally substituted with halogen or hydroxy, or halogen;

R8, R9, R, R', R10 and R11 are independently H or C ₁ -C ₆ alkyl optionally substituted with C ₁ -C ₆ alkoxy; or any two of R8, R9, R, R', R10 and R11, together with the carbon atom to which they are attached, may form a 3-6 membered saturated carbocyclic ring;

R12 is hydrogen or C ₁ -C ₆ alkyl optionally substituted with halogen or C 1 -C 6 alkoxy;

or R12 and any of R8, R9, R, R', R10, or R11, together with the atoms to which they are attached, may form a 4-, 5-, 6-, or 7-membered azacyclic ring, where the ring may optionally be substituted with halogen , cyano, hydroxyl, C ₁ -C ₆ alkyl or C ₁ -C ₆ alkoxy;

n is 0 or 1, and

R13 is C ₂ -C ₆ alkenyl optionally substituted with C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy or N,N-di-C ₁ -C ₆ alkylamino; C ₂ -C ₆ alkynyl optionally substituted with C ₁ -C ₆ alkyl or C 1 -C 6 alkoxy, or C ₂ -C ₆ alkylenyl oxide optionally substituted with C ₁ -C ₆ alkyl.

In some embodiments, the BTK inhibitor of Formula I is selected from:

- 181 040145

N-(3-(5-((1-acryloylazetidin-3-yl)oxy)-6·aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2fluorobenzamide; (E)-N-(3-(6-amino-5-((1-(but-2-enoyl)azetidin-3 yl)oxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl) -4-cyclopropyl-2fluorobenzamide; N-(3-(6-amino-5-((1-propioylazetidin-3 yl)oxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2fluorobenzamide; Ν-(3-(6-amino-5-((1-(but-2-inoyl)azetidin-3 yl)oxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl -2 fluorobenzamide; N-(3-(5-((1-acryloylpiperidin-4-yl)oxy)-6·aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2fluorobenzamide; N-(3-(6-amino-5-(2-(N-methylacrylamido)ethoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; (E)-N-(3-(6-amino-5-(2-(N-methylbut 2-enamido)ethoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4cyclopropyl-2- fluorobenzamide; N-(3-(6-amino-5-(2-(N-methylpropiolamido)ethoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; (E)-N-(3-(6-amino-5-(2-(4-methoxy N-methylbut-2-enamido)ethoxy)pyrimidin-4-yl)-5-fluoro-2methylphenyl)-4- cyclopropyl-2-fluorobenzamide; N-(3-(6-amino-5-(2·(N-methylbut-2-ynamido)ethoxy)pyrimidin-4-yl)-5-fluoro-2methylphenyl)-4-cyclopropyl-2-fluorobenzamide; Ν-(2-((4-amino-6-(3 (4-cyclopropyl-2-fluorobenzamido)-5-fluoro-2-methylphenyl)pyrimidine-5-182 040145 yl)oxy)ethyl)-N-methyloxirane -2-carboxamide; N-(2-((4-amino-6 ) (3-(6-cyclopropyl-8-fluoro-1-oxoisoquinoline-2(1H)yl)phenyl)pyrimidin-5-yl)oxy)ethyl)-N- methylacrylamide; N-(3-(5·(2-acrylamidoethoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; N-(3-(6-amino-5-(2·(N-ethylacrylamido)ethoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; N-(3-(6-amino-5-(2-(N-(2 fluoroethyl)acrylamido)ethoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)4-cyclopropyl-2-fluorobenzamide; N-(3-(5-((1·acrylamidocyclopropyl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2methylphenyl)-4-cyclopropyl-2-fluorobenzamide; (S)-N-(3-(5-(2·acrylamidopropoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)4-cyclopropyl-2-fluorobenzamide; (S)-N-(3-(6-amino-5-(2-(but-2·inamido)propoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; (S)-N-(3-(6-amino-5-(2-(N· methylacrylamido)propoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; (S)-N-(3-(6-amino-5-(2-(N-methylbut 2-ynamido)propoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4cyclopropyl-2- fluorobenzamide; N-(3-(6-amino-5-(3-(N-methylacrylamido)propoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; (S)-N-(3-(5-((1-acryloylpyrrolidin 2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide; (S)-N-(3-(6-amino-5-((1-(but-2 inoyl)pyrrolidin-2-yl)methoxy)pyrimidin-4-yl)-5-fluoro-2methylphenyl)-4 -cyclopropyl-2-fluorobenzamide; (S)-2- (3- (5- ( (1 acryloylpyrrolidin-2-yl) methoxy) - 6-aminopyrimidin-4-yl) -5-fluoro 2- (hydroxymethyl) phenyl) - 6-cyclopropyl-3, 4-dihydroisoquinoline 1(2H)-one; N-(2-((4-amino-6-(3-(6-cyclopropyl-1-oxo-3,4 dihydroisoquinolin-2 (1H)-yl)-5-fluoro-2(hydroxymethyl)phenyl)pyrimidine -5-yl)oxy)ethyl)-N-methylacrylamide; N-(3-(5-(((2S,4R)-1-acryloyl-4 methoxypyrrolidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2methylphenyl)-4-cyclopropyl -2-fluorobenzamide; N-(3-(6-amino-5 ) (((2S,4R)-1-(but-2-inoyl)-4-methoxypyrrolidin-2yl)methoxy)pyrimidin-4-yl)-5-fluoro-2 -methylphenyl)-4-cyclopropyl-2fluorobenzamide; 2-(3-(5-( ( (2S,4R)-1-acryloyl-4-methoxypyrrolidine

- 183 040145

2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2(hydroxymethyl)phenyl)-6-cyclopropyl-3,4-dihydroisoquinolin-1(2H)one; N-(3-(5-(((2S,4S)-1-acryloyl-4-methoxypyrrolidin-2yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4cyclopropyl-2 -fluorobenzamide; N-(3-(6-amino-5-(((2S,4S)-1-(but-2inoyl)-4-methoxypyrrolidin-2-yl)methoxy)pyrimidin-4-yl)-5-fluoro2-methylphenyl )-4-cyclopropyl-2-fluorobenzamide; N-(3-(5-(((2S,4R)1-acryloyl-4-fluoropyrrolidin-2-yl)methoxy)-6-aminopyrimidin-4yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl- 2-fluorobenzamide; N-(3-(6-amino-5-(((2S,4R)-1-(but-2-inoyl)-4-fluoropyrrolidin-2yl)methoxy)pyrimidin-4-yl)-5-fluoro-2 -methylphenyl)-4-cyclopropyl-2fluorobenzamide; (S)-N-(3-(5-((1-acryloylazetidin-2-yl)methoxy)-6aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2fluorobenzamide; (S)-N-(3-(6-amino-5-((1-propioylazetidin-2yl)methoxy)pyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2fluorobenzamide; (S)-2-(3-(5-((1-acryloylazetidin-2-yl)methoxy)-6aminopyrimidin-4-yl)-5-fluoro-2-(hydroxymethyl)phenyl)-6cyclopropyl-3,4- dihydroisoquinolin-1(2H)-one; (R)-N-(3-(5-((1acryloylazetidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2methylphenyl)-4-cyclopropyl-2-fluorobenzamide; (R)-N-(3-(5-((1acryloylpiperidin-3-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2methylphenyl)-4-cyclopropyl-2-fluorobenzamide; N-(3-(5-(((2R,3S)-1acryloyl-3-methoxypyrrolidin-2-yl)methoxy)-6-aminopyrimidin-4yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl-2 -fluorobenzamide; N-(3-(5(((2S,4R)-1-acryloyl-4-cyanopyrrolidin-2-yl)methoxy)-6aminopyrimidin-4-yl)-5-fluoro-2-methylphenyl)-4-cyclopropyl- 2fluorobenzamide or N-(3-(5-(((2S,4S)-1-acryloyl-4cyanopyrrolidin-2-yl)methoxy)-6-aminopyrimidin-4-yl)-5-fluoro-2methylphenyl)-4-cyclopropyl -2-fluorobenzamide.

Unless otherwise provided, the chemical terms used above in the description of the BTK inhibitor of formula I are used in accordance with their meanings as set forth in WO 2015/079417, which is incorporated herein in its entirety.

In one embodiment, the kinase inhibitor is an mTOR inhibitor selected from temsirolimus; ridaforolimus (1R,2R,4S)-4-[(2R)-2[{1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z,30S,32S,35R)- 1,18-dihydroxy-19,30-dimethoxy15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.0 ⁴ ' ⁹ ]hexatriacont-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyldimethylphosphinate, also known as AP23573 and MK8669; everolimus (RAD001); rapamycin (AY22989); simapimoda; (5-{2,4bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl}-2-methoxyphenyl)methanol (AZD8055); 2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methylpyrido[2,3d]pyrimidin-7(8H)-one (PF04691502) and N ² -[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2yl)morpholinium-4-yl]methoxy]butyl]-L-arginylglycyl-L -α-aspartyl-L-serine (SEQ ID NO: 64) internal salt (SF1126) and XL765.

In one embodiment, the kinase inhibitor is an mTOR inhibitor, e.g., rapamycin, and the rapamycin is administered at a dose of about 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg (e.g., 6 mg ) every day for a certain period of time, for example, every day for a cycle of 21 days or every day for a cycle of 28 days. In one embodiment, rapamycin is administered for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles. In one embodiment, the kinase inhibitor is an mTOR inhibitor, such as everolimus, and everolimus is administered at a dose of about 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 mg (for example, 10 mg) every day for a certain period of time, for example, every day for a cycle of 28 days. In one embodiment, everolimus is administered for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles.

In one embodiment, the kinase inhibitor is an MNK inhibitor selected from CGP052088; 4-amino-3-(lara-fluorophenylamino)pyrazolo[3,4-d]pyrimidine (CGP57380);

- 184 040145 cercosporamide; ETC-1780445-2 and 4-amino-5-(4-fluoroanilino)pyrazolo[3,4-d]pyrimidine.

In embodiments, a CAR expressing cell as described herein is administered to an individual in combination with a phosphoinositide 3-kinase (PI3K) inhibitor (e.g., a PI3K inhibitor as described herein, e.g., idelisib or duvelisib) and/or rituximab. In embodiments, a CAR-expressing cell as described herein is administered to an individual in combination with idelisib and rituximab. In embodiments, a CAR-expressing cell as described herein is administered to an individual in combination with duvelisib and rituximab. Idelalisib (also called GS-1101 or CAL-101; Gilead) is a small molecule compound that blocks the delta isoform of PI3K. The structure of idelicib (5-fluoro-3-phenyl-2-[(1S)-1-(7H-purin-6-ylamino)propyl]-4(3H)-quinazolinone) is shown below.

Duvelysib (also called IPI-145; Infinity Pharmaceuticals and Abbvie) is a small molecule compound that blocks PI3K-δ,γ. The structure of duvelisib (8-chloro-2-phenyl-3[(1S)-1 -(9H-purin-6-ylamino)ethyl]-1(2H)-isoquinolinone) is shown below.

In embodiments, the subject is suffering from CLL. In embodiments, the individual suffers from recurrent CLL, eg, the individual has previously received therapy against cancer (eg, previously administered anti-CD20 antibody or previously administered ibrutinib). For example, an individual has a deletion in the short arm of chromosome 17 (del(17p), for example, in a leukemic cell). In other examples, the individual does not have del(17p). In embodiments, the individual contains a leukemia cell containing a mutation in the immunoglobulin heavy chain variable region (IgVH) gene. In other embodiments, the individual does not contain a leukemia cell containing a mutation in the immunoglobulin heavy chain variable region (IgV _H ) gene. In embodiments, the individual has a deletion in the long arm of chromosome 11 (del(11q)). In other embodiments, the individual does not have del(11q). In embodiments, idelicib is administered at a dose of about 100-400 mg (e.g., 100-125, 125-150, 150-175, 175-200, 200-225, 225-250, 250-275, 275-300, 325-350 , 350-375 or 375-400 mg), for example, twice a day. In embodiments, duvelisib is administered at a dose of about 15-100 mg (eg, about 15-25, 25-50, 50-75, or 75-100 mg), eg, twice daily. In embodiments, rituximab is administered at a dose of about 350550 mg/m ² (eg, 350-375, 375-400, 400-425, 425-450, 450-475, or 475-500 mg/m ² ), eg, intravenously.

In one embodiment, the kinase inhibitor is a dual phosphatidylinositol 3-kinase (PI3K) and mTOR inhibitor selected from 2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy- 3-pyridinyl)-4-methylpyrido[2,3-d]pyrimidin-7(8H)-one (PF04691502); N-[4-[[4-(dimethylamino)-1-piperidinyl]carbonyl]phenyl]-N'-[4-(4,6-di-4-morpholinyl-1,3,5triazin-2-yl)phenyl ]urea (PF-05212384, PKI-587); 2-methyl-2-{4-[3-methyl-2-oxo-8-(quinolin-3-yl)2,3-dihydro-1H-imidazo[4,5-c]quinolin-1-yl]phenyl }propanenitrile (BEZ-235); apitolisib (GDC-0980, RG7422); 2,4-difluoro-N-{2-(methyloxy)-5-[4-(4-pyridazinyl)-6-quinolinyl]-3-pyridinyl}benzenesulfonamide (GSK2126458); maleic acid 8-(6-methoxypyridin-3-yl)-3-methyl-1-(4-(piperazin1-yl)-3-(trifluoromethyl)phenyl)-1H-imidazo[4,5-c]quinoline-2 (3H)-she (NVP-BGT226); 3-[4-(4morpholinylpyrido[3',2':4,5]furo[3,2-d]pyrimidin-2-yl]phenol (PI-103); 5-(9-isopropyl-8-methyl- 2morpholino-9H-purin-6-yl)pyrimidin-2-amine (VS-5584, SB2343) and N-[2-[(3,5-dimethoxyphenyl)amino]quinoxalin-3-yl]-4-[(4 -methyl-3-methoxyphenyl)carbonyl]aminophenylsulfonamide (XL765).

In embodiments, a CAR expressing cell as described herein is administered to an individual in combination with an anaplastic lymphoma kinase (ALK) inhibitor. Exemplary ALK kinases include, but are not limited to, crizotinib (Pfizer), ceritinib (Novartis), alectinib (Chugai), brigatinib (also referred to as AP26113; Ariad), entrectinib (Ignyta), PF-06463922

- 185 040145 (Pfizer), TSR-011 (Tesaro) (see, for example, clinical trial id no.

NCT02048488), CEP-37440 (Teva) and X-396 (Xcovery). In some embodiments, the subject is suffering from a solid cancer, such as a solid cancer as described herein, such as lung cancer.

The chemical name of crizotinib is 3-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]5-(1-piperidin-4-ylpyrazol1-4-yl)pyridin-2-amine. The chemical name of ceritinib is 5-chloro-N ² -[2-isopropoxy-5-methyl-4-(4-piperidinyl)phenyl]-N ⁴ -[2-(isopropylsulfonyl)phenyl]-

2,4-pyrimidinediamine. The chemical name of alectinib is 9-ethyl-6,6-dimethyl-8-(4morpholinopiperidin-1-yl)-11-oxo-6,11-dihydro-5H-benzo[b]carbazole-3-carbonitrile. The chemical name of brigatinib is 5-chloro-N ² -{4-[4-(dimethylamino)-1-piperidinyl]-2-methoxyphenyl}-N ⁴ -[2-(dimethylphosphoryl)phenyl]-2,4-pyrimidinediamine. The chemical name of entrectinib is N-(5-(3,5-difluorobenzyl)-1H-indazol-3-yl)-4-(4-methylpiperazin-1-yl)-2((tetrahydro-2H-pyran-4- yl)amino)benzamide. The chemical name of PF-06463922 is (10R)-7-amino-12-fluoro-2,10,16-trimethyl-15-oxo-10,15,16,17-tetrahydro-2H-8,4-(metheno) pyrazolo[4,3I][2,5,11]-benzoxadiazacyclotetradecin-3-carbonitrile. The chemical structure of CEP-37440 is (S)-2-((5-chloro-2-((6-(4-(2-hydroxyethyl)piperazin-1-yl)-1-methoxy-6,7,8, 9-tetrahydro-5Hbenzo[7]annulen-2-yl)amino)pyrimidin-4-yl)amino)-N-methylbenzamide. The chemical name of X-396 is (R)-6-amino-5-(1-(2,6-dichloro-3-fluorophenyl)ethoxy)-N-(4-(4-methylpiperazine-1carbonyl)phenyl)pyridazine- 3-carboxamide.

Drugs that inhibit calcium-dependent calcineurin phosphatase (cyclosporine and FK506) or inhibit p70S6 kinase, which is important for the growth factor-induced signaling cascade (rapamycin) can also be used (Liu et al., Cell, 66:807-815, 1991 ; Henderson et al., Immun. 73:316-321, 1991; Bierer et al., Curr. Opin. Immun., 5:763-773, 1993). In a further aspect, the cell compositions of the present invention may be administered to a patient in conjunction with (e.g., before, concurrently, or after) bone marrow transplantation, T-cell ablative therapy using chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide , and/or antibodies such as OKT3 or CAMPATH. In one aspect, the cell compositions of the present invention are administered after B-cell ablative therapy, such as agents that interact with CD20, such as Rituxan. For example, in one embodiment, individuals may receive standard treatment with high doses of chemotherapy followed by transplantation of peripheral blood stem cells. In certain embodiments, after transplantation, individuals receive an infusion of expanded immune cells of the present invention. In a further embodiment, the expanded cells are administered before or after surgery.

In embodiments, a CAR-expressing cell as described herein is administered to an individual in combination with an indolamine-2,3-dioxygenase (IDO) inhibitor. IDO is an enzyme that catalyses the breakdown of the amino acid L-tryptophan to kynurenine. Many cancers overexpress IDO, such as prostate cancer, colorectal cancer, pancreatic cancer, cervical cancer, gastric cancer, ovarian cancer, head cancer, and lung cancer. pDCs, macrophages and dendritic cells (DCs) can express IDO. Without wishing to be bound by theory, it is believed that a decrease in L-tryptophan (eg catalyzed by IDO) results in an immunosuppressive environment as a result of tolerance induction and T cell apoptosis. Thus, without being bound by theory, it is believed that an IDO inhibitor can enhance the efficacy of a CAR-expressing cell of the present invention, for example, by reducing the suppression or death of CAR-expressing immune cells. In embodiments, the subject is suffering from a solid tumor, such as a solid tumor as described herein, such as prostate cancer, colorectal cancer, pancreatic cancer, cervical cancer, gastric cancer, ovarian cancer, head cancer, and lung cancer. Exemplary IDO inhibitors include, but are not limited to, 1-methyltryptophan, indoxymod (NewLink Genetics) (see, e.g., Clinical Trial Identification No. NCT01191216; NCT01792050 and INCB024360 (Incyte Corp.) (See, e.g., Clinical Trial Identification No. No. NCT01604889; NCT01685255).

In embodiments, a CAR expressing cell as described herein is administered to an individual in combination with a myeloid suppressor cell (MDSC) modulator. MDSCs accumulate in the periphery and in tumor foci of many solid tumors. These cells suppress T-cell responses, thus hindering the effectiveness of therapy based on CAR-expressing cells. Without being bound by theory, it is believed that the introduction of an MDSC modulator will increase the efficiency of the CAR-expressing cell of the present invention. In one embodiment, the individual suffers from a solid tumor, such as a solid tumor as described herein, such as glioblastoma. Exemplary MDSC modulators include, but are not limited to, MCS110 and BLZ945. MCS110 is a monoclonal antibody (mAb) against macrophage colony stimulating factor (M-CSF). See, for example, clinical trial ID NCT00757757. BLZ945 is a low molecular weight

- 186 040145 colony stimulating factor receptor 1 (CSF1R) inhibitor. See, for example, Pyonteck et al., Nat.

Med., 19(2013):1264-72. The structure of BLZ945 is shown below.

In embodiments, a CAR expressing cell as described herein is administered to an individual in combination with an agent that inhibits or reduces the activity of immunosuppressive plasma cells. Immunosuppressive plasma cells have been shown to interfere with T-cell dependent immunogenic chemotherapy such as oxaliplatin (Shalapour et al., Nature, 2015, 521:94-101). In one embodiment, the immunosuppressive plasma cells may express one or more of IgA, interleukin (IL)-10, and PD-L1. In one embodiment, the agent is a CD19 CAR expressing cell or a CAR BCMA expressing cell.

In some embodiments, a CAR expressing cell as described herein is administered to an individual in combination with an interleukin 15 (IL-15) polypeptide, an interleukin 15 receptor alpha (IL-15Ra) polypeptide, or in combination with an IL-15 polypeptide and an IL15Ra polypeptide, e.g., hetIL-15 (Admune Therapeutics, LLC). hetIL-15 is a heterodimeric non-covalent complex of IL-15 and IL-15Ra. hetIL-15 is described, for example, in U.S. 8124084, U.S. 2012/0177598, U.S. 2009/0082299, U.S. 2012/0141413 and U.S. 2011/0081311 included in the present description by reference. In embodiments, het-IL-15 is administered subcutaneously. In embodiments, the subject is suffering from a cancer, such as a solid cancer, such as melanoma or colon cancer. In embodiments, the subject is suffering from a metastatic cancer.

In embodiments, an individual suffering from a disease described herein, e.g., a hematological disorder, e.g., AML or MDS, is administered a CAR-expressing cell described herein in combination with an agent, e.g., a cytotoxic or chemotherapeutic agent, biological therapy (e.g., , an antibody, such as a monoclonal antibody or cell therapy), or an inhibitor (eg, a kinase inhibitor). In embodiments, the individual is administered a CAR expressing cell as described herein in combination with a cytotoxic agent, e.g., CPX-351 (Celator Pharmaceuticals), cytarabine, daunorubicin, vosaroxin (Sunesis Pharmaceuticals), sapacitabine (Cyclacel Pharmaceuticals), idarubicin, or mitoxantrone. CPX-351 is a liposomal formulation containing cytarabine and daunorubicin in a 5:1 molar ratio. In embodiments, the individual is administered a CAR-expressing cell as described herein in combination with a hypomethylating agent, eg, a DNA methyltransferase inhibitor, eg, azacitidine or decitabine. In embodiments, an individual is administered a CAR expressing cell as described herein in combination with a biological therapy, e.g., antibody therapy or cell therapy, e.g. ), SGN-CD33A (Seattle Genetics), or gemtuzumab ozogamicin (Mylotarg; Pfizer). SGN-CD33A is an antibody drug conjugate (ADC) containing a pyrrolobenzodiazepine dimer that is linked to an anti-CD33 antibody. Actimab-A is an anti-CD33 antibody (lintuzumab) labeled with actinium. IPH2102 is a monoclonal antibody that targets the killer cell immunoglobulin-like receptor (KIR). In embodiments, an individual is administered a CAR expressing cell as described herein in combination with an FLT3 inhibitor, e.g. AKN-028 (Akinion Pharmaceuticals) or ASP2215 (Astellas). In embodiments, the individual is administered a CAR expressing cell as described herein in combination with an isocitrate dehydrogenase (IDH) inhibitor, such as AG-221 (Celgene/Agios) or AG-120 (Agios/Celgene). In embodiments, the individual is administered a CAR-expressing cell as described herein in combination with a cell cycle regulator, e.g., a polo-like kinase 1 (Plk1) inhibitor, e.g. for example, alvocidib (Tolero Pharmaceuticals/Sanofi Aventis). In embodiments, the individual is administered a CAR-expressing cell as described herein in combination with an inhibitor of the B-cell receptor signaling network, e.g., a B-cell lymphoma 2 (Bcl-2) inhibitor, e.g., venetoclax (Abbvie/Roche), or a Bruton's tyrosine kinase (Btk) inhibitor such as ibrutinib (Pharmacyclics/Johnson and Johnson Janssen Pharmaceuticals). In embodiments, the individual is administered a CAR expressing cell as described herein in combination with an aminopeptidase M1 inhibitor, eg, tosedostat (CTI BioPharma/Vernalis); a histone deacetylase (HDAC) inhibitor such as pracinostat (MEI Pharma); a multikinase inhibitor such as ri-187 040145 gosertib (Onconova Pharmaceuticals/Baxter/SymBio) or a reversible CXCR4 peptide agonist such as BL-8040 (BioLineRx).

In another embodiment, individuals receive an infusion of CAR expressing cell compositions of the present invention prior to cell transplantation, eg, allogeneic stem cell transplantation. In a preferred embodiment, CAR-expressing cells transiently express NKR-CAR, e.g. KIR-CAR, e.g., by electroporation of mRNA encoding NKR-CAR, e.g. KIR-CAR, such that tumor antigen expression is terminated before donor stem cell infusion to prevent deficiency. transplant.

Some patients may experience allergic reactions to the compounds of the present invention and/or other anti-cancer agent(s) during or after administration; thus, antiallergic agents are often administered to minimize the risk of an allergic reaction. Suitable antiallergic agents include corticosteroids such as dexamethasone (eg Decadron®), beclomethasone (eg Beclovent®), hydrocortisone (also known as cortisone, sodium hydrocortisone succinate, sodium hydrocortisone phosphate and sold under the trade names Ala-Cort®, hydrocortisone phosphate , Solu-Cortef®, Hydrocort Acetate® and Lanacort®), prednisone (sold under the trade names Delta-Cortel®, Orapred®, Pediapred® and Prelone®), prednisone (sold under the trade names Deltasone®, Liquid Red®, Meticorten® and Orasone®), methylprednisolone (also known as 6-methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, sold under the tradenames Duralone®, Medralone®, Medrol®, M-Prednisol® and Solu-Medrol®); antihistamines such as diphenhydramine (eg Benadryl®), hydroxyzine and cyproheptadine; and bronchodilators such as beta-adrenergic receptor agonists, albuterol (eg Proventil®) and terbutaline (Brethine®).

Some patients may experience nausea during and after administration of a compound of the present invention and/or other anti-cancer agent(s); thus, antiemetics are used to prevent nausea (upper stomach) and vomiting. Suitable antiemetics include aprepitant (Emend®), ondansetron (Zofran®), granisetron HCl (Kytril®), lorazepam (Ativan®), dexamethasone (Decadron®), prochlorpemazine (Compazine®), casopitant (Rezonic® and Zunrisa®), and their combinations.

In order to make the patient feel more comfortable, a drug is often prescribed to relieve the pain experienced during the treatment period. Typically, generic over-the-counter analgesics such as Tylenol® are used. However, for moderate to severe pain, opioid analgesics such as hydrocodone/paracetamol or hydrocodone/acetaminophen (e.g. Vicodin®), morphine (e.g. Astramorph® or Avinza®), oxycodone (e.g. OxyContin® or Percocet®), oxymorphone hydrochloride (Opana®), and fentanyl (eg Duragesic®).

To protect normal cells from drug toxicity and to limit organ toxicity, cytoprotective agents (such as neuroprotectors, free radical scavengers, cardioprotectors, anthracycline-based hemorrhagic agents, nutrients, etc.) can be used as adjuvant therapy. Suitable cytoprotective agents include amifostine (Ethyol®), glutamine, dimesna (Tavocept®), mesna (Mesnex®), dexrazoxane (Zinecard® or Totect®), xaliproden (Xaprila®), and leucovorin (also known as leucovorin calcium, citrovorum factor and folinic acid).

The structure of active compounds, which can be identified by code numbers, generic or trade names, can be obtained from the current edition of the standard reference book The Merck Index or from databases such as Patents International (eg IMS World publications).

The above compounds that can be used in combination with a compound of the present invention can be prepared and administered as described in the art, such as in the documents cited above.

In one embodiment, the present invention relates to pharmaceutical compositions comprising at least one compound of the present invention (e.g., a compound of the present invention), or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier suitable for administration to a human or animal subject, separately or together with other anti-cancer agents.

In one embodiment, the present invention provides methods for treating human or animal subjects suffering from a cell proliferative disease, such as cancer. The present invention relates to methods of treating a human or animal subject in need of such treatment, comprising administering to the subject a therapeutically effective amount of a compound of the present invention.

- 188 040145 (eg, compounds of the present invention) or a pharmaceutically acceptable salt thereof, alone or in combination with other anti-cancer agents.

In particular, the compositions are formulated together as a therapeutic or separately administered combination.

In combination therapy, the compound of the present invention and the other anti-cancer agent(s) may be administered simultaneously, simultaneously, or sequentially without any particular time limitation, wherein such administration provides therapeutically effective levels of the two compounds in the patient.

In a preferred embodiment, the compound of the present invention and the other anti-cancer agent(s) are generally administered sequentially in any order by infusion or orally. The dosage regimen may vary depending on the stage of the disease, the physical fitness of the patient, the safety performance of individual drugs and the tolerability of individual drugs, as well as other criteria well known to the attending physician(s) and physician(s)-therapist(s) administering combination. The compound of the present invention and the other anti-cancer agent(s) may be administered minutes apart, hours, days or even weeks apart, depending on the particular cycle used for treatment. In addition, a cycle may include administering one drug more frequently than another during a treatment cycle and at different doses per drug administration.

Another aspect of the present invention relates to kits that contain one or more compounds of the present invention and a combination partner as described herein. Representative kits contain (a) a compound of the present invention, or a pharmaceutically acceptable salt thereof, (b) at least one combination partner, for example, as indicated above, thus such a kit may contain a package insert or other label, including instructions for introduction.

The compound of the present invention can also be used advantageously in combination with known therapeutic methods, for example hormonal administration or in particular radiation. The compound of the present invention can in particular be used as a radiosensitizing agent, especially for the treatment of tumors which are characterized by low sensitivity to radiation therapy.

In one embodiment, an agent can be administered to an individual that reduces or improves a side effect associated with administration of a CAR expressing cell. Side effects associated with administration of a CAR expressing cell include, but are not limited to, CRS and hemophagocytic lymphohistiocytosis (HLH), also referred to as macrophage activation syndrome (MAS). The symptoms of CRS include high fevers, nausea, transient hypotension, hypoxia, and the like. CRS may include clinical constitutional signs and symptoms such as fever, fatigue, anorexia, myalgias, arthralgias, nausea, vomiting, and headache. CRS may include clinical skin signs and symptoms such as rash. CRS may include clinical gastrointestinal signs and symptoms such as nausea, vomiting, and diarrhea. CRS may include clinical respiratory signs and symptoms such as tachypnea and hypoxemia. CRS may include clinical cardiovascular signs and symptoms such as tachycardia, increased pulse pressure, hypotension, increased cardiac output (early) and potentially reduced cardiac output (late). CRS may include clinical signs and symptoms of blood clotting such as elevated d-dimer levels, hypofibrinogenemia with or without bleeding. CRS may include clinical renal signs and symptoms such as azotemia. CRS may include clinical hepatic signs and symptoms such as elevated transaminases and hyperbilirubinemia. CRS may include clinical neurological signs and symptoms such as headache, mental status changes, confusion, delirium, difficulty finding words or marked aphasia, hallucinations, tremors, dysmetria, altered gait, and seizures.

Thus, the methods of the present invention may include administering a CAR expressing cell of the present invention to an individual and further administering one or more agents for managing elevated levels of soluble factor resulting from treatment with the CAR expressing cell. In one embodiment, the soluble factor elevated in the individual is one or more of IFN-γ, TNFa, IL-2, and IL6. In one embodiment, the factor elevated in the individual is one or more of IL-1, GM-CSF, IL-10, IL-8, IL-5, and fractalkine. Thus, the agent administered to treat such a side effect may be an agent that neutralizes one or more of these soluble factors. In one embodiment, the agent that neutralizes one or more of these soluble forms is an antibody or an antigen-binding fragment thereof. Examples of such agents include, but are not limited to, a steroid (eg, a corticosteroid), a TNFa inhibitor, and an IL-6 inhibitor. An example of a TNFa inhibitor is an antibody molecule

- 189 040145 against TNFa such as infliximab, adalimumab, certolizumab pegol and golimumab. Another example of a TNFa inhibitor is a fusion protein such as entanercept. The small molecule TNFa inhibitor includes, but is not limited to, xanthine derivatives (eg, pentoxifylline) and bupropion. An example of an IL-6 inhibitor is an anti-IL-6 antibody molecule such as tocilizumab (toc), sarilumab, elsilimomub, CNTO 328, ALD518/BMS-945429, CNTO 136, CPSI-2364, CDP6038, VX30, ARGX-109, FE301 and FM101. In one embodiment, the anti-IL-6 antibody molecule is tocilizumab. An example of an IL-1R based inhibitor is anakinra.

In a particular embodiment, the subject is administered a corticosteroid, such as, for example, along with others methylprednisolone, hydrocortisone.

In some embodiments, the individual is administered a vasoconstrictor such as, for example, norepinephrine, dopamine, phenylephrine, epinephrine, vasopressin, or a combination thereof.

In one embodiment, an antipyretic may be administered to an individual. In one embodiment, an analgesic agent may be administered to an individual.

In one embodiment, an agent may be administered to an individual that enhances the activity of a CAR-expressing cell. For example, in one embodiment, the agent may be an agent that inhibits an inhibitory molecule, eg, the agent is a checkpoint inhibitor. inhibitory molecules such as

Programmed Death 1 (PD1), in some embodiments, may reduce the ability of a CAR-expressing cell to increase an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR beta. Inhibition of the inhibitory molecule, for example, through inhibition at the DNA, RNA or protein level, can optimize the performance of a CAR-expressing cell. In embodiments, an inhibitory nucleic acid, e.g., an inhibitory nucleic acid, e.g., dsRNA, e.g., siRNA or shRNA, regularly arranged short palindromic repeats (CRISPR), effector nuclease like activators transcription (TALEN), or zinc finger endonuclease (ZFN), for example, as described herein. In one embodiment, the inhibitor is shRNA. In one embodiment, the inhibitory molecule is inhibited in a CAR-expressing cell. In these embodiments, the dsRNA molecule that inhibits the expression of the inhibitory molecule is linked to a nucleic acid encoding component, eg, all components of a CAR.

In one embodiment, a nucleic acid molecule encoding a dsRNA molecule that inhibits the expression of a molecule that modulates or regulates, e.g., inhibits, T cell function is operably linked to a promoter, e.g., a promoter derived from H1 or U6, thus that a dsRNA molecule is expressed that inhibits the expression of a molecule that modulates or regulates, eg, inhibits T cell function, eg, is expressed in a CAR expressing cell. See, for example, Tiscornia G., Development of Lentiviral Vectors Expressing siRNA, Chapter 3, in Gene Transfer: Delivery and Expression of DNA and RNA (eds. Friedmann and Rossi). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2007; Brummelkamp T.R. et al. (2002) Science, 296: 550-553; Miyagishi M. et al., (2002) Nat. Biotechnol., 19: 497500. In one embodiment, a nucleic acid molecule encoding a dsRNA molecule that inhibits the expression of a molecule that modulates or regulates, e.g., inhibits T cell function, is contained in the same vector, e.g., a lentiviral vector, which contains a nucleic acid molecule encoding a component, for example, all components of CAR. In such an embodiment, a nucleic acid molecule encoding a dsRNA molecule that inhibits the expression of a molecule that modulates or regulates, e.g., inhibits T cell function, is located in a vector, e.g., a lentiviral vector, 5' or 3' end to a nucleic acid encoding a component, eg all components of CAR. A nucleic acid molecule encoding a dsRNA molecule that inhibits the expression of a molecule that modulates or regulates, e.g., inhibits T cell function, may be transcribed in the same or different direction as the nucleic acid encoding a component, e.g., all components of CAR.

In one embodiment, a nucleic acid molecule encoding a dsRNA molecule that inhibits the expression of a molecule that modulates or regulates, for example, inhibits T cell function, is contained in a vector other than a vector that contains a nucleic acid molecule encoding a component, for example, all CAR components. In one embodiment, a nucleic acid molecule encoding a dsRNA molecule that inhibits the expression of a molecule that modulates or regulates, eg, inhibits T cell function, is transiently expressed in a CAR expressing cell. In one embodiment, a nucleic acid molecule encoding a dsRNA molecule that inhibits the expression of a molecule that modulates or regulates, e.g., inhibits T cell function, is stably integrated into

- 190 040145 the genome of a CAR-expressing cell. In FIG. 44A-44E illustrate exemplary vectors for expressing a component, eg, all components, of a CAR with a dsRNA molecule that inhibits the expression of a molecule that modulates or regulates, eg, inhibits T cell function.

The following are examples of dsRNA molecules suitable for inhibiting the expression of a molecule that modulates or regulates, for example, inhibits T cell function, where the molecule that modulates or regulates, for example, inhibits T cell function, is PD-1.

In table. 7 below are the names of the PDCD1 RNAi (PD1) agents (derived from their positions in the mouse PDCD1 gene sequence NM_008798.2) along with SEQ ID NOs: 216-263. representing the DNA sequence. The sense (S) and antisense (AS) sequences are provided as containing 19 monomers and 21 monomer sequences in this table. It is also indicated that the position (PoS, eg 176) is derived from the position number in the sequence of the mouse PDCD1 gene NM_008798.2. SEQ ID NOs are listed in groups of 12, which correspond to semantic 19 of SEQ ID NOs: 65-76; semantic 21 SEQ ID NO: 77-88; antisense 21 SEQ ID NO: 89-100; antisense 19 SEQ ID NOs: 101-112.

Table 7

Mouse PDCD1 (PD1) shRNA sequences

Position in NM_0087 98.2 Region -target Smyslovaya 19 semantic 21 Antisense aya 21 Antisense aya 19 176 CDS GGAGGTCCCTCCA SSTTSTA (SEQ ID NO: 65) CTGGAGGTCCC TCACCTTCTA (SEQ ID NO: 77) TAGAAGGTGAG GGACCTCAG (SEQ ID NO: 89) TAGAAGGTGAG GGACCTC (SEQ ID NO: 101) 260 CDS CGGAGGATCTTA TGCTGAA (SEQ ID NO: 66) GTCGGAGGATC TTATGCTGAA (SEQ ID NO: 78) TTCAGCATAAG ATCCTCCGAC (SEQ ID NO: 90) TTCAGCATAAG ATCCTCCG (SEQ ID NO: 102) 359 CDS CCCGCTTCCAGA TCATACA (SEQ ID NO: 67) TGCCCGCTTCC AG AT CAT AC A (SEQ ID NO: 79) TGTATGATCGTG GAAGCGGGCA (SEQ ID NO: 91) TGTATGATCGTG GAAGCGGG (SEQ ID NO: 103)

- 191 040145

528 CDS GGAGACCTCAC AAGATAT (SEQ ID NO: 68) CTGGAGACCTC AACAAGATAT (SEQ ID NO: 80) ATATCTTGTTG AGGTCTCAG (SEQ ID NO: 92) ATATCTTGTTG AGGTCTC (SEQID NO: 104) 581 CDS AAGGCATGGTCA TTGGTAT (SEQ ID NO: 69) TCAAGGCATGG TCATTGGTAT (SEQ ID NO: 81) ATACCAATGAC CATGCCTTGA (SEQ ID NO: 93) ATACCAATGAC CATGCCTT (SEQ ID NO: 105) 584 CDS GCATGGTCATTG GTATCAT (SEQ ID NO: 70) AGGCATGGTCA TTGGTATCAT (SEQ ID NO: 82) ATGATACCAAT GACCATGCCT (SEQ ID NO: 94) ATGATACCAAT GACCATGC (SEQ ID NO: 106) 588 CDS GGTCATTGGTAT CATGAGT (SEQ ID NO: 71) ATGGTCATTGG TATCATGAGT (SEQ ID NO: 83) ATGGTCATTGG TATCATGAGT (SEQ ID NO: 95) ATGGTCATTGG TATCATGA (SEQ ID NO: 107) 609 CDS CCTAGTGGGTAT CCCTGTA (SEQ ID NO: 72) GCCCTAGTGGG TATCCCTGTA (SEQ ID NO: 84) GCCCTAGTGGG TATCCCTGTA (SEQ ID NO: 96) GCCCTAGTGGG TATCCCTG (SEQ ID NO: 108) 919 CDS GAGGATGGACAT TGTTTCTT (SEQ ID NO: 73) ATGAGGATGGA CATTGTTTCTT (SEQ ID NO: 85) ATGAGGATGGA CATTGTTTCTT (SEQ ID NO: 97) ATGAGGATGGA CATTGTTC (SEQ ID NO: 109) 1021 3'-UTR GCATGCAGGCTA CAGTTCA (SEQ ID NO: 74) GAGCATGCAGG CTACAGTTCA (SEQ ID NO: 86) GAGCATGCAGG CTACAGTTCA (SEQ ID NO: 98) GAGCATGCAGG CTACAGTT (SEQ ID NO: 110) 1097 3'-UTR CCAGCACATGCA CTGTTGA (SEQ ID NO: 75) TTCCAGCACAT GCACTGTTGA (SEQ ID NO: 87) TTCCAGCACAT GCACTGTTGA (SEQ ID NO: 99) TTCCAGCACAT GCACTGTT (SEQ ID NO: 111) 1101 3'-UTR CACATGCACTGT TGAGTGA (SEQ ID NO: 76) AGCACATGCAC TGTTGAGTGA (SEQ ID NO: 88) AGCACATGCAC TGTTGAGTGA (SEQ ID NO: 100) AGCACATGCAC TGTTGAGT (SEQ ID NO: 112)

In table. 8 below shows the names of PDCD1-based RNAi (PD1) agents derived from their position in the sequence of the human PDCD1 gene, along with SEQ ID NOs: 264-311 representing the DNA sequence. The sense (S) and antisense (AS) sequences are provided as containing 19 monomers and 21 sequence monomers. SEQ ID NOs are listed in groups of 12 which correspond to semantic 19 of SEQ ID NOs: 113-124; semantic 21 SEQ ID NO: 125-136; antisense 21 SEQ ID NOs: 137-148; antisense 19 SEQ ID NO: 149-160.

- 192 040145

Table 8

Human PDCD1 (PD1) shRNA sequences

Position in NM_0050 18.2 Region -target Smyslovaya 19 Antisense aya 19 semantic 21 Antisense aya 21 145 CDS GGCCAGGATGGT TCTTAGA (SEQ ID NO: 113) TCTAAGAACCA TCCTGGCC (SEQ ID NO: 125) GCGGCCAGGAT GGTTCTTAGA (SEQ ID NO: 137) TCTAAGAACCA TCCTGGCCGC (SEQ ID NO: 149) 271 CDS GCTTCGTGCTAA ACTGGTA (SEQ ID NO: 114) TACCAGTTTAG CACGAAGC (SEQ ID NO: 126) GAGCTTCGTGC TAAACTGGTA (SEQ ID NO: 138) TACCAGTTTAG CACGAAGCTC (SEQ ID NO: 150) 393 CDS GGGCGTGACTTC CACATGA (SEQ ID NO: 115) TCATGTGGAAG TCACGCCC (SEQ ID NO: 127) ACGGGCGTGAC TTCCACATGA (SEQ ID NO: 139) TCATGTGGAAG TCACGCCCGT (SEQ ID NO: 151) 1497 3'-UTR CAGGCCTAGAGA AGTTTC (SEQ ID NO: 116) TGAAACTTCTC TAGGCCTG (SEQ ID NO: 128) TGCAGGCCTAG AGAAGTTTTCA (SEQ ID NO: 140) TGAAACTTCTC TAGGCCTGCA (SEQ ID NO: 152) 1863 3'-UTR CTTGGAACCCAT TCCTGAA (SEQ ID NO: 117) TTCAGGAATGG GTTCCAAG (SEQ ID NO: 129) TCCTTGGAACC CATTCCTGAA (SEQ ID NO: 141) TTCAGGAATGG GTTCCAAGGA (SEQ ID NO: 153) 1866 3'-UTR GGAACCCATTCC TGAAATT (SEQ ID NO: 118) AATTTCAGGAA TGGGTTCC (SEQ ID NO: 130) TTGGAACCCAT TCCTGAAATT (SEQ ID NO: 142) AATTTCAGGAA TGGGTTCCAA (SEQ ID NO: 154) 1867 3'-UTR GAACCCATTCCT GAAATTA (SEQ ID NO: 119) TAATTTCAGGA ATGGGTTC (SEQID NO: 131) TGGAACCCATT CCTGAAATTA (SEQ ID NO: 143) TAATTTCAGGA ATGGGTTCCA (SEQ ID NO: 155) 1868 3'-UTR AACCCATTCCTG AAATTAT (SEQ ID NO: 120) ATAATTTTCAGG AATGGGTT (SEQ ID NO: 132) GGAACCCATTC CTGAAATTAT (SEQ ID NO: 144) ATAATTTTCAGG AATGGGTTCC (SEQ ID NO: 156) 1869 3'-UTR ACCCATTCCTGA AATTATT (SEQ ID NO: 121) AATAATTTCAG GAATGGGT (SEQ ID NO: 133) GAACCCATTCC TGAAATTATT (SEQ ID NO: 145) AATAATTTCAG GAATGGGTTC (SEQ ID NO: 157) 1870 3'-UTR CCCATTCCTGAA ATTATTT (SEQ ID NO: 122) AAATAATTTCA GGAATGGG (SEQ ID NO: 134) AACCCATTCCT GAAATTATTT (SEQ ID NO: 146) AAATAATTTCA GGAATGGGTT (SEQ ID NO: 158) 2079 3'-UTR CTGTGGTTCTAT TATATTA (SEQ ID NO: 123) TAATATAATAG AACCACAG (SEQ ID NO: 135) CCCTGTGGTTC TATTATATTA (SEQ ID NO: 147) TAATATAATAG AACCACAGGG (SEQ ID NO: 159) 2109 3'-UTR AAATATGAGGC ATGCTAA (SEQ ID NO: 124) TTAGCATGCTC TCATATTT (SEQ ID NO: 136) TTAAATATGAG AGCATGCTAA (SEQ ID NO: 148) TTAGCATGCTC TCATATTTAA (SEQ ID NO: 160)

In one embodiment, the inhibitor of the inhibitory signal may be a

- 193 040145 fight, for example, an antibody or antibody fragment that binds to an inhibitory molecule. For example, the agent may be an antibody or antibody fragment that binds to PD1, PD-L1, PD-L2, or CTLA4 (e.g., ipilimumab (also referred to as MDX-010 and MDX-101, and commercially available as Yervoy®; Bristol - Myers Squibb; tremelimumab (an IgG2 monoclonal antibody available from Pfizer, formerly known as ticilimumab CP-675,206). In one embodiment, the agent is an antibody or antibody fragment that binds to TIM3. In one embodiment, the agent is is an antibody or antibody fragment that binds to LAG3.In embodiments, an agent that enhances the activity of a CAR-expressing cell, e.g., an inhibitor of an inhibitory molecule, is administered in combination with an allogeneic CAR, e.g. the allogeneic CAR section in this specification).

PD-1 is an inhibitory member of the CD28 receptor family, which also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed on activated B cells, T cells and myeloid cells (Agata et al., 1996, Int. Immunol., 8:765-75). Two ligands for PD-1, PD-L1 and PD-L2, have been shown to suppress T cell activation when bound to PD-1 (Freeman et al., 2000 J. Exp. Med., 192:1027-34; Latchman et al., 2001 Nat. Immunol., 2:261-8; Carter et al., 2002 Eur. J. Immunol., 32:634-43). PD-L1 is abundant in human cancers (Dong et al., 2003 J. Mol. Med., 81:281-7; Blank et al., 2005 Cancer Immunol. Immunother., 54:307-314; Konishi et al., 2004 Clin Cancer Res. 10:5094). Immune suppression can be abolished by inhibiting the local interaction of PD-1 with PD-L1.

Antibodies, antibody fragments and other inhibitors of PD-1, PD-L1 and PD-L2 are available in the art and can be used in combination with the CAR of the present invention described in the present invention. For example, nivolumab (also referred to as BMS-936558 or MDX1106; Bristol-Myers Squibb) is a wholly human IgG4 monoclonal antibody that specifically blocks PD-1. Nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PD-1 are described in US 8008449 and WO 2006/121168. Pidilizumab (CT-011; Cure Tech) is a humanized IgG1k monoclonal antibody that binds to PD-1. Pidilizumab and other humanized anti-PD-1 monoclonal antibodies are described in WO 2009/101611. Pembrolizumab (formerly known as lambrolizumab and also referred to as MK03475; Merck) is a humanized IgG4 monoclonal antibody that binds to PD-1. Pembrolizumab and other humanized anti-PD-1 antibodies are described in US 8354509 and WO 2009/114335. MEDI4736 (medimmun) is a human monoclonal antibody that binds to PDL1 and inhibits ligand interaction with PD1. MDPL3280A (Genentech/Roche) is a human Fc optimized IgG1 monoclonal antibody that binds to PD-L1. MDPL3280A and other human monoclonal antibodies to PD-L1 are described in US Pat. No. 7,943,743 and US Publication No. 20120039906. Other anti-PD-L1 binders include YW243.55.S70 (heavy and light chain variable regions are shown in SEQ ID NOs 20 and 21 in WO 2010/077634) and MDX-1105 (also referred to as BMS-936559 and for example the anti-PD-L1 binders described in WO 2007/005874). AMP-224 (B7-DCIg; Amplimmune; eg described in WO 2010/027827 and WO 2011/066342) is a soluble receptor fused to Fc PD-L2 that blocks the interaction of PD-1 and B7-H1. Other anti-PD-1 antibodies include, along with other AMP 514 (Amplimmune), for example, anti-PD-1 antibodies described in US 8609089, US 2010028330 and/or US 20120114649.

In one embodiment, the anti-PD-1 antibody or fragment thereof is an anti-PD-1 antibody molecule as described in US 2015/0210769 entitled "Antibody Molecules to PD-1 and Uses Thereof", incorporated by reference in its entirety. In one embodiment, the anti-PD-1 antibody molecule comprises at least one, two, three, four, five, or six CDRs (or all CDRs in total) from the heavy and light chain variable region from an antibody selected from any of BAP049- hum01 BAP049-hum02 BAP049-hum03 BAP049-hum04 BAP049-hum05 BAP049-hum06 BAP049-hum07 BAP049-hum08 BAP049-hum09 BAP049-hum10 BAP049-hum11 BAP049-hum12 BAP049-hum14, BAP049-hum15, BAP049-hum16, BAP049-Clone-A, BAP049-Clone-B, BAP049-Clone-C, BAP049-Clone-D or BAP049-Clone-E; or as described in Table. 1 US 2015/0210769, or encoded by the nucleotide sequence in the table. 1 or a sequence substantially identical (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the above sequences; or closely related CDRs, eg CDRs that are identical or that contain at least one amino acid change, but no more than two, three or four changes (eg substitutions, deletions or insertions, eg conservative substitutions).

In yet another embodiment, an anti-PD-1 antibody molecule contains at least one, two, three, or four variable regions from an antibody described herein, e.g., an antibody selected from any of BAP049-hum01, BAP049-hum02, BAP049- hum03, BAP049hum04, BAP049-hum05, BAP049-hum06, BAP049-hum07, BAP049-hum08, BAP049-hum09, BAP049- 194 040145 BAP049hum16, BAP049-Clone-A, BAP049-Clone-B, BAP049-Clone-C, BAP049-Clone-D or BAP049-Clone-E; or as described in Table. 1 US 2015/0210769, or encoded by the nucleotide sequence in the table. 1;

or a sequence that is substantially identical (e.g., at least 80, 85, 90, 92, 95, 97,

98, 99% or more identical) to any of the above sequences.

TIM3 (T cell immunoglobulin 3) also negatively regulates T cell function, particularly in IFN-γ secreting CD4+ helper T cells 1 and CD8+ cytotoxic T cells 1 and plays a critical role in T cell depletion. Inhibition of the interaction of TIM3 and its ligands, such as galectin 9 (Gal9), phosphatidylserine (PS) and HMGB1, can increase the immune response. Antibodies, antibody fragments and other inhibitors of TIM3 and its ligands are available in the art and can be used in combination with the CD19 CAR of the present invention. For example, antibodies, antibody fragments, small molecule compounds, or peptide inhibitors that target TIM3 bind to the IgV domain of TIM3, inhibiting interaction with its ligands. Antibodies and peptides that inhibit TIM3 are described in WO 2013/006490 and US20100247521. Other anti-TIM3 antibodies include humanized variants of RMT3-23 (described in Ngiow et al., 2011, Cancer Res., 71:3540-3551) and clone 8B.2C12 (described in Monney et al., 2002, Nature, 415:536 -541). Bispecific antibodies that inhibit TIM3 and PD-1 are described in US20130156774.

In one embodiment, the anti-TIM3 antibody or fragment thereof is an anti-TIM3 antibody molecule as described in US 2015/0218274 entitled "Antibody Molecules to TIM3 and Uses Thereof", incorporated by reference in its entirety. In one embodiment, an anti-TIM3 antibody molecule contains at least one, two, three, four, five, or six

A CDR (or collectively all CDRs) from the heavy and light chain variable region from an antibody selected from any of ABTIM3, ABTIM3-hum01, ABTIM3-hum02, ABTIM3-hum03, ABTIM3-hum04, ABTIM3-hum05, ABTIM3-hum06, ABTIM3- hum07, ABTIM3-hum08, ABTIM3-hum09, ABTIM3-hum10, ABTIM3-hum11, ABTIM3-hum12, ABTIM3-hum13, ABTIM3-hum14, ABTIM3-hum15, ABTIM3-hum16, ABTIM3-hum17, ABTIM3-hum18, ABTIM3-hum19, ABTIM3-hum20, ABTIM3-hum21, ABTIM3-hum22, ABTIM3-hum23; or as described in Table. 1-4 US 2015/0218274; or encoded by the nucleotide sequence in Table. 1-4; or sequence substantially identical (e.g., at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the above sequences, or closely related CDRs, e.g., CDRs that are identical , or which contain at least one amino acid change, but no more than two, three or four changes (e.g. substitutions, deletions or insertions, e.g. conservative substitutions).

In another embodiment, an anti-TIM3 antibody molecule contains at least one, two, three, or four variable regions from an antibody described herein, e.g., an antibody selected from any of ABTIM3, ABTIM3-hum01, ABTIM3-hum02, ABTIM3- hum03, ABTIM3-hum04, ABTIM3-hum05, ABTIM3-hum06, ABTIM3-hum07, ABTIM3-hum08, ABTIM3-hum09, ABTIM3-hum10, ABTIM3-hum11, ABTIM3-hum12, ABTIM3-hum13, ABTIM3-hum14, ABTIM3-hum15, ABTIM3-hum16, ABTIM3-hum17, ABTIM3-hum18, ABTIM3-hum19, ABTIM3-hum20, ABTIM3-hum21,

ABTIM3-hum22, ABTIM3-hum23; or as described in Table. 1-4 US 2015/0218274; or encoded by the nucleotide sequence in the table. 1-4; or a sequence substantially identical (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to any of the above sequences.

In other embodiments, the agent that increases the activity of a CAR-expressing cell is a CEACAM inhibitor (eg, an inhibitor of CEACAM-1, CEACAM-3, and/or CEACAM-5). In one embodiment, the CEACAM inhibitor is an anti-CEACAM antibody molecule.

Illustrative antibodies against CEACAM-1 are described in WO 2010/125571, WO 2013/082366 WO 2014/059251 and WO 2014/022332, e.g. monoclonal antibody 34B1, 26H7 and 5F4 or its recombinant form, as described, for example, in US 2004/ 0047858, US 7132255 and WO 99/052552. In other embodiments, the anti-CEACAM antibody binds CEACAM-5 as described, for example, in Zheng et al., PLoS ONE. 2010 Sep 2; 5(9).pii: e12529 (DOI:10:1371/journal.pone.0021146), or cross-reacts with CEACAM-1 and CEACAM-5. as described, for example, in WO 2013/054331 and US 2014/0271618.

Without wishing to be bound by theory, it is believed that cancer embryonic antigen associated with cell adhesion molecules (CEACAM), such as CEACAM-1 and CEACAM-5, mediates at least in part the inhibition of the antitumor immune response (see e.g. Markel et al., J. Immunol., 2002 Mar 15;168(6):2803-10;Markel et al., J. Immunol., 2006 Nov 1;177(9):6062-71;Markel et al., Immunology, 2009 Feb;126(2):186-200;Markel et al., Cancer Immunol Immunother., 2010 Feb;59(2):215-30;Ortenberg et al., Mol.Cancer Ther., 2012 Jun; 11(6):1300-10;Stern et al., J. Immunol., 2005 Jun 1;174(11):6692-701;Zheng et al., PLoS ONE, 2010 Sep 2;5(9).pii : e12529). For example, CEACAM-1 has been described as a heterophilic TIM-3 ligand and as playing a role in TIM-3 mediated tolerance and T cell depletion (see e.g. WO 2014/022332; Huang, et al., (2014) Nature, doi: 10.1038/nature13848). IN

In embodiments, co-blockade of CEACAM-1 and TIM-3 has been demonstrated to enhance antitumor immune response in colorectal cancer xenograft models (see, for example, WO 2014/022332; Huang, et al. (2014), supra). In other embodiments, the implementation of the joint blockade of CEACAM-1 and PD-1 reduces the tolerance of T cells, as described, for example, in WO 2014/059251. Thus, CEACAM inhibitors can be used with other immunomodulators described herein (e.g., PD-1 and/or TIM-3 inhibitors) to enhance the immune response against cancer, e.g., melanoma, lung cancer (e.g., NSCLC), bladder cancer, colon cancer, ovarian cancer and other malignancies as described herein.

LAG3 (lymphocyte activation gene 3 or CD223) is a cell surface molecule expressed by activated T cells and B cells that have been shown to play a role in CD8+ T cell depletion. Antibodies, antibody fragments and other LAG3 inhibitors and ligands are available in the art and can be used in combination with the CD19 CAR of the present invention. For example, BMS-986016 (Bristol-Myers Squib) is a monoclonal antibody that targets t LAG3. IMP701 (Immutep) is an anti-LAG3 antagonist antibody, and IMP731 (Immutep and GlaxoSmithKline) is an anti-LAG3 depleting antibody. Other LAG3 inhibitors include IMP321 (Immutep), which is a recombinant LAG3-Ig soluble portion fusion protein that binds to MHC class II molecules and activates antigen presenting cells (APCs). Other antibodies are described, for example, in WO 2010/019570.

In some embodiments, an agent that increases the activity of a CAR-expressing cell may be, for example, a fusion protein containing a first domain and a second domain, where the first domain is an inhibitory molecule or fragment thereof, and the second domain is a polypeptide that is linked with a positive signal, for example, a polypeptide containing an intracellular signaling domain, as described in the present description. In some embodiments, a polypeptide that is associated with a positive signal may comprise a co-stimulatory domain of CD28, CD27, ICOS, e.g., an intracellular signaling domain of CD28, CD27, and/or ICOS, and/or a primary signaling domain, e.g., CD3-zeta, e.g., described in the present invention. In one embodiment, the fusion protein is expressed by the same cell that expresses the CAR. In another embodiment, the fusion protein is expressed by a cell, such as a T cell, that does not express the CD123 CAR.

In one embodiment, an agent that enhances the activity of a CAR-expressing cell of the present invention is miR-17-92.

In one embodiment, the agent that increases the activity of the CAR described herein is a cytokine. Cytokines have important functions related to T cell depletion, differentiation, survival, and homeostasis. Cytokines that can be administered to an individual receiving a CAR expressing cell as described herein include IL-2, IL-4, IL-7, IL-9, IL-15, IL-18, and IL-21, or a combination thereof. In preferred embodiments, the cytokine administered is IL-7, IL-15, or IL-21, or a combination thereof. The cytokine can be administered once a day or more than once a day, for example twice a day, three times a day, or four times a day. The cytokine can be administered over more than one day, for example, the cytokine is administered over 2, 3, 4, 5, 6 days, 1 week, 2, 3 or 4 weeks. For example, the cytokine is administered once a day for 7 days.

In embodiments, the cytokine is administered in combination with CAR-expressing T cells. The cytokine can be administered simultaneously or simultaneously with CAR-expressing T cells, for example, administered on the same day. The cytokine may be prepared in the same pharmaceutical composition as the CAR expressing T cells, or may be prepared in a separate pharmaceutical composition. Alternatively, the cytokine may be administered immediately after the administration of the CAR expressing T cells, for example 1, 2, 3, 4, 5, 6 or 7 days after the administration of the CAR expressing T cells. In embodiments where the cytokine is administered in a dosing regimen that includes more than one day, the first day of the cytokine dosing regimen may be on the same day as the administration of CAR-expressing T cells, or the first day of the cytokine dosing regimen may be on 1, 2, 3, 4, 5, 6 or 7 days after administration of CAR-expressing T cells. In one embodiment, on the first day, CAR expressing T cells are administered to the individual, and on the second day, the cytokine is administered once a day for the next 7 days. In a preferred embodiment, the cytokine to be administered in combination with CAR expressing T cells is IL-7, IL-15 or IL-21.

In other embodiments, the cytokine is administered at a time period following the administration of CAR-expressing cells, e.g., at least 2, 3, 4, 6, 8, 10, 12 weeks, 4 months, 5, 6, 7, 8, 9, 10 , 11 months or 1 year or more after administration of CAR-expressing cells. In one embodiment, the cytokine is administered after evaluating the individual's response to CAR-expressing cells. For example, an individual is administered CAR expressing cells according to the dosage and schedule

- 196 040145 mi of the treatment described in the present description. The individual's response to therapy with CAR-expressing cells is assessed at 2, 3, 4, 6, 8, 10, 12, 4 months, 5, 6, 7, 8, 9, 10, 11 months, or 1 year or more after administration of expressing CAR cells by any of the methods described herein, including tumor growth inhibition, reduction of circulating tumor cells, or tumor regression. Individuals who do not show a sufficient response to therapy with cells expressing CAR may be administered a cytokine. Administration of a cytokine to an individual with a suboptimal response to therapy with CAR-expressing cells improves the efficacy or anti-cancer activity of the CAR-expressing cell. In a preferred embodiment, the cytokine administered following the administration of the CAR expressing cells is IL-7.

Combination with CD19 inhibitors.

The methods and compositions described herein can be used in combination with a CD19 inhibitor. In some embodiments, CAR-containing cells as described herein, such as NKR-CAR-containing cells, and a CD19 inhibitor (e.g., one or more cells that express a CAR molecule that binds to CD19, e.g., a CAR molecule that binds to CD19 described in the present invention) is administered simultaneously or simultaneously or sequentially.

In some embodiments, the CAR-containing cells and the CD19 inhibitor are infused into the individual simultaneously or simultaneously, eg, mixed in the same infusion volume. For example, a population of CAR-containing cells and CD19CAR-containing cells are mixed with each other. Alternatively, a population of cells co-expressing the CAR of the present invention and CD19CAR is administered. In other embodiments, the simultaneous administration involves separately administering the CAR-containing cells and the CD19 inhibitor, eg, over a predetermined time interval (eg, within a 15, 30, or 45 minute interval).

In some embodiments, the onset of the CAR-containing cells and the onset of the CD19 inhibitor are within 1, 2, 3, 4, 6, 12, 18, or 24 hours of each other, or within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 60, 80 or 100 days apart. In some embodiments, the delivery end of CD123-containing CAR cells and the end of CD19 inhibitor delivery are within 1, 2, 3, 4, 6, 12, 18, or 24 hours of each other, or within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 60, 80 or 100 days apart. In some embodiments, the overlap between delivery of CAR-containing cells (e.g., infusion) and end of CD19 inhibitor delivery (e.g., infusion) is at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 30 minutes . In one embodiment, the CD19 inhibitor is administered prior to the CAR-containing cells. In other embodiments, CAR-containing cells are administered prior to the CD19 inhibitor.

In some embodiments, CAR-containing cells are administered while a CD19 inhibitor (eg, one or more cells that express the CD19 CAR molecule) is present (eg, cells are proliferating) in the individual. In other embodiments, a CD19 inhibitor (eg, one or more cells that expresses the CD19 CAR molecule) is administered while the CAR-containing cells are present (eg, cells in the proliferating stage) in the individual.

A CD19 inhibitor includes, but is not limited to, a CD19 CAR cell expressing, for example, a CD19 CART cell or an anti-CD19 antibody (eg, a mono- or bispecific anti-CD19 antibody), or a fragment or conjugate thereof.

In one embodiment, a CAR expressing cell as described herein is administered to an individual in combination with a CD19 CAR cell (e.g., CART cell) (e.g., CTL019, e.g., as described in WO 2012/079000, incorporated herein by reference). ).

In other embodiments, a CAR expressing cell as described herein is administered to an individual in combination with a CD19 CAR cell (e.g., CART cell) that contains a humanized antigen binding domain as described in WO 2014/153270 (e.g., Table 3 of WO 2014 /153270), included in the present description by reference.

The CD19 inhibitor (eg, the first CD19 expressing CAR cell) and the second expressing CAR cell may be expressed by the same cell type or by different cell types. For example, in some embodiments, the cell expressing the CD19 CAR is a CD4+ T cell and the cell expressing the CAR of the present invention is a CD8+ T cell, or the cell expressing the CD19 CAR is a CD8+ T cell and the cell expressing the CAR of the present invention is a CD4+ T cell. In other embodiments, the cell expressing the CD19 CAR is a T cell and the cell expressing the CAR of the present invention is an NK cell, or the cell expressing the CD19 CAR is an NK cell and the cell expressing The CAR described in the present invention is a T cell. In other embodiments, the cell expressing the CD19 CAR and the cell expressing the CAR of the present invention are both NK cells, or both are T cells, e.g., both are CD4+ T cells, or both are CD8+ T cells. In others

- 197 040145 embodiments, one cell expresses the CD19 CAR and the CAR of the present invention, and the cell is, for example, an NK cell or a T cell such as a CD4+ T cell or a CD8+ T cell.

In embodiments, the subject is suffering from acute myelogenous leukemia (AML), eg, CD19 positive AML or CD19 negative AML. In embodiments, the individual has a CD19+ lymphoma, eg, CD19+ non-Hodgkin's lymphoma (NHL), CD19+ FL, or CD19+ DLBCL. In embodiments, the subject is suffering from relapsed or refractory CD19+ lymphoma. In embodiments, the anti-lymphoma chemotherapy is administered to the individual before, simultaneously with, or after administration (eg, infusion) of CD19 CART cells. In an example, anti-lymphoma chemotherapy is administered to an individual prior to administration of CD19 CART cells. For example, anti-lymphoma chemotherapy ends 1-4 days (eg, 1, 2, 3, or 4 days) prior to CD19 CART cell infusion. In embodiments, multiple doses of CD19 CART cells are administered, for example, as described herein. For example, a single dose contains approximately 5x108 CD19 CART cells. In embodiments, anti-lymphoma chemotherapy is administered to the subject prior to, concurrent with, or after administration (eg, infusion) of a CAR expressing cell of the present invention, eg, a non-CD19 CAR expressing cell. In embodiments, CD19 CART is administered to the individual prior to, concurrent with, or after administration (eg, infusion) of a non-CD19 CAR expressing cell, eg, a non-CD19 CAR expressing cell, as described herein.

In some embodiments, a CAR expressing cell as described herein is administered to an individual in combination with a CD19 CAR expressing cell, e.g., CTL019, e.g., as described in WO 2012/079000, incorporated herein by reference, to treat the expression of a tumor antigen described in the present description, for example, a malignant tumor described in the present invention. Without wishing to be bound by theory, it is believed that the administration of a CD19 CAR expressing cell in combination with a CAR expressing cell improves the efficiency of the CAR expressing cell of the present invention by targeting early malignant cells, e.g. regulatory B cells and/or improving the tumor microenvironment. For example, a CD19-expressing CAR cell targets cancer cells that express early lineage markers, such as malignant stem cells and CD19-expressing cells, while a CAR-expressing cell described herein targets cancer cells that express late lineage markers. eg CD123. This preconditioning approach can improve the performance of the CAR expressing cell of the present invention. In such embodiments, the CD19 CAR expressing cell is administered prior to, simultaneously with, or after administration (eg, infusion) of the CAR expressing cell of the present invention.

In embodiments, the CAR-expressing cell described herein also expresses a CAR directed to CD19, eg, the CD19 CAR. In one embodiment, a cell expressing a CAR of the present invention and a CD19 CAR is administered to an individual for the treatment of a cancer of the present invention, eg, AML. In one embodiment, the configurations of one or both of the CAR molecules comprise a primary intracellular signaling domain and a co-stimulatory signaling domain. In another embodiment, the configurations of one or both CAR molecules comprise a primary intracellular signaling domain and two or more, eg 2, 3, 4 or 5 or more co-stimulatory signaling domains. In such embodiments, the CAR molecule described herein and the CD19 CAR may contain the same or different primary intracellular signaling domain, the same or different costimulatory signaling domains, or the same number or different number of costimulatory signaling domains. Alternatively, the CAR of the present invention and the CD19 CAR are configured as a cleaved CAR in which one of the CAR molecules contains an antigen-binding domain and a costimulatory domain (e.g., 4-1BB) while the other CAR molecule contains an antigen-binding domain and a primary intracellular signaling domain. (eg CD3 zeta).

In embodiments, the CAR-expressing cell described herein also expresses a CAR directed to CD19, eg, the CD19 CAR. In one embodiment, a cell expressing a CAR of the present invention and a CD19 CAR is administered to an individual for the treatment of a cancer of the present invention, eg, AML. In one embodiment, the configurations of one or both of the CAR molecules comprise a primary intracellular signaling domain and a co-stimulatory signaling domain. In another embodiment, the configurations of one or both of the CAR molecules comprise a primary intracellular signaling domain and two or more, eg 2, 3, 4 or 5 or more, co-stimulatory signaling domains. In such embodiments, the CAR molecule described herein and the CD19 CAR may contain the same or different primary intracellular signaling domain, the same or

- 198 040145 different costimulatory signaling domains, or the same number or a different number of costimulatory signaling domains. Alternatively, the CAR of the present invention and the CD 19 CAR are configured as a split CAR, in which one CAR molecule contains an antigen-binding domain and a co-stimulatory domain (e.g., 4-1BB), while the other CAR molecule contains an antigen-binding domain and a primary intracellular signaling domain. (for example, CD3zeta).

In one embodiment, the implementation of the CAR described in the present invention, and the second CAR, such as CD19 CAR, are in the same vector or are in two different vectors. In embodiments where the CAR of the present invention and the second CAR, such as the CD19 CAR, are in the same vector, the nucleic acid sequences encoding the CAR of the present invention and the second CAR, such as the CD19 CAR, are in the same reading frame and separated by one or more peptide cleavage sites, eg P2A.

In other embodiments, a CAR-expressing cell as described herein is administered in combination with an anti-CD19 antibody inhibitor. In one embodiment, the CD19 antibody is a humanized antigen binding domain as described in WO 2014/153270 (e.g., Table 3 of WO 2014/153270), incorporated herein by reference, or a conjugate thereof. Other illustrative anti-CD19 antibodies or fragments or conjugates thereof include, but are not limited to, blinatumomab, SAR3419 (Sanofi), MEDI-551 (MedImmune LLC), combotox, DT2219ARL (Masonic Cancer Center), MOR-208 (also called XmAb -5574; MorphoSys), XmAb-5871 (Xencor), MDX-1342 (Bristol-Myers Squibb), SGN-CD19A (Seattle Genetics), and AFM11 (Affimed Therapeutics). See, for example, Hammer, MAbs. 4.5(2012): 571-77. Blinatumomab is a bispecific antibody consisting of two scFvs, where one binds to CD19 and the other binds to CD3. Blinatumomab directs T cells to attack malignant cells. See, for example, Hammer et al. clinical trial with identification number NCT00274742 and NCT01209286. MEDI-551 is a humanized anti-CD19 antibody with an Fc engineered to have increased antibody-dependent cellular cytotoxicity (ADCC). See, for example, Hammer et al. and clinical trial ID NCT01957579. Combotox is a mixture of immunotoxins that bind to CD19 and CD22. Immunotoxins consist of scFv antibody fragments fused to a deglycosylated ricin A chain. See, for example, Hammer et al. and Herrera et al., J. Pediatr. Hematol. oncol. 31.12(2009):936-41; Schindler et al., Br. J. Haematol., 154.4(2011):471-6. DT2219ARL is a bispecific immunotoxin targeting CD19 and CD22 containing two scFvs and a truncated diphtheria toxin. See, for example, Hammer et al. and clinical trial ID NCT00889408. SGN-CD19A is an antibody-drug conjugate (ADC) consisting of a humanized anti-CD19 monoclonal antibody fused to the synthetic cytotoxic cell-killing agent monomethylauristatin F (MMAF). See, for example, Hammer et al. and clinical trials with identification no. NCT01786096 and NCT01786135. SAR3419 is an anti-CD19 antibody-drug (ADC) conjugate containing a humanized anti-CD19 monoclonal antibody conjugated to a maytansine derivative via a cleavable linker. See, for example, Younes et al., J. Clin. Oncol., 30.2(2012): 2776-82; Hammer et al., clinical trial ID NCT00549185 and Blanc et al., Clin. Cancer Res., 2011; 17:6448-58. XmAb-5871 is a humanized anti-CD19 antibody with engineered Fc. See, for example, Hammer et al. MDX-1342 is a human anti-CD19 antibody with a constructive Fc with increased ADCC. See, for example, Hammer et al. In embodiments, the antibody molecule is a bispecific anti-CD19 and anti-CD3 antibody molecule. For example, AFM11 is a bispecific antibody that targets CD19 and CD3. See, for example, Hammer et al. and clinical trial ID NCT02106091. In some embodiments, an anti-CD19 antibody described herein is conjugated or otherwise linked to a therapeutic agent, e.g., a chemotherapeutic agent, a peptide vaccine (such as described in Izumoto et al., 2008 J. Neurosurg., 108:963- 971), an immunosuppressive agent, or an immunoablative agent such as cyclosporine, azathioprine, methotrexate, mycophenolate, FK506, CAMPATH, anti-CD3 antibody, cytoxin, fludarabine, rapamycin, mycophenolic acid, steroid, FR901228, or cytokine.

Combination with low doses of an mTOR inhibitor.

The methods described herein use low immunostimulatory doses of mTOR inhibitors, for example allosteric mTOR inhibitors, including rapalogues such as RAD001. Administration of a low immunostimulatory dose of an mTOR inhibitor (eg, a dose that is insufficient to completely suppress the immune system, but sufficient to improve immune function) can optimize the characteristics of immune system effector cells, such as T cells or CAR expressing cells, in an individual. Methods for measuring mTOR inhibition, doses, treatment regimens, and suitable pharmaceutical compositions are described in the patent application.

- 199 040145

US No. 2015/01240036, thus incorporated by reference.

In one embodiment, administration of a low immunostimulatory dose of an mTOR inhibitor can result in one or more of the following:

i) reducing the number of PD-1 positive effector cells of the immune system;

ii) increasing the number of PD-1-positive effector cells of the immune system;

iii) increasing the ratio of PD-1 negative immune system effector cells/PD-1 positive immune system effector cells;

iv) increasing the number of naive T cells;

v) increasing the expression of one or more of the following markers: CD62L ^hlgh , CD127 ^hlgh , CD27+ and BCL2, eg in memory T cells, eg memory T cell progenitors;

vi) downregulating KLRG1 expression, eg in memory T cells, eg memory T cell progenitors; or vii) increasing the number of memory T cell precursors, eg, cells with any or a combination of the following characteristics: increased CD62L ^high , increased CD127 ^high , increased CD27+, increased KLRG1 and increased BCL2;

and where any of the above, for example, i), ii), iii), iv), v), vi) or vii) occurs, for example, at least temporarily, for example, compared to an untreated individual.

In another embodiment, administration of a low immunostimulatory dose of an mTOR inhibitor results in increased or prolonged proliferation of CAR expressing cells, eg in culture or in an individual, eg, compared to untreated CAR expressing cells or an untreated individual. In embodiments, the increased proliferation is associated with an increase in the number of CAR-expressing cells. Methods for measuring increased or prolonged proliferation are described in Examples 9 and 10. In another embodiment, administration of a low immunostimulatory dose of an mTOR inhibitor results in increased cytolysis of malignant cells by CAR expressing cells, e.g., in culture or in an individual, e.g., compared to untreated CAR expressing cells. or an untreated individual. In embodiments, increased cancer cell cytolysis is associated with a reduction in tumor volume. Methods for measuring increased cytolysis of malignant cells are described in Example 2.

In one embodiment, cells expressing a CAR molecule, such as the CAR molecule described herein, are administered in combination with a low immunostimulatory dose of an mTOR inhibitor, such as an allosteric mTOR inhibitor, such as RAD001 or a catalytic mTOR inhibitor. For example, administration of a low immunostimulatory dose of an mTOR inhibitor may be initiated prior to administration of the CAR expressing cell of the present invention; terminate before introducing the CAR-expressing cell of the present invention; start simultaneously with the introduction of expressing CAR cells described in the present invention; coincident with the time of introduction of the CAR-expressing cell described in the present invention; or continuing after administration of the CAR-expressing cell of the present invention.

Alternatively or additionally, administration of a low immunostimulatory dose of an mTOR inhibitor may optimize immune system effector cells that are engineered to express the CAR molecule of the present invention. In such embodiments, administration of a low immunostimulatory dose of an mTOR inhibitor, e.g., an allosteric inhibitor, e.g., RAD001, or a catalytic inhibitor, is initiated or continued prior to the collection of immune system effector cells, e.g., T cells or NK cells, which are engineered to express the CAR molecule described in the present invention, in an individual.

In another embodiment, immune system effector cells, e.g., T cells or NK cells, which are engineered to express the CAR molecule of the present invention, e.g., after harvest from an individual, or immune system CAR-expressing effector cells, e.g., T- cells or NK cells, for example, may be cultured in the presence of a low immunostimulatory dose of an mTOR inhibitor prior to administration to a subject.

In one embodiment, administering to an individual a low immunostimulatory dose of an mTOR inhibitor comprises administering, for example, once a week, for example, in an immediate release dosage form 0.1 to 20, 0.5 to 10, 2.5 to 7 ,5, 3 to 6 or approximately 5 mg of RAD001 or its equivalent dose. In one embodiment, administering to an individual a low immunostimulatory dose of an mTOR inhibitor comprises administering, for example, once a week, for example, a sustained release dosage form of 0.3 to 60, 1.5 to 30, 7.5 to 22, 5, 9 to 18 or approximately 15 mg of RAD001 or its equivalent dose.

In one embodiment, the dose of the mTOR inhibitor is associated with or provides mTOR inhibition by at least 5%, but not more than 90%, at least 10%, but not more than 90%, at least 15%, but not more than 90%, at least 20%, but not more than 90%, at least 30%, but not more than 90%, at least 40%, but not more than 90%, at least 200 040145 at least 50 but not more than 90%, at least 60 but not more than 90%, at least 70 but not more than 90%, at least 5 but not more than 80%, at least 10%, but not more than 80%, at least 15%, but not more than 80%, at least 20%, but not more than 80%, at least 30%, but not more than 80%, at least 40%, but not more than 80%, at least 50%, but not more than 80%, at least 60%, but not more more than 80%, at least 5 but not more than 70%, at least 10 but not more than 70% , at least 15%, but not more than 70%, at least 20%, but not more than 70%, at least 30%, but not more than 70%, at least 40%, but not more than 70%, at least 50%, but not more than 70%, at least 5%, but not more than 60%, at least 10%, but not more than 60%, at least 15% but not more than 60%, at least 20% but not more than 60%, at least 30% but not more than 60%, at least 40% but not more more than 60%, at least 5%, but not more than 50%, at least 10%, but not more than 50%, at least 15%, but not more than 50%, at least by 20, but not more than 50%, by at least 30, but not more than 50%, by at least 40, but not more than 50%, by at least 5, but not more than 40%, at least 10%, but not more than 40%, at least 15%, but not more than 40%, at least 20%, but not more than 40%, at least e 30 but not more than 40%, at least 35 but not more than 40%, at least 5 but not more than 30%, at least 10 but not more than 30%, at least 15% but not more than 30%, at least 20% but not more than 30%, or at least 25% but not more than 30%.

The degree of mTOR inhibition can be expressed as the degree of P70S6 kinase inhibition, or it can correspond to the degree of P70S6 kinase inhibition, for example, the degree of mTOR inhibition can be determined by the level of decrease in P70S6 kinase activity, for example, by the decrease in P70S6 kinase substrate phosphorylation. The level of mTOR inhibition can be assessed in various ways, such as measuring P70S6 kinase activity by the Boulay assay, as described in US Patent Application No. 2015/01240036, thus incorporated by reference, or as described in US Patent No. 7,727,950, thus incorporated by reference. ; measuring the level of phosphorylated S6 by Western blotting; or assessing the change in the ratio of PD1-negative immune system effector cells to PD1-positive immune system effector cells.

As used herein, the term mTOR inhibitor refers to a compound or ligand, or a pharmaceutically acceptable salt thereof, that inhibits mTOR kinase in a cell. In one embodiment, the mTOR inhibitor is an allosteric inhibitor. Allosteric mTOR inhibitors include the neutral tricyclic compound rapamycin (sirolimus), rapamycin-related compounds, ie. compounds with structure and functional similarities to rapamycin, including, for example, rapamycin derivatives, rapamycin analogs (also referred to as rapalogues), and other macrolide compounds that inhibit mTOR activity. In one embodiment, the mTOR inhibitor is a catalytic inhibitor.

Rapamycin is a known macrolide antibiotic produced by Streptomyces hygroscopicus having the structure shown in Formula A.

(A)

See, for example, McAlpine J.B. et al., J. Antibiotics, (1991) 44: 688; Schreiber S.L. et al., J. Am. Chem. Soc, (1991) 113: 7433; US patent No. 3929992. There are various numbering systems proposed for rapamycin. To avoid confusion, when specific rapamycin analogs are referred to herein, the names are given with reference to rapamycin using the formula A numbering system.

Rapamycin analogs useful in the invention are, for example, O-substituted analogs in which the hydroxyl group on the cyclohexyl ring of rapamycin is substituted with OR ₁ , where R ₁ is hydroxyalkyl, hydroxyalkoxyalkyl, acylaminoalkyl or aminoalkyl; for example, RAD001, also known as everolimus, as described in US 5665772 and WO 94/09010, the contents of which are incorporated by reference. Other suitable rapamycin analogs include

- 201 040145 analogues substituted in the 26- or 28-position. The rapamycin analog may be an epimer of an analog as defined above, in particular an epimer of an analog substituted at position 40, 28 or 26, and may optionally be further hydrogenated, for example as described in US 6015815, WO 95/14023 and WO 99/15530, contents of which are incorporated by reference, e.g., ABT578, also known as zotarolimus, or the rapamycin analog described in US 7091213, WO 98/02441 and WO 01/14387, the contents of which are incorporated by reference, e.g., AP23573, also known as ridaphorolimus.

Examples of rapamycin analogs suitable for use in the present invention from US 5,665,772 include, but are not limited to, 40-O-benzylrapamycin, 40-O-(4'hydroxymethyl)benzylrapamycin, 40-O-[4'-(1,2 -dihydroxyethyl)]benzylrapamycin, 40-O-allylrapamycin, 40-O-[3'-(2,2-dimethyl-1,3-duoxolαn-4(S)-yl)-pron-2'-en-1'-yl ]pαnαmycin, (2'E,4'S)-40O-(4',5'-dihydroxypent-2'-en-1'-yl)rapamycin, 40-O-(2-hydroxy)ethoxycarbonylmethylrapamycin, 40-O(2 -hydroxy)ethyl-rapamycin, 40-O-(3-hydroxy)propylrapamycin, 40-O-(6-hydroxy)hexylrapamycin, 40-O-[2-(2-hydroxy)ethoxy]ethylrapamycin, 40-O-[ (3S)-2,2-dimethyldioxolan-3-yl]methylrapamycin, 40O-[(2S)-2,3-dihydroxyprop-1-yl]rapamycin, 40-O-(2-acetoxy)ethylrapamycin, 40-O- (2-nicotinoyloxy)ethylrapamycin, 40-O-[2-(N-morpholino)acetoxy]ethylrapamycin, 40-O-(2-Nimidazolylacetoxy)ethylrapamycin, 40-O-[2-(N-methyl-N'-piperazinyl)acetoxy ]ethylrapamycin, 39-Odesmethyl-39,40-O,O-ethylenerapamycin, (26R)-26-dihydro-40-O-(2-hydroxy)ethylrapamycin, 40-O-(2aminoethyl)p apamycin, 40-O-(2-acetaminoethyl)rapamycin, 40-O-(2-nicotinamidoethyl)rapamycin, 40-O(2-(N-methylimidazo-2'-ylcarboxamido)ethyl)rapamycin, 40-O-(2 -ethoxycarbonylaminoethyl)rapamycin, 40-O-(2-tolylsulfonamidoethyl)rapamycin and 40-O-[2-(4',5'-dicarboethoxy-1',2',3'-thiazol-1'yl)ethyl]rapamycin .

Other analogs of rapamycin useful in the present invention are analogs where the hydroxyl group on the cyclohexyl ring of rapamycin and/or the hydroxyl group at position 28 is replaced by a hydroxyester group, for example rapamycin analogs found in US RE44.768, for example temsirolimus.

Other analogs of rapamycin useful in the present invention include analogs where the methoxy group at position 16 is replaced with another substituent, preferably (optionally substituted hydroxy) alkynyloxy, benzyl, ortho-methoxybenzyl or chlorobenzyl, and/or where the methoxy group at position 39 is removed together with carbon 39 , so that the cyclohexyl ring of rapamycin becomes a cyclopentyl ring containing no methoxy group at position 39; for example, as described in WO 95/16691 and WO 96/41807, the contents of which are incorporated by reference. The analogs can be further modified such that the hydroxy at position 40 of rapamycin is alkylated and/or the carbonyl at position 32 is reduced.

Rapamycin analogs from WO 95/16691 include, but are not limited to, 16-dimethoxy-16(pent-2-ynyl)oxyrapamycin, 16-dimethoxy-16-(but-2-ynyl)oxyrapamycin, 16-dimethoxy-16(propargyl )oxyrapamycin, 16-demethoxy-16-(4-hydroxy-but-2-ynyl)oxyrapamycin, 16-dimethoxy-16benzyloxy-40-O-(2-hydroxyethyl)rapamycin, 16-dimethoxy-16-benzyloxyrapamycin, 16-demethoxy -16ortho-methoxybenzylrapamycin, 16-demethoxy-40-O-(2-methoxyethyl)-16-pent-2-ynyl)oxyrapamycin, 39demethoxy-40-deoxy-39-formyl-42-norrapamycin, 39-demethoxy-40-deoxy -39-hydroxymethyl-42norrapamycin, 39-demethoxy-40-deoxy-39-carboxy-42-norrapamycin, 39-demethoxy-40-deoxy-39-(4methyl-piperazin-1-yl)carbonyl-42-norrapamycin, 39- demethoxy-40-deoxy-39-(morpholin-4yl)carbonyl-42-norrapamycin, 39-demethoxy-40-deoxy-39-[N-methyl,N-(2-pyridin-2-ylethyl)]carbamoyl-42- norrapamycin; and 39-demethoxy-40-deoxy-39-(p-toluenesulfonylhydrazonomethyl)42-norrapamycin.

Rapamycin analogs from WO 96/41807 include, but are not limited to, 32-deoxo-rapamycin, 16-O-pent-2-ynyl-32-deoxo-rapamycin, 16-O-pent-2-ynyl-32-deoxo- 40-O-(2-hydroxyethyl)rapamycin, 16-O-pent-2-ynyl-32-(S)-dihydro-40-O-(2-hydroxyethyl)rapamycin, 32(S)-dihydro-40 -O-(2methoxy)ethylrapamycin; and 32(S)-dihydro-40-O-(2-hydroxyethyl)rapamycin.

Another suitable analog of rapamycin is mirolimus as described in US2005/0101624, the content of which is incorporated by reference.

RAD001, also known as everolimus (Afinitor®), has the chemical name (1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28E,30S,32S,35R)-1,18- dihydroxy-12-{(1R)-2-[(1S,3R,4R)4-(2-hydroxyethoxy)-3-methoxycyclohexyl]-1-methylethyl}-19,30-dimethoxy-15,17,21,23 ,29,35hexamethyl-11,36-dioxa-4-aza-tricyclo[30.3.1.04.9]hexatriaconta-16,24,26,28-tetraen-2,3,10,14,20penta-one.

Additional examples of allosteric mTOR inhibitors include sirolimus (rapamycin, AY-22989), 40-[3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate]rapamycin (also called temsirolimus or CCI-779), and ridaphorolimus (AP-23573/MP -8669). Other examples of allosteric mTor inhibitors include zotarolimus (ABT578) and umirolimus.

Alternatively or additionally, catalytic ATP-competitive mTOR inhibitors have been found to target the mTOR kinase domain directly and target mTORCl and mTORC2. They are also more potent mTORCl inhibitors than allosteric mTOR inhibitors such as rapamycin due to their modulation of rapamycin-resistant mTORC1 output signals such as 4EBP1-T37/46 phosphorylation and cap-dependent translation.

Catalytic inhibitors include: BEZ235 or 2-methyl-2-[4-(3-methyl-2-oxo-8-quinolin-3-yl2,3-dihydro-imidazo[4,5-c]quinolin-1-yl) phenyl]propionitrile or the monotosylate salt form, the synthesis of BEZ235 is described in WO 2006/122806; CCG168 (also known as AZD-8055, Chresta C.M. et al., Cancer Res., 2010, 70(1), 288-298), whose chemical name is {5-[2,4-6uc-((S) -3methylmorpholin-4-yl)pyrido[2,3-d]pyrimidin-7-yl]-2-methoxyphenyl}methanol; 3-[2,4-6uc[(3S)-3methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl]-N-methylbenzamide (WO 09104019); 3-(2aminobenzo[d]oxazol-5-yl)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidine-4-amine (WO 10051043 and WO 2013023184); No.(3-(M-(3-((3,5-dimethoxyphenyl)amino)quinoxalin-2-yl)sulfamoyl)phenyl)-3-methoxy-4methylbenzamide (WO 07044729 and WO 12006552); PKI-587 (Venkatesan A.M., J. Med.Chem., 2010, 53, 26362645), whose chemical name is 1-[4-[4-(dimethylamino)piperidine-1carbonyl]phenyl]-3-[4-( 4,6-dimorpholino-1,3,5-triazin-2-yl)phenyl]urea; GSK-2126458 (ACS Med. Chem. Lett., 2010, 1, 39-43), whose chemical name is 2,4-difluoro-M-{2-methoxy5-[4-(4-pyridazinyl)-6- quinolinyl]-3-pyridinyl}benzenesulfonamide; 5-(9-isopropyl-8-methyl-2morpholino-9H-purin-6-yl)pyrimidin-2-amine (WO10114484); (E)-N-(8-(6-amino-5(trifluoromethyl)pyridin-3-yl)-1-(6-(2-cyanopropan-2-yl)pyridin-3-yl)-3-methyl- 1H-imidazo[4,5c]quinoline-2(3H)ylidene)cyanamide (WO12007926).

Additional examples of catalytic inhibitors include mTOR 2-one (WO 2006/122806) and Ku-0063794 (Garcia-Martinez J.M. et al., Biochem J., 2009, 421(1), 29-42. Ku-0063794 is a specific inhibitor of the mammalian target of rapamycin (mTOR) WYE-354 is another example of a catalytic mTor inhibitor (Yu K. et al. (2009). Biochemical, Cellular, and In vivo Activity of Novel ATP-Competitive and Selective Inhibitors of the Mammalian Target of Rapamycin. Cancer Res. 69( 15): 6232-6240).

mTOR inhibitors useful in the present invention also include prodrugs, derivatives, pharmaceutically acceptable salts or analogs thereof of any of the above compounds.

mTOR inhibitors, such as RAD001, can be formulated for delivery based on well-established methods in the art, based on the specific dosages described herein. In particular, US Pat. No. 6,004,973 (incorporated herein by reference) exemplifies formulations that can be used with the mTOR inhibitors described herein.

Pharmaceutical compositions and treatments.

Pharmaceutical compositions of the present invention may contain a CAR expressing cell, for example, a population of CAR expressing cells as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may contain buffers such as neutral buffered saline, phosphate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (eg aluminum hydroxide); and preservatives. In one aspect, the compositions of the present invention are formulated for intravenous administration.

The pharmaceutical compositions of the present invention may be administered in a manner suitable for the disease being treated (or prevented). The amount and frequency of administration is determined by such factors as the condition of the patient and the type and severity of the patient's illness, although appropriate doses may be determined by clinical trials.

In one embodiment, the pharmaceutical composition is substantially free of, e.g., no detectable levels of a contaminant, e.g., selected from the group consisting of endotoxin, mycoplasma, lentivirus replication component (RCL), p24, VSV-G nucleic acid, HIV gag , anti-CD3/anti-CD28 coated residual beads, mouse antibodies, pooled human serum, bovine serum albumin, calf serum, culture medium components, cell packaging vector components or plasmids, bacteria and fungus. In one embodiment, the bacterium is at least one selected from the group consisting of Alcaligenes faecalis, Candida albicans, Escherichia coli, Haemophilus influenza, Neisseria meningitides, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumonia, and Group A Streptococcus pyogenes.

When an immunologically effective amount, an antitumor effective amount, a tumor-inhibiting effective amount, or a therapeutic amount is indicated, the exact amount of compositions of the present invention to be administered can be determined by the physician, taking into account individual differences in age, weight, tumor size, grade

- 203 040145 infection or metastasis, and the condition of the patient (individual). In general, it can be stated that the pharmaceutical composition containing the T cells described in the present invention can be administered at a dose of 10 ⁴ to 10 ⁹ cells/kg body weight, preferably 10 ⁵ to 10 ⁶ cells/kg body weight , including all integer values in those ranges. T cell compositions can also be administered multiple times at these doses. Cells can be administered by infusion methods that are well known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med., 319:1676, 1988). One skilled in the art can readily determine the optimal dosage and treatment regimen for a particular patient by monitoring the patient for signs of disease and thus adjusting treatment.

In some embodiments, the dose of CAR cells (e.g., NKR-CAR cells, e.g., KIR-CAR cells) is approximately 1 x 10 ⁶ , 1.1 x 10 ⁶ , 2 x 10 ⁶ , 3.6 x 10 ⁶ , 5 x 10 ⁶ , 1 x 10 ⁷ , 1 , 8x10 ⁷ , 2x10 ⁷ , 5x10 ⁷ , 1x10 ⁸ , 2x10 ⁸ or 5x10 ⁸ cells/kg. In some embodiments, the dose of CAR cells (e.g., NKR-CAR cells, e.g., KIR-CAR cells) is at least about 1 x 10 ⁶ , 1.1 x 10 ⁶ , 2 x 10 ⁶ , 3.6 x 10 ⁶ , 5 x 10 ⁶ , 1 x 10 ⁷ . 1.8x10 ⁷ , 2x10 ⁷ , 5x10 ⁷ , 1x10 ⁸ , 2x10 ⁸ or 5x10 ⁸ cells/kg. In some embodiments, the dose of CAR cells (e.g., NKR-CAR cells, e.g., KIR-CAR cells) is up to about 1 x 10 ⁶ , 1.1 x 10 ⁶ , 2 x 10 ⁶ , 3.6 x 10 ⁶ , 5 x 10 ⁶ , 1 x 10 ⁷ , 1, 8x10 ⁷ , 2x10 ⁷ , 5x10 ⁷ , 1x10 ⁸ , 2x10 ⁸ or 5x10 ⁸ cells/kg. In some embodiments, the dose of CAR cells (eg, NKR-CAR cells, eg, KIR-CAR cells) is about 1.1 x 10 ⁶ -1.8 x 10 ⁷ cells/kg. In some embodiments, the dose of CAR cells (e.g., NKR-CAR cells, e.g., KIRCAR cells) is approximately 1x10 ⁷ , 2x10 ⁷ , 5x10 ⁷ , 1x10 ⁸ , 2x10 ⁸ , 5x10 ⁸ , 1x10 ⁹ , 2x10 ⁹ , or 5x10 ⁹ cells . In some embodiments, the dose of CAR cells (e.g., NKR-CAR cells, e.g., KIR-CAR cells) is at least about 1x10 ⁷ , 2x10 ⁷ , 5x10 ⁷ , 1x10 ⁸ , 2x10 ⁸ , 5x10 ⁸ , 1x10 ⁹ , 2x10 ⁹ or 5x10 ⁹ cells. In some embodiments, the dose of CAR cells (e.g., NKR-CAR cells, e.g., KIR-CAR cells) is up to about 1x10 ⁷ , 2x10 ⁷ , 5x10 ⁷ , 1x10 ⁸ , 2x10 ⁸ , 5x10 ⁸ , 1x10 ⁹ , 2x10 ⁹ , or 5x10 ⁹ cells.

Cells can be administered using infusion techniques that are well known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med., 319:1676, 1988).

In certain aspects, it may be desirable to administer activated CAR expressing cells to an individual and then rebleed (or perform apheresis), activate the cell thereafter according to the present invention, and reinfuse such activated and expanded cells to the patient. This process can be repeated many times over several weeks. In certain embodiments, the implementation of T cells can be activated from the selected blood samples from 10c to 400cc. In certain embodiments, T cells are activated from 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, or 100cc blood samples. Without wishing to be bound by theory, the use of such a multiple draw/multiple reinfusion protocol may serve to exclude certain T cell populations.

Administration of the compositions to an individual may be by any suitable means, including by aerosol inhalation, injection, ingestion, transfusion, implantation, or transplantation. The compositions of the present invention may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (IV) injection, or intraperitoneally. In one embodiment, a CAR-expressing cell (eg, T cell or NK cell) composition of the present invention is administered to a patient via intradermal or subcutaneous injection. In another embodiment, the CAR expressing cell (eg, T cells or NK cells) compositions of the present invention are preferably administered by intravenous injection. CAR-expressing cell compositions (eg, T cells or NK cells) can be injected directly into a tumor, lymph node, or site of infection.

In certain embodiments of the present invention, cells activated and expanded by the methods described herein, or by other methods known in the art, where T cells are expanded to therapeutic levels, are administered to the patient in combination with (for example, before, simultaneously or after) any number suitable treatments including, but not limited to, treatment with agents such as antiviral therapy, treatment with cidofovir and interleukin 2, cytarabine (also known as ARA-C), or natalizumab for patients with MS, or treatment with efalizumab for patients with psoriasis, or others types of treatment for patients with PML. In additional embodiments, the T cells of the invention may be used in combination with chemotherapy, radiation therapy, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunodestructive agents such as CAMPATH, CD3 antibodies, or other species. therapies with antibodies, cytotoxin, fludaribine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and radiation. These drugs inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit p70S6 kinase, which is important for induced

- 204 040145 signaling growth factor (rapamycin) (Liu et al., Cell, 66:807-815, 1991; Henderson et al., Immun., 73:316-321, 1991; Bierer et al., Curr. Opin Immun., 5:763-773, 1993). In a further embodiment, the cell compositions of the present invention are administered to a patient in combination with (e.g., before, concurrently, or after) bone marrow transplantation, T-cell ablative therapy using chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide or antibodies such as OKT3 or CAMPATH. In another embodiment, the cell compositions of the present invention are administered following B cell ablative therapy, such as agents that interact with CD20, such as Rituxan. For example, in one embodiment, individuals may receive standard treatment with high doses of chemotherapy followed by transplantation of peripheral blood stem cells. In certain embodiments, after transplantation, individuals receive an infusion of expanded immune cells of the present invention. In a further embodiment, the expanded cells are administered before or after surgery.

In a specific exemplary aspect, individuals may undergo leukapheresis where leukocytes are harvested, enriched or depleted ex vivo to select and/or isolate cells of interest, eg, immune system effector cells (eg, T cells or NK cells). Such isolates of immune system effector cells (e.g., T cells or NK cells) can be propagated by methods known in the art and processed such that one or more CAR constructs of the invention can be administered, thereby creating a CAR expressing cell (e.g., , CAR T cell or CAR expressing NK cell) according to the invention. Individuals in need can then receive standard high dose chemotherapy treatment followed by peripheral blood stem cell transplantation. In certain aspects, after or concomitantly with transplantation, individuals receive an infusion of a proliferated CAR-expressing cell (eg, CAR T cell or CAR-expressing NK cell) of the present invention. In a further aspect, the expanded cells are administered before or after surgery.

The dosage of the above treatments to be administered to a patient will vary depending on the exact nature of the condition being treated and the recipient of the treatment. Dose scaling for human administration can be performed in accordance with accepted practices in the art. Dosing strategies and administration schedules for CAR T cells have been described (Ertl et al., 2011, Cancer Res, 71:3175-81; Junghans, 2010, Journal of Translational Medicine, 8:55).

In one embodiment, CARs are introduced into effector cells of the immune system (e.g., T cells or NK cells), e.g., using in vitro transcription, and the individual (e.g., human) receives an initial administration of a CAR-expressing cell (e.g., CAR T cell). or a CAR-expressing NK cell) of the invention and one or more subsequent administrations of a CAR-expressing cell (e.g., a CAR T cell or a CAR-expressing NK cell) of the invention, wherein one or more subsequent administrations are administered less than 15 days, e.g., 14 , 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 days after the previous injection. In one embodiment, more than one administration of a CAR expressing cell (e.g., a CAR T cell or a CAR expressing NK cell) of the invention is administered to an individual (e.g., a human) per week, e.g., 2, 3, or 4 administrations of a CAR expressing cell (e.g., , CAR T cell or CAR expressing NK cell) according to the invention is administered weekly. In one embodiment, the subject (e.g., a human subject) receives more than one administration of a CAR expressing cell (e.g., a CAR T cell or a CAR expressing NK cell) per week (e.g., 2, 3, or 4 administrations per week) (also referred to herein as a cycle) followed by a week without injections of a CAR expressing cell (e.g., a CAR T cell or a CAR expressing NK cell), followed by one or more additional injections of a CAR expressing cell (e.g., a CAR T cell or a CAR expressing NK cell). cells) (eg, more than one administration of a CAR expressing cell (eg, CAR T cell or CAR expressing NK cell) per week) to an individual. In another embodiment, the individual (e.g., a human being) receives more than one cycle of a CAR expressing cell (e.g., a CAR T cell or a CAR expressing NK cell) and the time period between cycles is less than 10, 9, 8, 7, 6 , 5, 4 or 3 days. In one embodiment, a CAR expressing cell (eg, a CAR T cell or a CAR expressing NK cell) is administered every other day in 3 injections per week. In one embodiment, a CAR expressing cell (eg, a CAR T cell or a CAR expressing NK cell) of the invention is administered for at least two, three, four, five, six, seven, eight or more weeks.

In one aspect, a CD123 expressing CAR cell (eg, a CAR T cell or a CAR expressing NK cell) is generated using lentiviral vectors such as a lentivirus. The CAR-expressing cell (eg, CAR T cell or CAR-expressing NK cell) thus obtained is characterized by stable CAR expression.

In one aspect, CAR-expressing cells, e.g., CART or CAR-expressing NK cells, are generated using a viral vector, such as a gamma retroviral vector,

- 205 040145 for example, the gamma-retroviral vector described in the present invention. expressing

CAR cells, such as CART or CAR expressing NK cells, obtained using these vectors, can be characterized by stable expression of CAR.

In one aspect, a CAR expressing cell (e.g., a CAR T cell or a CAR expressing NK cell) transiently expresses CAR vectors for a time period of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 15 days after transduction. Transient expression of CAR can be obtained by delivering a vector with CAR RNA. In one aspect, the CAR RNA is transduced into a cell (eg, T cell or NK cell) by electroporation.

A possible problem that may occur in patients treated with a transiently expressing CAR cell (e.g., CAR T cell or CAR expressing NK cell) (particularly with CAR-bearing scFv mice) is anaphylaxis after several cycles of treatment.

Without being bound by this theory, it is believed that such an anaphylactic reaction may be caused by a developing anti-CAR humoral response in the patient, ie. anti-CAR antibodies with anti-IgE isotype. The patient's antibody-producing cells are believed to undergo a class switch from an IgG isotype (which does not cause anaphylaxis) to an IgE isotype, followed by a ten to fourteen day break in antigen exposure.

If a patient is at high risk of developing a pro-CAR antibody response during therapy with transiently expressed CARs (such as CARs resulting from RNA transductions), interruptions in infusion of CAR-expressing cells (eg, CAR T cells or CAR-expressing NK cells ) should not last more than ten to fourteen days.

Experimental examples

The invention is further described in detail with reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise indicated. Thus, the invention should not be interpreted in any way as being limited to the following examples, but rather should be interpreted as including any and all of the variations that will become apparent from the teachings provided herein.

Without further description, it is believed that a person skilled in the art using the foregoing description and the following illustrative examples can make and use the compounds of the present invention and practice the methods described in the application. The following illustrative examples, therefore, specifically indicate the preferred embodiments of the present invention, and should not be interpreted as limiting in any way the remainder of the invention.

Example 1 Chimeric NK receptors.

The results presented herein demonstrate an alternative design approach for T cell CARs that can be more finely tuned compared to existing CAR structures. Planned experiments to develop a new regulated system CAR, which contains at least two or three chimeric fusion proteins. The primary activating and inhibitory signals of T cells are based on natural activating and inhibitory NK cell receptors known as killer cell immunoglobulin-like receptors (KIRs).

KIRs exist in activating and inhibitory forms that depend on the intracellular domain of the receptor. Activating KIRs deliver their signals through interaction with an immunoreceptor tyrosine activating motif (ITAM) containing the membrane protein DAP12, which is assembled from residues in the transmembrane domains of these proteins. Inhibitory KIRs carry a cytoplasmic domain that contains immunoreceptor tyrosine inhibitory motifs (ITIMs) that abolish the activating signal, resulting in inhibition of NK cytolytic activity and cytokine production activity. Like TCRs, KIRs belong to the immunoglobulin family of protein receptors and can bind to invariant MHC ligands and MHC-like ligands. Without wishing to be bound by any particular theory, it is believed that such interactions are used to distinguish the nature of normal cells (generally expressing MHC class I in high density) from malignant or virus-infected cells (often with low MHC class I or no MHC class I).

KIR-like chimeric antigen receptors (KIR-CARs) have been constructed that fuse scfv to presenting target antigen with an activating and inhibitory KIR as shown in FIG. 1. Conditional T cell activation is obtained by interaction of an activating KIR-CAR (actKIR-CAR) or a standard TCR-zeta CAR carrying scfv to an antigen on the cancer cell of interest. Inhibitory CAR (inhCAR) carrying a scFv directed against an antigen that is present on normal but non-malignant tissue provides attenuation of the primary activating CAR signal when the T cell encounters normal cells. Examples of antigens that have come up as useful targets for inhibitory CARs include ephrin receptors (Pasquale, 2010, Nat. Rev. Cancer, 10(3):165-80) and claudins (Singh et al., 2010, J. Oncol. , 2010:541957), which are expressed by epithelial cells from normal tissues but are often selectively lost by malignant tumors (eg, EPHA7).

Example 2 Construction and activity of an activating KIR-CAR.

Experiments were planned to develop activating KIR-CARs based on anti-CD19 or anti-mesothelin (SS-1) scFv fusion, which had previously been introduced into CARs based on the cytoplasmic domain of TCR-zeta, which is currently in clinical trials. The human activating KIR KIR2DS2 receptor was chosen as the initial main receptor for actKIR-CAR. Coexpression of DAP12, which is not normally expressed in T cells, is required for delivery of activating signals for actKIR-CAR. Thus, a lentiviral vector was constructed that expresses actKIR-CAR with human DAP12 using a bicistronic gene cassette based on a 2A ribosome skipping peptide. The lentiviral vector map is illustrated in FIG. 2. Initial studies demonstrated that actKIR-CAR was efficiently expressed in primary human T cells and actKIR-CAR SS1 bound to mesothelin (FIG. 3). Similar to the previously developed and published CAR scFv SS1 CD3 zeta (SS1-Z) (Carpenito et al., 2009, Proc. Natl. Acad. Sci. USA, 106(9):3360-5), for T cells expressing actKIR -CAR SS1 showed cytotoxic activity against K562 target cells engineered to express the mesothelin ligand (KT-meso) as shown in FIG. 4. None of the receptors show cytolysis of wild-type K562, which lacks the mesothelin target.

Because the cytotoxic activity of KIR-CAR SS1 against mesothelin-positive target cells was lower than that of a standard TCR-zeta-based CAR targeting the same antigen, with comparable surface CAR expression, it is believed that mesothelin CARs may contain an extracellular hinge (based on wild-type KIR2DS2) that is not optimal for CD45 segregation due to its length. It is believed that the kinetic segregation of ITAM-based activating receptors from CD45 is the key mechanism of TCR activation and depends on the scale of distances between the membranes of the T cell and the target cell ~14-15 nm (Choudhuri et al., 2005, Nature, 436(7050): 578-82). The distance scale of KIR-CAR SS1 based on KIR2DS2 is estimated to be greater than 20 nm depending on the specific crystal structure of mesothelin, demonstrating that the SS1 epitope is likely ~10 nm away from the target cell membrane (Ma et al., 2012, J Biol. Chem., 287(40): 33123-31), and CAR, which is estimated to be ~10 nm, suggesting that each Ig-like domain is ~3.5 nm in the KIR2DS2 hinge in addition to scFv. Thus, an activating KIR-CAR was constructed in which the hinge KIR2DS2 (CAR KIRS2) was removed, as schematically represented in FIG. 5. KIRS2 CAR based on scFv SS1 was shown to have increased cytolytic activity against mesothelin-expressing target cells compared to CAR formed by fusion of scFv SS1 with full-length wild-type KIR2DS2 (FIG. 6). For this optimized CAR, KIRS2 also showed increased activity compared to the TCR zeta CAR based on scFv SS1 containing the CD8 alpha extracellular hinge.

Example 3 Construction and activity of InhKIR-CAR.

An inhibitory KIR-CAR was constructed based on the fusion of SS1 scFv against mesothelin with an inhibitory backbone of the KIR2DL3 receptor. Initial studies have demonstrated that inhKIRCAR is efficiently expressed in primary human T cells. CD19 actKIR-CAR, SS1 actKIR-CAR and SS1 inhKIR-CAR alone or in combination were injected into Jurkat T cells carrying the dsGFP reporter under the control of an NFAT-dependent promoter to monitor the activation of this critical T cell signaling pathway. Although Jurkat T cells expressing CD19 actKIR-CAR or SS1 actKIR-CAR were separately efficiently activated by K562 expressing CD19 and mesothelin (KTmeso/CD19), for Jurkat T cells co-expressing CD19 actKIR-CAR and SS1 inhKIR-CAR showed markedly reduced activation by the same 9 KT-meso/CD1 target cells (FIG. 7A); however, analysis of CD19 surface expression and scFv mesothelin binding using idiotype-specific reagents unexpectedly demonstrated that the expression of scFvs with different target specificities were mutually exclusive (FIG. 7B).

Example 4. Sensitivity of constructs activating KIR-CAR to natural inhibitory receptor systems.

Because CAR co-expression with two scFvs is limited, a strategy was developed to assess the sensitivity of KIR-based activating CARs to inhibitory signals derived from the PD-1 receptor. PD-1 is a natural receptor in T cells that is used by ITIMs in the cytoplasmic domain, similar to inhibiting KIRs to recruit phosphatase, which negatively regulates TCR signaling. A schematic representation is shown in FIG. 19. The results presented herein demonstrate that wild-type PD-1 can be expressed at elevated levels with an activating KIR-based CAR and a TCR-zeta-based CAR targeting mesothelin (FIGS. 8A and 8C). The results also demonstrate that this combination resulted in PD-1 ligand 1 (PDL-1) dependent inhibition of the cytotoxicity of the mesothelin-specific activating KIR-CAR (FIG. 9). With respect to normal expression of PD-1 by T cells (i.e., T cells not transfected with PD-1), KIR-CAR show less inhibition when encountering PD-L1 overexpressing target cells compared to CAR based on TCR-zeta. Without wishing to be bound by any particular theory, it is believed that this may be an advantage of KIR-CAR when faced with tumors that in most cases express inhibitory receptor ligands.

Example 5 Costimulation-Dependent Activation of KIR-CAR.

Experiments were designed to evaluate the effects of chimeric costimulatory receptors (CCRs) in the KIR-CAR system compared to the CCRs described for standard CARs by Kloss et al. (Kloss et al., 2013, Nat. Biotechnol., 31(1):71-5). Experiments were also planned to evaluate the necessary conditions for costimulation dependent activation of KIR by introducing the endogenous CD28 receptor into a T cell using an agonist antibody, clone 9.3. As shown in FIG. 14, CAR KIRS2 were characterized by sustained proliferation in response to mesothelin-positive targets in the absence of CD28 costimulation. This proliferation is superior to that seen with CAR TCR-zeta, where costimulation has been shown to be critical for proliferation. These data suggest that for antigen-specific proliferation, KIR-based CARs may not have the same necessary costimulation conditions as TCR-zeta CARs (Milone et al., 2009, Mol. Ther., 17(8):145364; Carpenito et et al. al., 2009, Proc. Natl. Acad. Sci. USA, 106(9):3360-5), and this cost-independence may be a significant advantage of KIR-based CARs over existing TCR-zeta-based CARs. Experiments were also designed to evaluate KIR-based CAR in humanized mice to test KIR-based CARs against CARs with and without co-stimulatory domains in preclinical in vivo studies (data and experiments in Example 5 are also provided in Example 6).

Example 6 Chimeric antigen receptors (CAR) based on the killer cell immunoglobulin-like receptor (KIR) induce sustained cytotoxic activity in solid tumors.

Chimeric antigen receptors (CARs) carrying an antigen-binding domain cis-linked to CD3-Z cytoplasmic domains and costimulatory receptors provide an efficient way to construct T-cell cytotoxicity towards tumors (Grupp et al., The New England journal of medicine, 368 (16):1509-18, 2013; Brentjens et al., Science translational medicine, 5(177):177ra38, 2013; Porter et al., The New England journal of medicine, 365(8):725-33, 2011 ). Described herein is an alternative chimeric receptor in which a single chain variable fragment (scFv) targeting mesothelin (SS1) is fused to the transmembrane and cytoplasmic domain of KIR2DS2, a stimulatory killer cell immunoglobulin-like receptor (KIR) normally expressed by natural killer (NK) cells. This KIR-based SS1-KIRS2 CAR induces robust antigen-specific cytotoxic activity and in vitro effector function such as cytokine secretion and proliferation when administered to human T cells in combination with a DAP12 adapter molecule. T cells modified to express KIR-CAR and DAP12 have significantly increased antitumor activity in a resistant tumor xenograft model compared to T cells transduced with standard CD3Z-based CAR, suggesting that KIR-based CAR can attenuate inhibitory signals in tumors that limit second and third generation CD3Z-based CARs. The data provided herein substantiate the forthcoming clinical evaluation of KIR-based CARs in malignant tumors, including solid tumors.

First-generation CARs were constructed by introducing a cytoplasmic domain containing an immunoreceptor tyrosine activating motif (ITAM) into a single chimeric receptor that uses a single-chain variable fragment (scFv) from an antibody for antigen-specific targeting (Sadelain et al., Cancer discovery, 3(4 ):388-98, 2013). Subsequently, a number of different additional signaling domains from costimulatory receptors, such as CD28, ICOS, 4-1BB, and OX-40, were sequentially introduced into these receptors to increase T cell proliferation, survival, and function (Finney H.M. et al., J. Immunol ., 1998; 161:2791-2797; Maher J. et al., Nat. Biotech., 2002; 20:70-75; Finney H. M. et al., J. Immunol., 2004; 18:676-684; Milone et al., 2009, Mol. Ther., 17(8):1453-64; Carpenito et al., 2009, Proc. Natl. Acad. Sci. USA, 106(9):3360-5). These second generation (one costimulatory domain) and third generation (2 costimulatory domain) CARs are characterized by enhanced function in preclinical animal models of cancer, and some enhanced costimulation CARs are currently in the early phase of human clinical trials for the treatment of cancer (review). see Barrett D.M. et al., Ann. Rev. Med., 2014; 65:333-347).

Although single-chain CARs induce robust antigen-specific cytotoxic activity, natural receptors using highly conserved ITAM domains are typically structured into multi-chain complexes consisting of single ligand-binding and ITAM-containing signaling chains, such as the T-cell receptor (TCR)-CD3 complex, B cell receptor (BCR)-Iga/e complex; and Fc receptor (FcR) complex. Potential benefits of the multichain immunoreceptor complex lie in diversity, including a greater variety of signals available as a result of many interactions between ligand-binding and signaling molecules and long-term ITAM signaling that is decoupled from the internalization of the ligand-binding chain (Sigalov et al., Advances in experimental medicine and biology, 640:ix-xi, 2008). The implications of the combination of several receptor components commonly found in heterologous receptors in CARs have not been fully elucidated; however, anergy and antigen-independent signaling have been observed for some constructs (Brocker, Blood, 96(5):1999-2001, 2000; Brocker et al., The Journal of experimental medicine, 181(5):1653-9, 1995; Milone et al., Molecular therapy: the journal of the American Society of Gene Therapy, 17(8):1453-64, 2009).

The invention disclosed herein relates to CARs constructed from a more natural multi-chain immunoreceptor construct having greater activity in T cell activation due to naturally selected interactions between subunits in the receptor complex and other receptors in immune cells. The killer cell immunoglobulin-like receptor (KIR) and the DAP12 multichain immunoreceptor complex were chosen as the basis for CAR (Thielens et al., Current opinion in immunology, 24(2):239-45, 2012). Although they are expressed by natural killer cells where they contribute to their natural cytotoxicity, KIR expression has also been observed in CD4+ and CD8+ T cells (Moretta et al., Immunological reviews, 155:105-17, 1997; Falk et al ., Human immunology, 61(12):1219-32, 2000; Remtoula et al., Journal of immunology, 180(5):2767-71, 2008). Activating KIRs such as KIR2DS2 contain a short cytoplasmic domain with no known endogenous signaling activity. However, KIRs form a non-covalent complex with DAP12 dimers, an ITAM-containing adapter molecule capable of binding to Syk and Zap70 kinases in NK cells (Lanier et al., Nature, 391(6668):703-7, 1998). In addition to stimulating cytotoxicity upon ligand binding, KIRs have also been shown to exhibit co-stimulatory activities in T cells in the absence of DAP12, suggesting that these molecules might be capable of conferring primary initiating activity and costimulation in T cells (Snyder et al. ., Journal of immunology, 173(6):3725-31, 2004).

A KIR-based CAR was constructed by splicing the mesothelin-specific scFv SS1 onto the transmembrane and short cytoplasmic domain of an activating KIR, KIR2DS2 (SS1-KIRS2), as schematically illustrated in FIG. 1 (Hassan et al., Clinical cancer research: an official journal of the American Association for Cancer Research, 8(11):3520-6, 2002). The ITAM-containing DAP12 adapter molecule is constitutively expressed in natural killer (NK) cells, but it is expressed only in a subpopulation of human T cells (Moretta et al.). Thus, to obtain co-expression of both molecules, a bicistronic lentiviral vector encoding CAR based on mesothelin-specific KIR (SS1-KIRS2) and a DAP12 molecule separated by Thoseaasigna 2A (T2A) virus sequence (Fig. 2) was obtained. Transduction of primary human T cells SS1-KIRS2 and DAP12 with bicistronic lentivirus after activation with anti-CD3 and CD28 antibodies showed consistent expression on the surface of SS1-KIRS2 that was comparable to CD3Z-based CAR SS1Z (Fig. 6). SS1-KIRS2/DAP12 co-transduced T cells proliferated after stimulation with anti-CD3/anti-CD28 polyclonal antibodies with kinetics that were comparable to those observed with sham-transduced T cells or T cells transduced with mesothelin-specific CAR containing CD3Z cytoplasmic domain (data not shown). The cytotoxic activities of KIR-based CAR T cells were compared with those of CD3Z (SS1-z) CAR T cells. SS1-KIRS2/DAP12 transduced T cells showed high cytotoxic activity against K562 cells that expressed human mesothelin, (Kmeso), similar in magnitude to the SS1Z construct. None of the engineered T cells showed lytic activity against wild-type K562 (Kwt), suggesting specific activation of the SS1-KIRS2 receptor by the cognate target antigen mesothelin (FIG. 6).

Because KIR2DS2 expression has been described in T cells in the absence of detectable DAP12 expression, expression and function of the SS1-KIRS2 receptor was evaluated with or without DAP12 co-delivery. Using a lentiviral vector that co-expresses DAP12 with the red fluorescent protein, dsRed (DAP12-dsRed) or a dsRed-expressing control vector (dsRed), T cells were transduced with a lentiviral vector with DAP12 or a control vector followed by transfection with an in vitro transcribed RNA expressing SS1-KIRS2 . SS1KIRS2 was expressed on the surface of T cells without the addition of DAP12; however, surface expression of SS1-KIRS2 increased by ~1 log when DAP12 was added (FIG. 12A). Despite expression of SS1KIRS2, T cells lacking DAP12 did not lyse mesothelin-expressing target cells, demonstrating the need for DAP12 in SS1-KIRS2-induced T cell cytotoxic activity (FIG. 12B). These data do not exclude the possibility that the chimeric KIR may signal independently of its association with DAP12, as previously reported for the natural receptor KIR2DS2 in T cell clones (Snyder et al.).

Non-covalent bonding of natural KIR2DS2 and DAP12 depends on electrostatic interactions

- 209 040145 between an aspartic acid residue in the transmembrane (TM) domain of KIR and a lysine residue in the TM domain of DAP12 (Feng et al., PLoS biology, 4(5):e142, 2006). Although it is believed that the configuration of these ionizable amino acid residues in the TM domains of the TCR and CD3 subunits is different from that of KIR and DAP12, providing some specificity of interactions, the possibility that SS1KIRS2 might interact with components of the CD3 complex instead of the co-delivered DAP12 was investigated. Because the association between the CD3 complex and TCR chains is essential for TCR expression on the cell surface, competition of KIRs for CD3 components is expected to interfere with normal TCR expression, as previously observed with expression of cloned TCRs. Introduction of an ectopic Ve strand into T cells has been shown to downregulate endogenous TCR Ve surface expression due to competition during complex assembly (Varela-Rohena et al., Nature medicine, 14(12):1390-5, 2008). The similar frequency and intensity of TCR Ve 13.1+ in KIRS2-transduced polyclonal T cells compared to sham-transduced control T cells confirms the absence of a significant interaction between SS1-KIRS2 and members of the endogenous CD3 complex (Fig. 13).

Although cytotoxic activity is an important effector function of in vivo antitumor activity of T cells, the ability of an antigen receptor to induce cytokine secretion and T cell proliferation is also an important characteristic that generally correlates with high in vivo antitumor activity. Thus, antigen-induced secretion of interferon-γ (IFN-γ) and interleukin 2 (IL-2) by SS1-KIRS2/DAP12 modified T cells was compared to T cells bearing CD3Z-based CAR without a costimulatory domain (SS1-Z) or with CD28 or 4-1BB costimulatory domains (SS1-28Z and SS1-BBZ), respectively. The SS1-Z construct stimulates lower secretion of IFN-γ and IL-2 (FIGS. 10, 11). Interferon-y production is increased and is comparable in T cells expressing SS1-KIRS2/DAP12 or SS1-BBZ, while T cells expressing CAR SS1-28Z are characterized by significantly higher production of IL-2 and IFN-γ (Fig. . 10). Analysis of a larger panel of cytokines and chemokines demonstrates that SS1-KIRS2/DAP12 stimulates an expression profile that is qualitatively similar to CD3Z-based CARs with antigen-induced cytokine and chemokine secretion that is comparable to SS1-Z and SS1-BBZ CARs (Fig. . eleven).

The SS1-KIRS2/DAP12 receptor was also an effective stimulator of T cell proliferation in response to the cognate antigen (FIG. 14). In contrast to standard SS1-Z CAR, which depends on additional costimulatory signals for sustained proliferation, SS1-KIRS2/DAP12 T cells proliferate that is comparable to SS1Z when an anti-CD28 agonist antibody is added. The costimulation mechanism provided by SS1-KIR2 in the absence of DAP12 may be related to interactions of KIR with other adapter molecules (Synder et al.). Additional receptors naturally expressed by T cells may also be able to use co-delivered DAP12, further contributing to T cell activation and proliferation. In particular, integrins can provide costimulatory signals to T cells (Brunmark et al. (1996) PNAS USA, 93(25):14736-41; Zuckerman et al. (1998) J. Immunol., 160(7):3259- 68). DAP12 is critical for external integrin signaling to macrophages and neutrophils (Jakus et al. (2007) Trends in Cell Biol., 17(10):493-501; Mocsai et al. (2006) Nature Immunol., 7(12) :1326-33) and confer unique signaling activity on LFA-1 and other integrins in T cells by contributing to SS1-KIRS2/DAP12 activity.

CD28 and 4-1BB were introduced into CARs to increase CAR T cell activity in vivo (Carpenito et al. (2009) PNAS USA 106(9):3360-65); however, costimulation is not always able to overcome the immunosuppressive tumor microenvironment. It has recently been reported that CAR SS1-BBZ T cells injected into immunodeficient NOD-SCID-y _c ' ^/ ' (NSG) mice harboring an EMmeso cell xenograft (a cell line derived from the pleural effusion of a patient with malignant mesothelioma) proliferate in vivo, but become hypofunctional in the tumor microenvironment due to failure to eliminate tumors (Moon et al. (2014) Clinical Cancer Res., 20:4262-4273). The activity of modified SS1-KIRS2/DAP12 T cells was evaluated in this highly resistant model of mesothelioma. CARs based on SS1-KIRS2/DAP12 and CD3Z with or without costimulation are able to lyse EM-meso cells in vitro with comparable efficiency. Sham-transduced T cells and DAP12-dsRed-transduced T cells show minimal lytic activity against EM-meso cells (FIG. 36). A single intravenous injection of sham-transduced, transduced SS1Z and SS1BBZ T cells produced no observable antitumor effect on established EM-meso xenografts (Fig. 15A). Tumor growth was significantly slowed down by T cells with CAR SS1-28Z; however, only SS1-KIRS2/DAP12 modified T cells induced tumor regression with significant suppression of EM-meso tumor growth at day 52 (p<0.001, ANOVA with Scheffe's post hoc F-test). Second experiment comparing T cells expressing SS1-KIRS2/DAP12 to DAP12 alone or SS1-28Z engineered T

- 210 040145 cells, showed a similar increased antitumor activity of SS1KIRS2/DAP12 T cells (Fig. 38). Sustained KIR-based CAR activity is not unique to mesothelin specificity. KIR-based CD19-specific CARs with in vitro activity comparable to second generation CD3Z CARs were also designed (FIG. 16B). Testing in the NALM-6 leukemia xenograft model also demonstrated superior efficacy of KIR-CAR to first-generation CARs and comparable to second-generation CARs with the 4-1BB costimulatory domain.

T cell engraftment and tumor infiltrating lymphocyte (TIL) assays were performed to investigate the mechanism of increased antitumor activity of SS1-KIRS2/DAP12 T cells in the EM-meso xenograft model. Only mice receiving CAR SS1-BBZ T cells showed detectable human CD45 (hCD45)-positive cells in blood and spleen, consistent with the previously observed effect of the 4-1BB co-stimulatory domain on maintenance of CAR+ T cells in vivo (Milone et al.). Several hCD45+ tumor infiltrating lymphocytes (TILs) were detected in mice treated under simulated conditions or treated with SS1Z. In contrast, tumors treated with T cells with CAR SS1-KIRS2/DAP12, SS1-28Z, and SS1-41BBZ had hCD45+ TILs that accounted for 2-4% of all viable cells with comparable frequencies for each group (Fig. 15B) .

Immunohistochemical staining demonstrated CD8+ and CD4+ TIL (data not shown) in mouse tumors treated with CAR T cells SS1-KIRS2/DAP12, SS1-28Z, and SS1-41BBZ, confirmed by flow cytometry analysis. Thus, the increased efficiency of SS1-KIRS2/DAP12 T cells is not associated with the frequency of TIL in tumors in the late stages of tumor growth. Because TIL comparison is limited by large differences in tumor volume at late time points, evaluations were made at early time points after T cell injection. At day 10 post T cell injection, comparable frequencies of hCD45+ TIL were observed in the SS1-28Z and SS1-KIRS2/DAP12 CAR T cell treated groups, but several CD45+ TILs were present in the SS1BBZ CAR T cell group (Figure 15B). . A limited analysis of these isolated TILs demonstrated that only T cells with CAR SS1-KIRS2/DAP12 were capable of exhibiting in vitro lytic activity against EM-meso cells (data not shown). These results indicate that the delayed accumulation of SS1-BBZ T cells in the tumor, along with tumor-induced hypofunction, underlies the weak antitumor activity of these cells, despite their high frequency in the late stages of tumor development. A re-experiment was performed comparing T cells with CAR SS1-28Z and SS1KIRS2/DAP12 with isolated TILs 18 days post T cell injection to obtain more TILs for phenotypic and functional analysis. Isolated SS1-28Z TILs were characterized by markedly reduced cytotoxic activity and antigen-specific IFN-γ production compared to cryopreserved T cells used for treatment. In contrast, TILs from tumors treated with T cells with CAR SS1-KIRS2/DAP12 had comparable in vitro cytotoxicity and IFN-γ production with cryopreserved cells (Fig. 15E and Fig. 37). Immunoblotting of protein lysates from TIL and cryopreserved cells for CAR protein showed loss of CAR expression (data not shown). The absence of CAR in TIL may be due to suppression of expression or poor survival of T cells with SS1-28Z CAR relative to non-transduced T cells in the tumor microenvironment.

In conclusion, the data provided herein demonstrates that the combination of KIR-based CAR and DAP12 provides a highly efficient antigen-specific receptor system for conferring artificial antigen specificity on T cells. Despite relative equivalent activity in vitro, such a KIR-based CAR has also been shown to have significantly improved antitumor efficacy compared to a CD3Z-based CAR with one or more co-stimulatory domains in the tumor model system used herein, possibly due to increased resistance to inactivation. Further research into the mechanisms of this increased efficiency and chimeric receptor designs based on other DAP12-associated ligand-binding receptors as well as additional natural ITAM-containing receptor systems such as FcRy will be pursued.

Example 7 A KIR-based CAR can be co-expressed with a naturally occurring inhibitory KIR, allowing regulation through HLA expression on target cells.

Preparation and characterization of the K562-meso cell line that expresses the HLA-Cw ligand KIR2DL3.

Materials and method. Wild-type K562 cells or a K562 line previously engineered to express mesothelin (K562-meso) were transduced with a lentiviral vector encoding the HLA-Cw3 allele. Cells were sorted for uniform expression of mesothelin and HLA-Cw3 by fluorescence-activated cell sorting. HLA-Cw3 expression was confirmed by flow cytometry after staining with APC-conjugated anti-HLA A, B, C antibody W6/32.

Result. You can get cell lines K562 expressing alone or in combination mesothelin or HLA-Cw3 (Fig. 20).

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Co-expression of SS1-KIRS2 and KIR2DL3 in primary human T cells.

Materials and method. Primary human T cells were stimulated with anti-CD3/28 antibody microbeads followed by transduction with a bicistronic lentiviral vector expressing DAP12 and SS1-KIRS2 alone or in combination with a lentiviral vector expressing KIR2DL3 on day 1 post-activation. SS1-KIRS2 CAR expression was assessed by flow cytometry using a biotinylated goat anti-mouse F(ab)2 polyclonal antibody followed by SA-APC. KIR2DL3 expression was determined using a KIR2D-specific monoclonal antibody.

Result. Primary human T cells can be generated expressing a mesothelin-specific KIR-based CAR with one DAP12 (KIRS2), one KIR2DL3, or a combination of two receptors (FIG. 21).

KIR2DL3 co-expressed with KIR-CAR can suppress antigen-specific cytotoxicity in the presence of HLA-Cw on target cells.

Materials and method. Primary human T cells were stimulated with anti-CD3/28 antibody microbeads followed by transduction with a bicistronic lentiviral vector expressing DAP12 and SS1-KIRS2. 5 μg of in vitro transcribed mRNA encoding KIR2DL3 was introduced into lentivirus transduced T cells by electroporation after 10 days of ex vivo expansion. These T cell populations were mixed with ⁵¹ Cr-labeled K562 target cells (K562, K562meso, K562-HLACw and K562-meso/HLACw) as indicated in various ratios of effector T cells to K562 target cells (E:T ratio ). Cytotoxicity was determined by measuring the fraction of ⁵¹ Cr released into the supernatant after 4 hours.

Result. SS1-KIRS2/DAP12 expressing T cells were able to induce cytolysis of K562 target cells that express mesothelin, independent of HLA-Cw3 expression. In contrast, T cells co-expressing the SS1-KIRS2/DAP12 receptor complex and inhibiting KIR, KIR2DL3, were unable to exhibit sustained cytotoxicity against K562 expressing mesothelin with HLA-Cw3; however, these cells showed cytotoxic activity against K562 cells expressing mesothelin alone, which was comparable to modified SS1-KIRS2/DAP12 T cells. These results demonstrate the ability of inhibitory KIR receptors to regulate the functional activity of KIR-based activating CAR (FIG. 22).

Example 8 KIR-based CARs with CD19 specificity can activate antigen-specific cytotoxicity against target cells in vitro and in vivo.

KIR-based CARs with specificity for CD19 can activate antigen-specific cytotoxicity against target cells in vitro.

Materials and method. After activation with anti-CD3/CD28 antibody beads, T cells were transduced with a bicistronic lentiviral vector expressing DAP12 along with a CD19-specific KIR-based CAR in which the FMC63-derived scFv is fused to the full-length KIR2DS2 (CD19-KIR2DS2), or to the CAR on based on KIR obtained by fusion of scFv FMC63 with the transmembrane and cytoplasmic domain of KIR2DS2 through a short linker [GlyF-Ser-linker(CD19-KIRS2).

Transduced T cells were cultured to the end of the logarithmic growth phase and CD19-specific CAR expression was assessed based on KIR by flow cytometry using a biotinylated goat anti-mouse F(ab)2 polyclonal antibody followed by SA-PE. ⁵¹ Cr-labeled K562 target cells with or without CD19 expression (K562-CD19) (K562-wt) were mixed in various ratios of T cells to target cells (E:T ratio). Cytotoxicity was determined by measuring the fraction of ⁵¹ Cr released into the supernatant after 4 hours. Also included as negative and positive controls were control T cells that were either sham-transduced (NTD) or transduced with CD19-specific CD3Z-based CAR (CD19- z) respectively.

Result. Flow cytometry analysis demonstrates expression of CD19-specific scFv on the surface of T cells transduced with CD19-KIr2dS2, CD19-KIRS2 and CD19-Z (FIG. 16A). Cells expressing DAP12 with CD19-KIR2DS2 or CD19-KIRS2 were capable of inducing cytolysis of target cells in an antigen-specific manner (FIG. 16B). The cytotoxicity exhibited by modified KIR-based CAR T cells was comparable to or greater than that of T cells expressing the CD19-specific CD3Z-based CAR.

T cells transduced with CD19-KIRS2/DAP12 induce tumor regression in a human leukemia xenograft.

Materials and method. NOD-SCID-yc'/' (NSG) mice were inoculated intravenously via the tail vein on day 0 with 1 million Nalm-6 CBG tumor cells, a CD19-expressing leukemic cell line. In the experiment, T cells were stimulated with anti-CD3/CD28 antibody stimulatory beads followed by lentivirus transduction on day 1 with a series of CD3-based CARs with costimulatory

- 212 040145 domain (CD19-Z, CD19-BBz) or without or CD19-specific CAR based on KIR, CD19KIRS2 with DAP12 as indicated in the figure. Sham transduced (NTD) T cells were used as controls. T cells were expanded to the end of the ex vivo logarithmic growth phase and injected intravenously on day 5 after injection of the leukemic cell line with 2 million CAR T cells per mouse. Tumor burden was assessed by bioluminescent imaging. For each T cell condition, 5 animals were analyzed (FIG. 17).

Result. In the present in vivo experiment (FIG. 17), NTD T cells had no effect on tumor growth, while transduced CD19z, CD19BBz and CD19-KIRS2 T cells exhibited various antitumor effects. Mice infused with CD19z T cells exhibited a slight reduction in tumor burden but retention of detectable levels of luminescence. In contrast, tumor cell luminescence in mice infused with CD19BBZ or CD19KIRS2 T cells fell to the lower detection limit (Fig. 17B, dotted line) only 7 days after T cell injection, demonstrating complete clearance outside a small reservoir. leukemic cells in the root of the tooth inaccessible to T-cells. On day 15, the tumor load in the T cell group under simulated conditions exceeded the end point (2×10 ¹⁰ photons/s) and the animals were sacrificed, while the luminescence in the CD19BBZ and CD19KIRS2 groups remained at the lower limit of detection.

Example 9 Camelid VHH single domain CAR can be expressed on the surface of T cells in combination with scFv CAR without noticeable receptor interaction.

Materials and method. Jurkat T cells expressing GFP under the control of an NFAT-dependent promoter (NF-GFP) were transduced with mesothelin-specific activating CAR (SS1-CAR), CD19-specific activating (19-CAR), or CAR derived using the camel VHH domain , specific to EGFR (VHH-CAR). After being transduced with an activating CAR, the cells were then transduced with an additional inhibitory CAR recognizing CD19 (19-PD1) to obtain cells co-expressing the activating and inhibitory CAR (SS1+19PD1, 19+19PD1 or VHH+19PD1). Transduced Jurkat T cells were cultured for 24 h in association with various cell lines that 1) lack all target antigens (K562), 2) express mesothelin (K-meso), CD19 (K-19), or only EGFR (A431) 3) express a combination of EGFR and mesothelin (A431-mesothelin) or CD19 (A431-CD19) or 4) express a combination of CD19 and mesothelin (K19/meso). Additional conditions were also included as negative and positive controls for NFAT activation, which included the absence of stimulating cells (no stim) or K562 with 1 μg/ml OKT3 (OKT3), respectively. GFP expression as a marker of NFAT activation was assessed by flow cytometry.

Result. Camels and related species (eg, llamas) naturally produce antibodies that contain a single heavy chain variable domain. This domain, known as the camel VHH domain, has evolved to exist without pairing with the light chain variable domain. In FIG. 27A is a schematic representation of the possibility that two heterologous scFvcan molecules dissociate and re-associate with each other when exposed to the cell surface, as demonstrated by the observed disruption of scFv binding to the cognate ligand during receptor co-expression (FIG. 25 and FIG. 26). In FIG. 27B is a schematic representation of the expected reduced interaction of cell surface delivered scFv CARs in combination with a VHH domain based CAR. In FIG. 28 demonstrates that co-expression of two scFv-based CARs (SS1-z activating CAR and CD19-PD1 inhibitory CAR) on the surface of Jurkat results in the inability of the activating CAR (SS1-z) to recognize its cognate ligand on the target cell and triggers T cell activation despite for the absence of an inhibitory receptor ligand. This is consistent with the observed reduced surface ligand binding (FIG. 25). In contrast, co-expression of the same inhibitory CAR (CD19-PD1) with an activating camelid VHH-based CAR (VHH-z) did not affect the ability of the activating VHH-based CAR to recognize its cognate EGFR ligand. These data confirm the model illustrated in Fig. 27B that VHH-based activating CA can be expressed from scFv-based CARs without significant interaction between receptors due to the reduced ability of the scFv and VHH domains to interact.

Example 10 NKp46-based NCR CARHa with specificity for mesothelin activates antigen-specific cytotoxicity.

Materials and method. After activation with anti-CD3/CD28 antibody beads, T cells were transduced with a bicistronic lentiviral vector expressing DAP12 and SS1-KIRS2 (control) or FcεRγ and a mesothelin-specific NKp46-based CAR (SS1-NKp46) or FcεRγ and a mesothelin-specific NKp46 CAR , in which the native extracellular domain of NKp46 was truncated (SS1-TNKp46). Expression of mesothelin-specific CARs was assessed by flow cytometry using a biotinylated goat anti-mouse F(ab)2 polyclonal antibody followed by SA-PE (FIG. 18). T cells were mixed with ⁵¹ Cr-labeled K562 target cells expressing

- 213 040145 mesothelin, in various ratios of effector T cells to K562 target cells (E:T ratio).

Cytotoxicity was determined by measuring the ⁵¹ Cr fraction released into the supernatant after 4 hours compared to spontaneous release.

Result. Both SS1-NKp46 and SS1-NKp46 receptors show expression on the surface of T cells. SS1-NKp46 transduced T cells show robust target cell cytolysis that is comparable to KIR-based CAR SS1-KIRS2. For SS1-NKp46, weaker cytotoxic activity was demonstrated, which was only apparent at high ratios of effector to target cell (FIG. 18). These data demonstrate that an antigen-specific chimeric immunoreceptor for use in redirected T cell cytolytic activity can be derived from natural cytotoxicity receptors (NCRs) using a construct similar to that used to generate KIR-based CARs.

Example 11 Interaction of scFv domains.

Materials and method. In FIG. 24 Jurkat T cells were transduced with a lentiviral vector encoding a mesothelin-specific KIR-based inhibitory CAR (SS1-KIR2DL3). These transduced cells were then transduced with lentiviral vectors encoding a CD19-specific KIR-based activating CAR (CD19-KIR2DS2) at various dilutions. These KIR-CARs are shown schematically in FIG. 23. After transduction with both CARs, flow cytometry assessed the frequency of cells expressing on the surface of CARs with intact scFv capable of binding its target ligand after staining with mesothelin-Fc fusion protein, followed by staining with a secondary anti-Fc antibody labeled with PE and specific for CD19 (clone FMC63 ) anti-idiotypic monoclonal antibody labeled with APC. In FIG. 25 Anti-CD3/28 antibody-activated primary human T cells were transduced with various lentiviral vectors encoding a mesothelin-specific CAR based on CD3Z carrying an mCherry C-terminal fusion (SS1z-mCh), a CD19 CAR specific to CD3Z and 4-1BB cytoplasmic domain (19bbz) or a combination of both SS1z-mCh and 19bbz. mCherry expression and functional scFv SS1 were assessed by flow cytometry after staining with mesothelin-Fc fusion protein followed by staining with FITC-labeled anti-Fc secondary antibody. In FIG. 26 Anti-CD3/28 antibody-activated primary human T cells were transduced with various lentiviral vectors encoding CD3Z-based mesothelin-specific CAR (SS1z), CD19-specific CAR carrying FMC63 scFv (19bbz), or CD19-specific CAR carrying 21d4 scFv (21d4bbz), or a CD19 specific CAR carrying a BL22 scFv (BL22bbz) in which the scFv consisted of a heavy chain variable domain (VH) 5' to a light chain variable domain (VL) in scFv (H2L) or a VL localized at 5 '-end to VH (L2H). After transduction with each of the CD19-specific CARs, T cells were co-transduced with SS1z. SS1z binding to mesothelin and surface expression of anti-CD19 scFv was assessed by flow cytometry after staining with mesothelin-Fc fusion protein followed by staining with FITC labeled anti-Fc secondary antibody or biotinylated L protein followed by streptavidin-conjugated APC staining.

Result. In FIG. 24 demonstrates that co-expression of CARs based on two intact ligand-binding scFvs (SS1-KIR2DL3 and CD19-KIR2DS2) on the cell surface is mutually exclusive. In FIG. 26 demonstrates that loss of ligand binding occurs despite CAR expression in the cell, as illustrated by the presence of mCherry expressed by cells with reduced mesothelin binding in cells co-transduced with SS1z-mCh and 19bbz. In FIG. 26 demonstrates that scFv-to-scFv interaction leading to loss of scFv binding function can be observed using various scFv-based CARs, confirming the universal nature of this effect. These observations are consistent with the model illustrated in FIG. 27 panel A, in which the variable domain of a single scFv can undergo intermolecular pairing with a heterologous scFv-based chimeric receptor, resulting in loss of scFv binding to a single CAR.

Example 12 A chimeric antigen receptor (CAR) based on the killer cell immunoglobulin-like receptor (KIR) triggers cytotoxic activity in solid tumors.

Chimeric antigen receptors (CARs), based on a single chimeric molecule carrying an antigen-binding domain cis-linked to CD3Z cytoplasmic domains and costimulatory CD28 or 4-1BB receptors, provide an efficient way to construct T-cell cytotoxicity against tumors. A chimeric multichain receptor based on killer cell immunoglobulin-like receptors (KIRs), typically expressed by natural killer (NK) cells, and T cells were used. Constructed by fusion of a single-chain variable fragment (scFv) with the transmembrane and cytoplasmic domain of KIR, the mesothelin-targeted KIR-based CAR (SS1-KIR) has been shown to trigger antigen-specific cytotoxic activity and cytokine production that is comparable to CD3Z-based CAR with antigen-induced proliferation that does not depend on additional costimulation. Using a mesothelioma xenograft model resistant to T-cell immunotherapy,

- 214 040145 further demonstrated that mesothelin-targeted KIR-based CARs have more potent antitumor activity compared to T cells carrying mesothelin-specific CD3Z-based CARs with costimulation despite in vivo resistance of the latest CAR-modified T cells. Evaluation of tumor infiltrating lymphocytes demonstrates that KIR-based CAR+ T cells are resistant to acquired hypofunction in the tumor microenvironment compared to CD3Z-based CARs with costimulatory receptor domains. The ability of KIR-based CARs to induce tumor regression, against which second-generation CD3Z-based CARs show limited activity, warrants further clinical evaluation of KIR-based CARs in mesothelioma and other tumors, such as other solid tumors.

Example 13 In vivo antitumor activity of mesothelin-specific CARs.

Similar to the xenograft experiment described in Example 6, a series of mesothelin-based CAR constructs were tested in a mesothelioma model to evaluate in vivo antitumor activity. Three mesothelin-specific CAR constructs were tested (as shown in Fig. 39): a 2nd generation CAR construct containing the SS1 antigen-binding domain and a cytoplasmic domain containing the CD28 and CD3-zeta intracellular signaling domains (SS1-28z, Fig. 39C), a multi-stranded KIR-CAR containing an SS1 antigen-binding domain and a KIR2DS2 cytoplasmic domain on one strand and a DAP12 adapter molecule on the other strand (SS1-KIRS2/DAP12, Fig. 39A), and a single-stranded KIR-CAR containing an SS1 antigen-binding domain and a cytoplasmic domain containing DAP12 (SS1-DAP12, Fig. 39B). The transmembrane domain of the SS1-DAP12 construct used was the CD8 transmembrane domain.

Adult NSG mice were injected subcutaneously with 2x106 EM-meso cells as described previously and in Example 6. Primary human T cells were transduced with SS1-28z, SS1-KIRS2/DAP12, SS1-DAP12 achieving 90% transduction efficiency or were transduced under simulated conditions. . 4.3x106 transduced CARs or mimic transduced primary human T cells were injected intravenously on day 20 after tumor implantation. Tumor volume was measured with a caliper according to the formula (n/6)x(length)x(width) ² at the indicated time points (n=5 mice per group). Tumor volume was compared between CAR T cell treatment groups at day 40 (lowest tumor regression) and day 52 (end of experiment) by one-way ANOVA (p<0.001) with intergroup comparisons by Scheffe's post hoc F test. * means statistically significant difference from sham control at both time points (p<0.001).

As shown in FIG. 40, SS1-DAP12 had anti-tumor activity resulting in a reduction in tumor volume compared to those transduced under sham controls and controls without T cells. SS1-KIRS2/DAP12, similar to the results described in example 6, had a stable antitumor activity equivalent to the activity obtained by construct SS1-28z.

Example 14 In vitro activity of NKR-CARs containing human antigen-binding domains that bind to mesothelin.

Received NKR-CAR containing antigennegative domains of a person that binds to mesothelin. The human scFv sequences that bind to mesothelin, M-5, M-11, M-12, M-14, M-16, M-17, M-21, and M-23, are shown in Table 1. 4 was fused with a transmembrane domain and a cytoplasmic domain derived from KIR2DS2, hereinafter referred to as huMesoKIRS2. Lentiviral constructs were generated as described in the previous examples, for example, a bicistronic lentiviral vector further containing DAP12 linked to the NKR-CAR cleavage site of the T2A peptide. Various in vitro assays were performed to evaluate the CAR activity of mesothelin-specific NKR-CARs containing scFv against human mesothelin.

surface expression.

An assay was performed to determine surface expression of huMeso-KIRS2 constructs on primary human T cells. Primary human T cells were stimulated with anti-CD3/CD28 T cell activating beads (Dynabeads® CD3/CD28 CTS™, Life Technologies). After 24 h of stimulation, T cells were transduced with a lentiviral vector encoding huMeso-KIRS2 CARs: M5, M-11, M-12, M-14, M-16, M-17, M-21, and M23 or SS1-PLENS controls and SS1-PTRPE. Cells were expanded for 7-8 days and analyzed by flow cytometry for expression of these CARs using soluble mesothelin-V5-Hisx12 followed by FITC-conjugated anti-V5 epitope antibody or biotinylated goat anti-human antibody followed by PE-conjugated SA. Surface expression of the huMeso-KIRS2 constructs was quantified by flow cytometry analysis as demonstrated in FIG. 41F. For huMeso-KIRS2 constructs, expression was demonstrated on the surface of primary human T cells.

Cytotoxicity.

The target-specific cytotoxic activity of CAR cells expressing huMeso-KIRS2 was also evaluated. Primary human T cells expressing huMeso-KIRS2 were mixed with

- 215 040145 ⁵¹ Cr-labeled mesothelin-expressing target cells (K562 cells engineered to express mesothelin, K562-meso) or control target cells that do not express mesothelin (K562, K562 cells) in varying ratios of effector T cells to cells- targets (0:1, 10:1, 20:1 and 30:1). Cytotoxicity was determined by measuring the fraction of ⁵¹ Cr released into the supernatant after 4 hours. As expected in the control experiment with control cells that do not express mesothelin (K562), none of the CAR expressing cells showed a significant amount of cytotoxicity. In Figure 42B, non-transduced (NTD) cells show no antigen-specific cytotoxicity. HuMesoKIRS2 containing scFv M-23 and M-12 showed negligible antigen-specific cytotoxicity. However, huMeso-KIRS2 containing M-5, M-11, M-16, M-17 and M-21 showed significant antigen-specific cytotoxicity at levels similar to those of controls containing SS1.

Cytokine production.

Cytokine production by huMeso-KIRS2 expressing CAR cells was also assessed. Primary human T cells were stimulated, transduced with huMes-KIRS2 and expanded as previously described. After expansion, transduced T cells were mixed with K562 (mesothelin negative cells, empty bars) or K562 cells engineered to express mesothelin (black bars) at a ratio of 2:1. The concentrations of cytokines (IFNy and IL-2) were determined in the supernatants after 24 h of stimulation by ELISA for these cytokines. As shown in FIG. 43A containing M-5, M-11, M-14, M-16, M-17 and M-23 expressing huMeso-KIRS2 CAR cells produced IFNy. As shown in FIG. 43B containing M-5, M-11, M1b and M17 expressing huMes-KIRS2 cells produced IL-2.

Example 15 A low dose of RAD001 stimulates CART proliferation in a cell culture model.

The effect of low doses of RAD001 on CART cell proliferation in vitro was assessed by co-culture of CART expressing cells with target cells in the presence of various concentrations of RAD001.

Materials and methods.

Obtaining transduced CAR T cells.

A lentiviral transfer vector with a humanized anti-human CD19 CAR (huCART19) was used to generate genomic material packaged in pseudotyped lentiviral VSVg particles. The amino acid and nucleotide sequence of a humanized anti-human CD19 CAR (huCART19) is CAR 1, ID 104875, as described in PCT Publication WO 2014/153270, filed March 15, 2014, and referred to herein as SEQ ID NO. 85 and 31.

The lentiviral DNA transfer vector was mixed with the three packaging components VSVg env, gag/pol and rev in combination with the Lenti-X 293T cell transfection lipofectamine reagent. Subsequently, the medium is changed after 24 and 30 hours, the virus-containing medium is collected, filtered and stored at 80°C. CART is obtained by transduction of fresh or frozen naive T cells obtained by negative magnetic selection of healthy donor blood or Leuko Pak. T cells are activated by incubation with anti-CD3/anti-CD28 antibody beads for 24 hours, after which virus-containing supernatant or concentrated virus (MOI=2 or 10, respectively) is added to the cultures. The modified T cells are allowed to proliferate for approximately 10 days. The percentage of transduced cells (expressing CAR on the cell surface) and the level of CAR expression (relative fluorescence intensity, Geo Mean) are determined by flow cytometric analysis on days 7 to 9. The combination of slowing the growth rate and approaching T cell size to ~350 fl determines the condition T cells for cryopreservation for further analysis.

Assessment of CART proliferation.

To evaluate CART functionality, T cells are thawed and counted and assessed for viability with a Cellometer. The number of CAR-positive cells in each culture is normalized using non-transduced T cells (UTD). The effect of RAD001 on CART was tested in titrations with RAD001 starting at 50 nM. The target cell line used in all co-culture experiments is Nalm-6, a human pre-B-cell acute lymphoblastic leukemia (ALL) cell line expressing CD19 and transduced to express luciferase.

To measure CART proliferation, T cells are cultured with target cells in a 1:1 ratio. The assay is run for 4 days when cells are stained for CD3, CD4, CD8 and CAR expression. The number of T cells is estimated by flow cytometry using the bead count as a reference value.

Results.

The proliferative capacity of CART cells was tested on day 4 of the coculture assay. Number of CAR-positive CD3-positive T cells (black bars) and total

- 216 040145

CD3 positive T cells (white bars) were evaluated after culturing CAR transduced and non-transduced T cells with Nalm-6 (FIG. 39). huCART19 cells proliferated when cultured in the presence of less than 0.016 nM RAD001 and to a lesser extent at higher concentrations of the compound. It should be noted that at 0.0032 and 0.016 nM RAD001, proliferation was higher than that observed without the addition of RAD001. For non-transduced T cells (UTD) did not show detectable expansion.

Example 16 A low dose of RAD001 stimulates the expansion of CART in vivo.

This example evaluates the ability of huCAR19 cells to proliferate in vivo at various concentrations of RAD001.

Materials and methods.

NALM6-luc cells. The human acute lymphoblastic leukemia (ALL) cell line NALM6 was established from the peripheral blood of a patient with recurrent ALL. The cells were then labeled with firefly luciferase. These cell suspensions were grown in RPMI supplemented with 10% heat-inactivated fetal calf serum.

Mice. NSG mice (NOD.Cg-Prkdc ^scld I12rg ^tm1w '1/SzJ) at 6 weeks of age were obtained from Jackson Laboratory (stock no. 005557).

Tumor implantation. NALM6-luc cells were grown and expanded in vitro in RPMI supplemented with 10% heat-inactivated fetal calf serum. The cells were then transferred to a 15 ml conical tube and washed twice with cold sterile PBS. NALM6-luc cells were then counted and resuspended at a concentration of 10x106 cells per milliliter of PBS. Cells were placed on ice and implanted into mice immediately (within 1 h). NALM6-luc cells were injected intravenously into the tail vein in a volume of 100 μl for a total of 1x106 cells per mouse.

Dosing of CAR T cells. Mice were injected with 5x106 CAR T cells 7 days after tumor implantation. The cells were partially thawed in a 37°C water bath and then completely thawed by adding 1 ml of cold sterile PBS to the tube containing the cells. Thawed cells were transferred to a 15 ml Falcon tube and made up to a final volume of 10 ml with PBS. Cells were washed twice at 1000 rpm for 10 min each time and then counted on a hemocytometer. The T cells were then resuspended at 50x106 CAR T cells per ml cold PBS and kept on ice until dosing to mice. Mice were injected intravenously via the tail vein with 100 μl of CAR T cells at a dose of 5x106 CAR T cells per mouse. Eight mice per group were treated with 100 μl of PBS alone or humanized CD19 CAR T cells.

Dosing RAD001. A concentrated microemulsion of 50 mg equivalent to 1 mg RAD001 was formulated and then resuspended in D5W (5% dextrose in water) at the time of dosing. Mice were orally dosed every day (by oral force-feeding) with 200 μl of the desired doses of RAD001.

PK analysis. Mice were dosed every day with RAD001 starting from day 7 after tumor implantation. Dosing groups were as follows: 0.3, 1, 3 and 10 mg/kg. Mice received blood samples on days 0 and 14 after the first and last dose of RAD001 at the following time points for PK analysis: 15, 30 min, 1 h, 2, 4, 8, 12 and 24 h.

Results.

The reproduction and pharmacokinetics of RAD001 were tested in NSG mice with NALM6-luc tumors. Daily oral dosing of RAD001 alone had no effect on the growth of NALM6-luc tumors (FIG. 47). Pharmacokinetic analysis of RAD001 demonstrates that it is quite stable in the blood of tumor-bearing mice (FIGS. 48A and 48B). PK analysis on day 0 and day 14 showed that blood concentrations of RAD001 are above 10 nM even 24 hours after dosing the smallest dose tested (0.3 mg/kg).

Based on these doses, huCAR19 CAR T cells were dosed with RAD001 and not dosed with RAD001 to determine the proliferative capacity of these cells. The highest dose used was 3 mg/kg based on blood levels of RAD001 24 hours after dosing. Due to the RAD001 concentration being above 10 nM 24 hours after the last dose of RAD001, several lower doses of RAD001 were used in the in vivo study with CAR T cells. CAR T cells were dosed iv the day before the start of oral dosing with RAD001 every day. Mice were monitored by FACS for T cell expansion.

For the lowest doses, RAD001 show increased proliferation of CAR T cells (FIGS. 49A-49B). This increased proliferation is more pronounced and prolonged with CD4 ⁺ CAR T cells than with CD8+ CAR T cells. However, with CD8+ CAR T cells, increased proliferation can be observed at early time points after a dose of CAR T cells. In embodiments, CART cell RNA can also be used in combination with immune checkpoint inhibitors.

The disclosures of each and every patent, patent application, and publication cited in this specification are hereby incorporated by reference in their entirety. Despite

- 217 040145 While this invention has been described with reference to specific embodiments, it is obvious that other embodiments and variations of the present invention may be developed by others skilled in the art without departing from the spirit and scope of the invention. The appended claims are to be construed as including all such embodiments and equivalent modifications.

Claims (20)

CLAIM 1. An isolated nucleic acid molecule encoding a chimeric antigen receptor (CAR) polypeptide, wherein the nucleic acid molecule contains a nucleotide sequence encoding said CAR polypeptide and said CAR polypeptide contains: (a) an extracellular antigen-binding domain that binds to mesothelin, wherein the antigen-binding domain comprises a heavy chain complementarity determining region 1 (HC CDR1), a heavy chain complementarity determining region 2 (HC CDR2), a heavy chain complementarity determining region 3 (HC CDR3), light chain complementarity region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2), and light chain complementarity determining region 3 (LC CDR3), where: (i) HC CDR1, HC CDR2 and HC CDR3 are parts of the amino acid sequence of the heavy chain of the human anti-mesothelin antibody SEQ ID NO: 234, and LC CDR1, LC CDR2 and LC CDR3 are parts of the amino acid sequence of the light chain of the human anti-mesothelin antibody of SEQ ID NO: 234; or (ii) HC CDR1, HC CDR2, and HC CDR3 are parts of the heavy chain amino acid sequence of the human anti-mesothelin antibody of SEQ ID NO: 240, and LC CDR1, LC CDR2, and LC CDR3 are parts of the amino acid sequence of the human anti-mesothelin antibody light chain of SEQ ID NO: 240; (b) natural killer cell immune function receptor (NKR) transmembrane domain; and (c) the cytoplasmic domain of NKR. 2. An isolated CAR polypeptide for modulating immune function, comprising: (a) an extracellular antigen-binding domain that binds to mesothelin, wherein the antigen-binding domain comprises a heavy chain complementarity determining region 1 (HC CDR1), a heavy chain complementarity determining region 2 (HC CDR2), a heavy chain complementarity determining region 3 (HC CDR3), light chain complementarity region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2), and light chain complementarity determining region 3 (LC CDR3), where: (i) HC CDR1, HC CDR2 and HC CDR3 are parts of the amino acid sequence of the heavy chain of the human anti-mesothelin antibody SEQ ID NO: 234, and LC CDR1, LC CDR2 and LC CDR3 are parts of the amino acid sequence of the light chain of the human anti-mesothelin antibody of SEQ ID NO: 234; or (ii) HC CDR1, HC CDR2, and HC CDR3 are parts of the heavy chain amino acid sequence of the human anti-mesothelin antibody of SEQ ID NO: 240, and LC CDR1, LC CDR2, and LC CDR3 are parts of the amino acid sequence of the human anti-mesothelin antibody light chain of SEQ ID NO: 240; (b) NKR transmembrane domain; and (c) NKR cytoplasmic domain. 3. An isolated nucleic acid molecule according to claim 1 or an isolated CAR polypeptide according to claim 2, where: (a) an antigen-binding domain that binds to mesothelin comprises a heavy chain variable region comprising the amino acid sequence of the heavy chain variable region of an anti-mesothelin antibody in any of SEQ ID NOs: 234 and 240, and/or a light chain variable region comprising the amino acid sequence of the anti-mesothelin antibody heavy chain light chain regions of an anti-mesothelin antibody in any of SEQ ID NOs: 234 and 240; and/or (b) an antigen-binding domain that binds to mesothelin contains the amino acid sequence of any of SEQ ID NOs: 234 and 240. 4. The isolated nucleic acid molecule according to claim 1, 2 or the isolated CAR polypeptide according to claim 2 or 3, where the CAR polypeptide contains an immunoglobulin-like killer cell receptor (KIR)-CAR, where the KIR-CAR contains one or both of the transmembrane domain of KIR or a cytoplasmic domain containing a functional signaling domain from KIR (KIR cytoplasmic domain); a natural cytotoxic receptor (NCR)-CAR, wherein the NCR-CAR contains one or both of a transmembrane domain from NCR or a cytoplasmic domain containing a functional signaling domain from NCR; chimeric antigen receptor of the lymphocyte activation signaling molecule family (SLAMF) - 218 040145 CAR, where SLAMF-CAR contains one or both of a transmembrane domain from SLAMF or a cytoplasmic domain containing a functional signaling domain from SLAMF; Fc receptor (Fc)R-CAR, where the FcR-CAR contains one or both of a transmembrane domain from an FcR selected from CD16 or CD64, or a cytoplasmic domain containing a functional signaling domain from an FcR selected from CD16 or CD64; or Ly49 receptor (Ly49)-CAR, where Ly49-CAR contains one or both of a transmembrane domain from Ly49 or a cytoplasmic domain containing a functional signaling domain from Ly49. 5. An isolated nucleic acid molecule according to claim 4 or an isolated CAR polypeptide according to claim 4, where: (a) the transmembrane domain of KIR 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; (b) a cytoplasmic domain containing a functional signaling domain from KIR contains 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; (c) the KIR-CAR further comprises one or more of a KIR D0 domain, a KIR D1 domain, and/or a KIR D2 domain; (d) the transmembrane domain of the NCR contains the transmembrane domain of a protein selected from the group consisting of NKp46, NKp30 and NKp44; (e) a cytoplasmic domain containing a functional signaling domain from the NCR contains a functional signaling domain of a protein selected from the group consisting of NKp46, NKp30 and NKp44; (f) the transmembrane domain of SLAMF comprises the transmembrane domain of a protein selected from the group consisting of SLAM, CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME and CD2F-10; (g) a cytoplasmic domain containing a functional signaling domain from SLAMF contains 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; (h) the transmembrane domain of Ly49 contains the transmembrane domain of a protein selected from the group consisting of Ly49A, Ly49C, Ly49H and Ly49D; and/or (i) a cytoplasmic domain containing a functional signaling domain from Ly49 contains a functional signaling domain of a protein selected from the group consisting of Ly49A, Ly49C, Ly49H and Ly49D. 6. An isolated nucleic acid molecule according to any one of claims 1 or 2-5 or an isolated CAR polypeptide according to any one of claims 2-5, where: a) the transmembrane domain contains the amino acid sequence of SEQ ID NO: 357, 358 or 359; or b) the nucleotide sequence encoding the transmembrane domain comprises nucleotides 771-830 of SEQ ID NO: 342, nucleotides 773-832 of SEQ ID NO: 344, or nucleotides 803-875 of SEQ ID NO: 346. 7. An isolated nucleic acid molecule according to any one of claims 1 or 2-6 or an isolated CAR polypeptide according to any one of claims 2-6, where: (a) the cytoplasmic domain comprises an NKR cytoplasmic domain containing one or more functional protein signaling domains selected from the group consisting of KIR2DS2, KIR2DL3, NKp46, DAP12, KIR, NCR, SLAMF, FcR, and Ly49; (b) the cytoplasmic domain contains the amino acid sequence of SEQ ID NO: 360, 361 or 362; or (c) the nucleotide sequence encoding the cytoplasmic domain comprises nucleotides 831-947 of SEQ ID NO: 342, nucleotides 833-1060 of SEQ ID NO: 344, or nucleotides 876-949 of SEQ ID NO: 346. 8. An isolated nucleic acid molecule according to any one of claims 1 or 2-7, or an isolated CAR polypeptide according to any one of claims 2-7, wherein the CAR polypeptide further comprises a leader sequence containing the amino acid sequence of SEQ ID NO: 1. 9. An isolated nucleic acid molecule according to any one of claims 1 or 2-8 or an isolated CAR polypeptide according to any one of claims 2-8, wherein the extracellular antigen-binding domain that binds to mesothelin is linked to the transmembrane domain by a hinge domain. 10. An isolated nucleic acid molecule according to claim 9 or an isolated CAR polypeptide according to claim 9, where: (a) 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; (b) the hinge domain contains the amino acid sequence of SEQ ID NO: 5, 2, 3 or 4; and/or (c) the nucleotide sequence encoding the hinge domain contains a nucleotide sequence - 219 040145 the sequence of SEQ ID NO: 356, 16, 13, 14 or 15. 11. An isolated nucleic acid molecule according to any one of claims 1 or 2-10, or an isolated CAR polypeptide according to any one of claims 2-10, wherein the transmembrane domain and the cytoplasmic domain together comprise: a) 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 (b) the nucleotide sequence encoding the transmembrane domain and the cytoplasmic domain comprises nucleotides 1237-1464 of SEQ ID NO: 332 or nucleotides 1156-1365 of SEQ ID NO: 334. 12. An isolated nucleic acid molecule according to any one of claims 1 or 2-11, wherein the nucleic acid molecule is a DNA molecule or an RNA molecule. 13. CAR complex for modulating immune function, containing: (a) a CAR polypeptide encoded by a nucleic acid molecule according to any one of claims 1 or 2-12, or a CAR polypeptide according to any one of claims 2-11, and (b) an adapter molecule selected from DAP12 or FceRy. 14. The CAR complex of claim 13, wherein the CAR polypeptide interacts with the adapter molecule by binding the extracellular antigen-binding domain of the CAR polypeptide to mesothelin. 15. An expression vector containing a nucleic acid molecule according to any one of claims 1 or 2-12, wherein the vector is a DNA vector or an RNA vector. 16. A cell for modulating immune function, where the cell contains a nucleic acid molecule according to any one of claims 1 or 2-12, a CAR polypeptide according to any one of claims 2-11, an expression vector according to claim 15, or a CAR complex according to any one of claims. 13, 14. 17. A method for producing a cell, comprising introducing into the cell a nucleic acid molecule according to any one of claims 1 or 2-12 or an expression vector according to claim 15. 18. A method of providing an antitumor immune response in a mammal, comprising administering to the mammal an effective amount of a cell containing a nucleic acid molecule according to any one of claims 1 or 2-12, a CAR polypeptide according to any one of claims 2-11, an expression vector according to claim 15, or CAR complex according to any one of claims 13, 14. 19. A method of treating a mammal suffering from a disease or disorder associated with mesothelin expression, comprising administering to the mammal an effective amount of a cell containing a nucleic acid molecule according to any one of claims 1 or 2-12, a CAR polypeptide according to any one of claims 2-11, a vector expression according to claim 15 or a CAR complex according to any one of claims 13, 14. 20. The method of claim 19, 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, metastases in pancreas, ovarian cancer, colorectal cancer and bladder cancer, or any combination thereof.