Chimeric antigen receptors containing the interleukin-9 receptor signaling domain

Abstract

The present disclosure provides a CAR comprising a tumor antigen binding domain, a transmembrane domain, and an intracellular domain comprising an intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra), and a modified cell, i.e., an immune cell or a precursor thereof, engineered to express the CAR. Also provided are methods and uses of the modified cell, for example, for treating at least one sign and / or symptom of cancer. Related nucleic acids, vectors, and pharmaceutical compositions are also provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under U.S. Provisional Patent Application No. 63 / 245,400, filed September 17, 2021, and U.S. Provisional Patent Application No. 63 / 245,386, filed September 17, 2021, each of which is incorporated by reference in its entirety. [Background technology]

[0002] 2. Background of the Invention Current immunotherapy advances have been revolutionary for the treatment of hematological malignancies, as evidenced by the FDA approval of CD19-targeted chimeric antigen receptor T cells (CAR-T cells) for the treatment of acute lymphoblastic leukemia and diffuse large B-cell lymphoma. However, the greatest unsolved challenge for cancer therapy is solid tumors. CAR-T cells have lacked efficacy in fighting solid tumors due to a number of challenges, including lack of tumor-specific antigens, treatment resistance, tumor heterogeneity, poor proliferation and persistence, and overcoming the obstacles of extrinsic functional impairment and physical barriers to T cell infiltration caused by the dense immunosuppressive tumor microenvironment (TME). One major limitation is that adoptively transferred T cells expand and persist in vivo poorly, necessitating lymphodepleting conditioning chemotherapy, a toxic regimen that limits patient eligibility. Even T cells that proliferate and persist eventually become differentiated and dysfunctional. T cells with stem-like phenotype can overcome these limitations and show excellent antitumor activity in mouse models and humans, but therapeutic manipulations to select or expand stem-like T cells are limited to the cell manufacturing stage and cannot be performed in vivo. There is a need in the art for novel cell-based therapies that overcome these obstacles and challenges. The present invention addresses this need. Summary of the Invention

[0003] In some aspects, the present invention provides a chimeric antigen receptor (CAR) comprising a tumor antigen binding domain, a transmembrane domain, and an intracellular domain comprising the intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra).

[0004] In some embodiments, the tumor antigen is alpha fetoprotein (AFP) / HLA-A2, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA, claudin 18.2, c-Met, DLL3, DR5, EGFR, EGFRvIII, EpC Selected from AM, EphA2, FAP, folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, glycolipid F77, glypican-2 (GPC2), glypican-3 (GPC3), HER2, HLA-A2, ICAM1, IL3Ra, IL13Ra2, LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan-A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, nectin4 / FAP, NKG2D ligands (MIC-A, MIC-B, and ULBP 1-6), NY-ESO-1, P16, PD-L1, PSCA, PSMA, ROR1, ROR2, TIM-3, TM4SF1, TnMuc1, VEGFR2, and any combination thereof.

[0005] In some embodiments, the tumor antigen is selected from mesothelin, GD2, HER2, TnMuc1, CD70, PMSA, and EGFRvIII.

[0006] In some embodiments, the tumor antigen binding domain is selected from the group consisting of a full length antibody or antigen-binding fragment thereof, a monospecific antibody, a bispecific antibody, a Fab, a Fab', a F(ab')2, an Fv, a single chain variable fragment (scFv), a linear antibody, a single domain antibody (sdAb) and an antibody mimetic (e.g., a designed ankyrin repeat protein (DARPin), an affibody, a monobody (adnectin), an affilin, an affimer, an affitin, an alphabody, an avimer, a Kunitz domain peptide, anticalin, and a syntherin).

[0007] In some embodiments, the tumor antigen binding domain is a single chain variable fragment (scFv).

[0008] In some embodiments, the tumor antigen binding domain comprises: (a) an anti-mesothelin scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from SEQ ID NO:49 and SEQ ID NO:65; (b) an anti-GD2 scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:108; (c) an anti-HER2 scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:110 or SEQ ID NO:112; (d) an anti-TnMuc1 scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:114; (e) an anti-CD70 scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:116; (f) an anti-PMSA scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:120; and (g) an anti-EGFRvIII scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:122. is selected from.

[0009] In some embodiments, the intracellular domain of the CAR further comprises a costimulatory domain of a protein selected from the group consisting of the TNFR superfamily of proteins, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3 (CD276), or a variant thereof, or an intracellular domain derived from a killer immunoglobulin-like receptor (KIR).

[0010] In some embodiments, the intracellular domain of the CAR further comprises an intracellular signaling domain of a protein selected from the group consisting of CD3 zeta chain (CD3ζ), FcγRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor with an immunoreceptor tyrosine-based activation motif (ITAM), TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or a variant thereof.

[0011] In some embodiments, the intracellular domain of the CAR further comprises a costimulatory domain of CD28, a costimulatory domain of 4-1BB, an intracellular signaling domain of CD3 zeta, or any combination thereof.

[0012] In some embodiments, the CAR further comprises a hinge domain.

[0013] In some embodiments, the CAR comprises: (a) anti-human mesothelin scFv, human CD8 hinge domain, human CD28 transmembrane domain, human CD28 costimulatory domain, human IL9Ra intracellular signaling domain and human CD3z signaling domain; (b) anti-human mesothelin scFv, human CD8 hinge domain, human CD8 transmembrane domain, human 4-1BB costimulatory domain, human IL9Ra intracellular signaling domain and human CD3z signaling domain; (c) anti-mouse mesothelin scFv, mouse CD8 hinge domain, mouse CD28 transmembrane domain, mouse CD28 costimulatory domain, mouse IL9Ra intracellular signaling domain and mouse CD3z signaling domain; or (d) anti-mouse mesothelin scFv, mouse CD8 hinge domain, mouse CD8 transmembrane domain, mouse 4-1BB costimulatory domain, mouse IL9Ra intracellular signaling domain, and mouse CD3z signaling domain Includes.

[0014] In some embodiments, the CAR comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from SEQ ID NOs:81, 83, 85, and 87.

[0015] In some embodiments, the CAR is encoded by a nucleic acid comprising a nucleotide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from SEQ ID NOs:82, 84, 86, and 88.

[0016] In another aspect, an isolated nucleic acid is provided that comprises a nucleotide sequence encoding a chimeric antigen receptor (CAR) comprising a tumor antigen binding domain, a transmembrane domain, and an intracellular domain that comprises the intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra).

[0017] In some embodiments, the tumor antigen is alpha fetoprotein (AFP) / HLA-A2, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA, claudin 18.2, c-Met, DLL3, DR5, EGFR, EGFRvIII, EpC AM, EphA2, FAP, folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, glycolipid F77, glypican-2 (GPC2), glypican-3 (GPC3), HER2, HLA-A2, ICAM1, IL3Ra, IL13Ra2, LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan-A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, nectin4 / FAP, NKG2D ligands (MIC-A, MIC-B, and ULBP 1-6), NY-ESO-1, P16, PD-L1, PSCA, PSMA, ROR1, ROR2, TIM-3, TM4SF1, TnMuc1, VEGFR2, and any combination thereof.

[0018] In some embodiments, the tumor antigen is selected from mesothelin, GD2, HER2, TnMuc1, CD70, PMSA, and EGFRvIII.

[0019] In some embodiments, the tumor antigen binding domain is selected from the group consisting of a full length antibody or antigen-binding fragment thereof, a monospecific antibody, a bispecific antibody, a Fab, a Fab', a F(ab')2, an Fv, a single chain variable fragment (scFv), a linear antibody, a single domain antibody (sdAb) and an antibody mimetic (e.g., a designed ankyrin repeat protein (DARPin), an affibody, a monobody (adnectin), an affilin, an affimer, an affitin, an alphabody, an avimer, a Kunitz domain peptide, anticalin, and a syntherin).

[0020] In some embodiments, the tumor antigen binding domain is a single chain variable fragment (scFv).

[0021] In some embodiments, the tumor antigen binding domain comprises: (a) an anti-mesothelin scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from SEQ ID NO:49 and SEQ ID NO:65; (b) an anti-GD2 scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:108; (c) an anti-HER2 scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:110 or SEQ ID NO:112; (d) an anti-TnMuc1 scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:114; (e) an anti-CD70 scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:116; (f) an anti-PMSA scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:120; and (g) an anti-EGFRvIII scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:122. is selected from.

[0022] In some embodiments, the intracellular domain of the CAR further comprises a costimulatory domain of a protein selected from the group consisting of the TNFR superfamily of proteins, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3 (CD276), or a variant thereof, or an intracellular domain derived from a killer immunoglobulin-like receptor (KIR).

[0023] In some embodiments, the intracellular domain of the CAR further comprises an intracellular signaling domain of a protein selected from the group consisting of CD3 zeta chain (CD3ζ), FcγRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor with an immunoreceptor tyrosine-based activation motif (ITAM), TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or a variant thereof.

[0024] In some embodiments, the intracellular domain of the CAR further comprises a costimulatory domain of CD28, a costimulatory domain of 4-1BB, an intracellular signaling domain of CD3 zeta, or any combination thereof.

[0025] In some embodiments, the isolated nucleic acid of the invention further comprises a hinge domain.

[0026] In some embodiments, the CAR comprises: (a) anti-human mesothelin scFv, human CD8 hinge domain, human CD28 transmembrane domain, human CD28 costimulatory domain, human IL9Ra intracellular signaling domain and human CD3z signaling domain; (b) anti-human mesothelin scFv, human CD8 hinge domain, human CD8 transmembrane domain, human 4-1BB costimulatory domain, human IL9Ra intracellular signaling domain and human CD3z signaling domain; (c) anti-mouse mesothelin scFv, mouse CD8 hinge domain, mouse CD28 transmembrane domain, mouse CD28 costimulatory domain, mouse IL9Ra intracellular signaling domain and mouse CD3z signaling domain; or (d) anti-mouse mesothelin scFv, mouse CD8 hinge domain, mouse CD8 transmembrane domain, mouse 4-1BB costimulatory domain, mouse IL9Ra intracellular signaling domain, and mouse CD3z signaling domain Includes.

[0027] In some embodiments, the CAR comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from SEQ ID NOs:81, 83, 85, and 87.

[0028] In some embodiments, the CAR is encoded by a nucleic acid comprising a nucleotide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from SEQ ID NOs:82, 84, 86, and 88.

[0029] In another aspect, a vector is provided that includes an isolated nucleic acid of the invention.

[0030] In some embodiments, the vector is a retroviral vector or a lentiviral vector.

[0031] In another aspect, there is provided a modified cell comprising: The cell is an immune cell or a precursor thereof, and The cells are engineered to express a chimeric antigen receptor (CAR) comprising a tumor antigen binding domain, a transmembrane domain, and an intracellular domain comprising the intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra), Modified cells are provided.

[0032] In some embodiments, the tumor antigen is alpha fetoprotein (AFP) / HLA-A2, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA, claudin 18.2, c-Met, DLL3, DR5, EGFR, EGFRvIII, EpC AM, EphA2, FAP, folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, glycolipid F77, glypican-2 (GPC2), glypican-3 (GPC3), HER2, HLA-A2, ICAM1, IL3Ra, IL13Ra2, LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan-A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, nectin4 / FAP, NKG2D ligands (MIC-A, MIC-B, and ULBP 1-6), NY-ESO-1, P16, PD-L1, PSCA, PSMA, ROR1, ROR2, TIM-3, TM4SF1, TnMuc1, VEGFR2, and any combination thereof.

[0033] In some embodiments, the tumor antigen is selected from mesothelin, GD2, HER2, TnMuc1, CD70, PMSA, and EGFRvIII.

[0034] In some embodiments, the tumor antigen binding domain is selected from the group consisting of a full length antibody or antigen-binding fragment thereof, a monospecific antibody, a bispecific antibody, a Fab, a Fab', a F(ab')2, an Fv, a single chain variable fragment (scFv), a linear antibody, a single domain antibody (sdAb), and an antibody mimetic (e.g., a designed ankyrin repeat protein (DARPin), an affibody, a monobody (adnectin), an affilin, an affimer, an affitin, an alphabody, an avimer, a Kunitz domain peptide, anticalin, and a syntherin).

[0035] In some embodiments, the tumor antigen binding domain is a single chain variable fragment (scFv).

[0036] In some embodiments, the tumor antigen binding domain comprises: (a) an anti-mesothelin scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from SEQ ID NO:49 and SEQ ID NO:65; (b) an anti-GD2 scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:108; (c) an anti-HER2 scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:110 or SEQ ID NO:112; (d) an anti-TnMuc1 scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:114; (e) an anti-CD70 scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:116; (f) an anti-PMSA scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:120; and (g) an anti-EGFRvIII scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:122. is selected from.

[0037] In some embodiments, the intracellular domain of the CAR further comprises a costimulatory domain of a protein selected from the group consisting of the TNFR superfamily of proteins, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3 (CD276), or a variant thereof, or an intracellular domain derived from a killer immunoglobulin-like receptor (KIR).

[0038] In some embodiments, the intracellular domain of the CAR further comprises an intracellular signaling domain of a protein selected from the group consisting of CD3 zeta chain (CD3ζ), FcγRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor with an immunoreceptor tyrosine-based activation motif (ITAM), TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or a variant thereof.

[0039] In some embodiments, the intracellular domain of the CAR further comprises a costimulatory domain of CD28, a costimulatory domain of 4-1BB, an intracellular signaling domain of CD3 zeta, or any combination thereof.

[0040] In some embodiments, the modified cells of the present invention further comprise a hinge domain.

[0041] In some embodiments, the CAR comprises: (a) anti-human mesothelin scFv, human CD8 hinge domain, human CD28 transmembrane domain, human CD28 costimulatory domain, human IL9Ra intracellular signaling domain and human CD3z signaling domain; (b) anti-human mesothelin scFv, human CD8 hinge domain, human CD8 transmembrane domain, human 4-1BB costimulatory domain, human IL9Ra intracellular signaling domain and human CD3z signaling domain; (c) anti-mouse mesothelin scFv, mouse CD8 hinge domain, mouse CD28 transmembrane domain, mouse CD28 costimulatory domain, mouse IL9Ra intracellular signaling domain and mouse CD3z signaling domain; or (d) anti-mouse mesothelin scFv, mouse CD8 hinge domain, mouse CD8 transmembrane domain, mouse 4-1BB costimulatory domain, mouse IL9Ra intracellular signaling domain, and mouse CD3z signaling domain Includes.

[0042] In some embodiments, the CAR comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from SEQ ID NOs:81, 83, 85, and 87.

[0043] In some embodiments, the CAR is encoded by a nucleic acid comprising a nucleotide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from SEQ ID NOs:82, 84, 86, and 88.

[0044] In some embodiments, the cells are T cells, autologous cells, human cells, or any combination thereof.

[0045] In some embodiments, the cells are capable of activating STAT1, STAT3, STAT5, or any combination thereof.

[0046] In another aspect, there is provided a pharmaceutical composition comprising a population of modified cells of any one of the preceding embodiments and at least one pharma- ceutically acceptable carrier.

[0047] In another aspect, there is provided a method of treating cancer in a subject in need thereof, comprising administering to the subject a population of modified cells, wherein the cells are immune cells or precursor cells thereof, and the cells are engineered to express a chimeric antigen receptor (CAR) comprising a tumor antigen binding domain, a transmembrane domain, and an intracellular domain comprising the intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra).

[0048] In some embodiments, the tumor antigen is alpha fetoprotein (AFP) / HLA-A2, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA, claudin 18.2, c-Met, DLL3, DR5, EGFR, EGFRvIII, EpC AM, EphA2, FAP, folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, glycolipid F77, glypican-2 (GPC2), glypican-3 (GPC3), HER2, HLA-A2, ICAM1, IL3Ra, IL13Ra2, LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan-A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, nectin4 / FAP, NKG2D ligands (MIC-A, MIC-B, and ULBP 1-6), NY-ESO-1, P16, PD-L1, PSCA, PSMA, ROR1, ROR2, TIM-3, TM4SF1, TnMuc1, VEGFR2, and any combination thereof.

[0049] In some embodiments, the tumor antigen is selected from mesothelin, GD2, HER2, TnMuc1, CD70, PMSA, and EGFRvIII.

[0050] In some embodiments, the tumor antigen binding domain is selected from the group consisting of a full length antibody or antigen-binding fragment thereof, a monospecific antibody, a bispecific antibody, a Fab, a Fab', a F(ab')2, an Fv, a single chain variable fragment (scFv), a linear antibody, a single domain antibody (sdAb) and an antibody mimetic (e.g., a designed ankyrin repeat protein (DARPin), an affibody, a monobody (adnectin), an affilin, an affimer, an affitin, an alphabody, an avimer, a Kunitz domain peptide, anticalin, and a syntherin).

[0051] In some embodiments, the tumor antigen binding domain is a single chain variable fragment (scFv).

[0052] In some embodiments, the tumor antigen binding domain comprises: (a) an anti-mesothelin scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from SEQ ID NO:49 and SEQ ID NO:65; (b) an anti-GD2 scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:108; (c) an anti-HER2 scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:110 or SEQ ID NO:112; (d) an anti-TnMuc1 scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:114; (e) an anti-CD70 scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:116; (f) an anti-PMSA scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:120; and (g) an anti-EGFRvIII scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:122. is selected from.

[0053] In some embodiments, the intracellular domain of the CAR further comprises a costimulatory domain of a protein selected from the group consisting of the TNFR superfamily of proteins, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3 (CD276), or a variant thereof, or an intracellular domain derived from a killer immunoglobulin-like receptor (KIR).

[0054] In some embodiments, the intracellular domain of the CAR further comprises an intracellular signaling domain of a protein selected from the group consisting of CD3 zeta chain (CD3ζ), FcγRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor with an immunoreceptor tyrosine-based activation motif (ITAM), TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or a variant thereof.

[0055] In some embodiments, the intracellular domain of the CAR further comprises a costimulatory domain of CD28, a costimulatory domain of 4-1BB, an intracellular signaling domain of CD3 zeta, or any combination thereof.

[0056] In some embodiments of the methods of the present invention, the CAR further comprises a hinge domain.

[0057] In some embodiments, the CAR comprises: (a) anti-human mesothelin scFv, human CD8 hinge domain, human CD28 transmembrane domain, human CD28 costimulatory domain, human IL9Ra intracellular signaling domain and human CD3z signaling domain; (b) anti-human mesothelin scFv, human CD8 hinge domain, human CD8 transmembrane domain, human 4-1BB costimulatory domain, human IL9Ra intracellular signaling domain and human CD3z signaling domain; (c) anti-mouse mesothelin scFv, mouse CD8 hinge domain, mouse CD28 transmembrane domain, mouse CD28 costimulatory domain, mouse IL9Ra intracellular signaling domain and mouse CD3z signaling domain; or (d) anti-mouse mesothelin scFv, mouse CD8 hinge domain, mouse CD8 transmembrane domain, mouse 4-1BB costimulatory domain, mouse IL9Ra intracellular signaling domain, and mouse CD3z signaling domain Includes.

[0058] In some embodiments, the CAR comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from SEQ ID NOs:81, 83, 85, and 87.

[0059] In some embodiments, the CAR is encoded by a nucleic acid comprising a nucleotide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from SEQ ID NOs:82, 84, 86, and 88.

[0060] In some embodiments of the methods of the invention, the population of cells comprises T cells, autologous cells, human cells, or any combination thereof.

[0061] In some embodiments, the population of cells is capable of activating STAT1, STAT3, STAT5, or any combination thereof.

[0062] In some embodiments, the subject is a human.

[0063] In some embodiments, the cancer is selected from B-cell malignancies (such as B-cell lymphoma or leukemia), lung cancer, non-small cell lung cancer, small cell lung cancer, Merkel cell carcinoma, melanoma, Hodgkin lymphoma, non-Hodgkin lymphoma, urothelial carcinoma, gastric cancer, cervical cancer, cutaneous squamous cell carcinoma, renal cell carcinoma, breast cancer, triple-negative breast cancer, colon cancer, esophageal cancer, gastric cancer, liver cancer, kidney cancer, pancreatic cancer, prostate cancer, brain cancer, lung adenocarcinoma, glioblastoma, hepatocellular carcinoma, gallbladder cancer, cervical cancer, cervical squamous cell carcinoma, colorectal cancer, ovarian cancer, and renal cancer. [Brief description of the drawings]

[0064] The above and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings. [Figure 1A]FIG. 1A is a schematic diagram showing a chimeric antigen receptor (CAR) comprising an antigen-binding scFv extracellular domain, a CD8 hinge domain, a transmembrane domain, and an intracellular domain comprising a 4-1BB and / or CD28 costimulatory domain, an IL9Ra signaling domain, and a CD3z signaling domain. [Figure 1B] Figure IB provides flow cytometry data illustrating expression of CAR on transduced mouse T cells compared to untransduced (UTD) cells. The CAR comprises an anti-mouse mesothelin (anti-mMSLN) A03 scFv extracellular domain, a mouse CD8 hinge, a transmembrane domain, and an intracellular domain comprising a mouse 4-1BB costimulatory domain, a mouse IL9Ra signaling domain, and a mouse CD3z signaling domain. [Figure 2A]Figures 2A-2D provide data on wild-type mouse IL9Ra cytokine receptor co-expressed with an exemplary CAR on transduced mouse CD3+ T cells. Figure 3A provides data showing the co-expression of mouse IL9Ra and mouse CAR on transduced mouse CD3+ T cells compared to untransduced (UTD) cells. Figure 2B provides flow cytometry analysis of surface markers CD44, C62L and Fas (CD95), illustrating the finding that transduced cells exhibit a Tscm phenotype 24 hours after stimulation with 100 ng / mL wild-type mIL9 or wild-type mIL2. Figure 2C provides global gene expression profile data in transduced mouse CAR T cells expressing mIL9Ra 24 hours after stimulation with wild-type mIL9 or wild-type mIL2. Total RNA was extracted from transduced T cells cultured for 24 hours in the presence of mIL-2 or mIL-9. RNA was analyzed with the Nanostring nCounter Mouse Immunology Panel (562 genes) and plotted using nSolver 4.0 software. Figure 2D provides a graph showing in vitro expression of mIL9 via the adenoviral vector construct Ad-mIL9. Mouse pancreatic cancer cell line PDA7940b (10,000 cells / well) was infected with Ad-mIL9 at 100 viral particles / cell, and cell culture supernatants were analyzed for mIL-9 by ELISA at the indicated time points. [Figure 2B] Please see the legend to FIG. 2A. [Figure 2C] Please see the legend to FIG. 2A. [Figure 2D] Please see the legend to FIG. 2A. [Figure 3A] FIG. 3A provides a schematic of the gene expression constructs for expressing human IL9Ra and a human anti-mesothelin CAR (M5), as well as flow cytometry data showing co-expression of IL9Ra and CAR in human T cells. [Figure 3B]Figure 3B provides a schematic of the gene expression constructs for expressing mouse IL9Ra and mouse anti-mesothelin CAR (A03), as well as flow cytometry data showing co-expression of IL9Ra and CAR in mouse cells 5 days post-transduction. [Figure 4] FIG. 4 provides flow cytometry data illustrating the finding that IL9Ra signaling in T cells leads to a Tscm phenotype. [Diagram 5] Figure 5 provides phospho-flow cytometry data illustrating the finding that IL9Ra signaling in T induces phosphorylation of STAT1, STAT3, and STAT5. The log2 of MFI (fold change) is shown. [Figure 6-1] Figure 6 provides quantified cytokine secretion data for the indicated cytokines in mouse T cells incubated with IL9. T cells were transduced to express A03 CAR (left side of each panel) or A03 CAR and IL9Ra (right side of each panel). [Figure 6-2] Please refer to the description of Figure 6-1. [Figure 6-3] Please refer to the description of Figure 6-1. [Figure 7] FIG. 7 shows the finding that IL9Ra signaling in mouse T cells enhances tumor cell killing. [Figure 8A]Figures 8A-8C illustrate the finding that IL9a signaling induces similar gene expression profiles in T cells engineered to express anti-meso CAR and IL9Ra or anti-meso CAR and an orthogonal chimeric cytokine receptor (ortho-IL2Rβ-IL9Ra chimeric cytokine receptor (o9R)). Figure 8A shows the top 20 up- and down-regulated genes for T cells expressing anti-meso CAR and IL9Ra pre-incubated with either IL9 or IL2. Figure 8B shows the top 20 up- and down-regulated genes for T cells expressing anti-meso CAR and o9R pre-incubated with ortho-IL2 or IL-2. Figure 8C shows the common up- and down-regulated genes. [Figure 8B] Please see the legend to FIG. 8A. [Figure 8C] Please see the legend to FIG. 8A. [Figure 9A-1]Figures 9A-9F show gene set mutation analysis (GSVA) ​​and gene set enrichment analysis (GSEA) data for T cells expressing anti-meso CAR and IL9Ra preincubated with either IL9 or IL2, and for T cells expressing anti-meso CAR and ortho-IL2Rβ-IL9Ra chimeric cytokine receptor (o9R) preincubated with ortho-IL2 or IL-2. The data in Figure 9A compares pathways significantly enriched in CAR T cells stimulated with IL9 versus IL2. Figure 9B provides a table of enriched pathways along with GSEA statistics. Figure 9C provides enrichment plots and analysis for interferon gamma responses in T cells expressing anti-meso CAR and IL9Ra preincubated with IL9 compared to IL2. Figure 9D provides enrichment plots and analysis for interferon alpha responses in T cells expressing anti-meso CAR and IL9Ra preincubated with IL9 compared to IL2. Figure 9E provides enrichment plots and analysis for interferon gamma responses in T cells expressing anti-meso CAR and ortho IL2Rβ-IL9Ra chimeric cytokine receptor (o9R) preincubated with ortho IL2 compared to IL-2. Figure 9F provides enrichment plots and analysis for interferon alpha responses in T cells expressing anti-meso CAR and ortho IL2Rβ-IL9Ra chimeric cytokine receptor (o9R) preincubated with ortho IL2 compared to IL-2. [Figure 9A-2] Please refer to the description of FIG. 9A-1. [Figure 9B] Please refer to the description of FIG. 9A-1. [Figure 9C-1] Please refer to the description of FIG. 9A-1. [Figure 9C-2] Please refer to the description of FIG. 9A-1. [Figure 9D] Please refer to the description of FIG. 9A-1. [Figure 9E-1] Please refer to the description of FIG. 9A-1. [Figure 9E-2] Please refer to the description of FIG. 9A-1. [Figure 9F] Please refer to the description of FIG. 9A-1. [Figure 10A-1] Figures 10A-10D relate to the establishment of an in vivo syngeneic mouse model of PDA. Figure 10A shows a schematic of the protocol, a chart of tumor volume and mesothelin expression data of PDA7940b cells. Figure 10B shows the experimental design for dose-finding of an adenoviral vector expressing mIL9 (Ad-mIL9) in a syngeneic PDA mouse model. Figure 10C provides transduction efficiency data of Ad-mIL9. Figure 10D provides dose-finding tumor growth data for the indicated conditions in a syngeneic PDA mouse model. [Figure 10A-2] Please refer to the description of FIG. 10A-1. [Figure 10B-1] Please refer to the description of FIG. 10A-1. [Figure 10B-2] Please refer to the description of FIG. 10A-1. [Figure 10C-1] Please refer to the description of FIG. 10A-1. [Figure 10C-2] Please refer to the description of FIG. 10A-1. [Figure 10D-1] Please refer to the description of FIG. 10A-1. [Figure 10D-2] Please refer to the description of FIG. 10A-1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0065] Detailed Description The present disclosure provides a CAR comprising an extracellular tumor antigen binding domain, a transmembrane domain, and an intracellular domain comprising an intracellular signaling domain of IL9Ra, and its use to improve CAR cell immunotherapy for treating cancer by (1) utilizing tumor antigens in the tumor to enhance immune stimulatory signals in immune cells (e.g., T cells), (2) changing the phenotype of immune cells expressing the CAR, and (3) enabling IL-9 signaling in immune cells expressing the CAR to improve effector function in situ. By repurposing IL-9R signaling in T cells using a CAR comprising an IL9Ra intracellular signaling domain, these cells gain new functions through the concomitant activation of STAT1, STAT3, and STAT5. Such CAR T cells take on characteristics of stem cell memory (Tscm) with improved trafficking and effector function, thereby resulting in improved antitumor activity against difficult-to-treat solid tumors.

[0066] It is contemplated herein that the IL-9R intracellular domain (ICD)-containing receptors (e.g., CARs) of the present disclosure are distinct from IL-4R ICD, IL-7R ICD, or IL-21R ICD-containing receptors (e.g., CARs) because the orthogonal chimeric cytokine receptors containing the IL-9R ICD resulted in strong activation (e.g., phosphorylation) of STAT1, STAT3, and STAT5 in T cells expressing the orthogonal chimeric cytokine receptor. See Kalbasi, et al. Nature, 607:360-365 (2022). Indeed, CAR T cells expressing oIL2Rβ-IL9Rα chimeric cytokine receptors took on characteristics of stem cell memory and effector T cells, and showed superior anti-tumor efficacy in two refractory syngeneic mouse solid tumor models of melanoma and pancreatic cancer when compared to cells expressing orthogonal receptors containing IL-2, ICD. Moreover, the antitumor effect of receptors containing the IL-9R ICD was effective in the absence of lymphodepletion conditioning, and CAR T cells expressing orthogonal chimeric cytokine receptors containing the IL-9 ICD proliferated less than cells expressing, for example, the IL-2 ICD.

[0067] Thus, the present disclosure provides novel CARs, CAR-expressing cells (e.g., CAR T cells), and novel processes for engineering CAR T cells with stem-like phenotypes that do not require the administration of orthogonal or any exogenous cytokines. It is envisioned herein that the cells of the present invention exhibit excellent anti-tumor activity. A stem-like phenotype in T cells is demonstrated herein by expressing wild-type IL9Ra together with the CAR, which is associated with activation of STAT1, STAT3, and STAT5, as well as CD62L. + This resulted in enrichment of the population and higher expression of Fas (CD95) and Sca-1. + is known for its excellent antitumor activity in adoptive cellular therapy (ACT).

[0068] The novelty of the CARs and CAR-expressing cells disclosed herein is highlighted by the fact that IL-9 naive T cells are insensitive to IL-9 and T cell development is not impaired in IL-9-deficient mice. Mouse T cells do not express the IL-9R receptor. Therefore, IL-9 may not be a key natural cytokine in T cell biology. Indeed, IL-9R is naturally expressed by mast cells, memory B cells, innate lymphoid cells and hematopoietic progenitor cells. However, T cell subsets that produce IL-9 have been described. However, the effect of IL-9R signaling on T cells has not been fully characterized. The identification of unique signaling properties of IL-9 (a lesser known cytokine in the γc cytokine receptor family) in T cells, such as a unique STAT signaling profile (e.g., strong activation) and the expression of IL-9R in stem cell memory T (T SCM ) The acquisition of cellular characteristics was surprising and unexpected.

[0069] Thus, in one aspect, the present invention provides a chimeric antigen receptor (CAR) comprising a tumor antigen binding domain, a transmembrane domain, and an intracellular domain comprising the intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra).

[0070] In another aspect, the present invention provides an isolated nucleic acid comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) comprising a tumor antigen binding domain, a transmembrane domain, and an intracellular domain comprising the intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra).

[0071] In another aspect, the present invention provides a modified cell comprising: The cell is an immune cell or a precursor thereof, and The cells are engineered to express a chimeric antigen receptor (CAR) comprising a tumor antigen binding domain, a transmembrane domain, and an intracellular domain comprising the intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra), Modified cells are provided.

[0072] In another aspect, the present invention provides a method of treating cancer in a subject in need thereof, comprising administering to said subject a population of modified cells, wherein said cells are immune cells or precursor cells thereof, and said cells have been engineered to express a chimeric antigen receptor (CAR) comprising a tumor antigen binding domain, a transmembrane domain, and an intracellular domain comprising the intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra).

[0073] In other aspects, related compositions (eg, pharmaceutical compositions) and kits are provided herein.

[0074] It is to be understood that the methods described in this disclosure are not limited to the particular methods and experimental conditions disclosed herein, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0075] Furthermore, the experiments described herein use conventional molecular and cell biology and immunological techniques that are within the skill of the person skilled in the art, unless otherwise indicated.Such techniques are well known to those skilled in the art and are fully described in the literature.See, for example, Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley&Sons, Inc., NY, NY(1987-2008), Molecular Cloning: A Laboratory Manual (Fourth Edition) by MR Green and J.Sambrook and Harlow et al., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor(2013, 2nd edition), including all supplements.

[0076] Methods and techniques using immune cells bearing chimeric antigen receptors (e.g., CAR T cells) are described, for example, in Ruella, et al., J.Clin.Invest., 126(10):3814-3826(2016) and Kalos, et al., 3(95), 95ra73:1-11(2011), the contents of which are incorporated by reference herein in their entireties.

[0077] A. Definition Unless otherwise defined, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In the event of potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. Unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. The use of "or" means "and / or" unless otherwise specified. The use of the term "including" as well as other forms such as "includes" and "included" is not limiting.

[0078] In general, the nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein is well known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification, unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications as commonly accomplished in the art or as described herein. The nomenclature used in connection with analytical chemistry, synthetic organic chemistry, and medicinal chemistry and drug discovery chemistry described herein, as well as the experimental techniques and techniques thereof, are well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and treatment of patients.

[0079] In order that this disclosure may be more readily understood, select terms are defined below.

[0080] The article "a" or "an" is used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0081] As used herein, "about" when referring to a measurable value, such as an amount, duration, and the like, is intended to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value, such variations being appropriate for practicing the disclosed methods.

[0082] As used herein, "activation" refers to the state of T cells that are sufficiently stimulated to induce detectable cell proliferation. Activation can also be associated with the induction of cytokine production and detectable effector function. The term "activated T cells" refers, inter alia, to T cells undergoing cell division.

[0083] As used herein, "alleviating" a disease means reducing the severity of one or more symptoms of the disease.

[0084] The term "antigen" as used herein is defined as a molecule that elicits an immune response. This immune response may include antibody production, or activation of specific immunocompetent cells, or both. Those skilled in the art will appreciate that virtually any macromolecule, including any protein or peptide, may function as an antigen.

[0085] Furthermore, the antigen may be derived from recombinant or genomic DNA. Thus, one skilled in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response will encode an "antigen" as the term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded only by the full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of multiple genes, and that these nucleotide sequences are arranged in various combinations to elicit a desired immune response. Furthermore, one skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen may be synthetically produced or derived from a biological sample. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0086] As used herein, the term "autologous" is intended to refer to any material that is derived from an individual and that is later reintroduced into the same individual.

[0087] "Costimulatory molecule" refers to the 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 include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors.

[0088] As used herein, a "costimulatory signal" refers to a signal that, in combination with a primary signal, such as TCR / CD3 ligation, leads to T cell proliferation and / or up- or down-regulation of key molecules.

[0089] "Disease" is an animal's health condition in which the animal is unable to maintain homeostasis and if the disease is not improved, the animal's health will continue to deteriorate.In contrast, a "disorder" in an animal is a health condition in which the animal is able to maintain homeostasis, but the animal's health condition is less favorable than if the disorder does not exist.If left untreated, the disorder does not necessarily cause further deterioration of the animal's health condition.

[0090] As used herein, the term "downregulation" refers to the reduction or elimination of gene expression of one or more genes.

[0091] "Effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, material, or composition described herein that is effective to achieve a particular biological result or provides a therapeutic or prophylactic benefit. Such results may include, but are not limited to, an amount that, when administered to a mammal, causes a detectable level of immune suppression or tolerance compared to the immune response detected in the absence of the composition of the present invention. The immune response can be readily assessed by a number of methods recognized in the art. Those skilled in the art will understand that the amount of the composition administered herein will vary and can be readily determined based on many factors, such as the disease or condition being treated, the age and health and physical condition of the mammal being treated, the severity of the disease, the particular compound being administered, and the like.

[0092] "Encode" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the biological properties resulting therefrom. Thus, a gene codes for a protein if transcription and translation of the mRNA corresponding to that gene produces a protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to code for the protein or other product of that gene or cDNA.

[0093] As used herein, "endogenous" refers to any substance that is produced from or within an organism, cell, tissue or system.

[0094] The term "epitope" as used herein is defined as a small chemical molecule on an antigen that can elicit an immune response and induce a B cell and / or T cell response. An antigen can have one or more epitopes. Most antigens have many epitopes; i.e., they are multivalent. Generally, an epitope is roughly the size of about 10 amino acids and / or sugars. Preferably, an epitope is about 4-18 amino acids, more preferably about 5-16 amino acids, even more preferably about 6-14 amino acids, more preferably about 7-12 amino acids, and most preferably about 8-10 amino acids. Those skilled in the art will understand that, in general, the overall three-dimensional structure, rather than the specific linear sequence of the molecule, is the primary criterion for antigen specificity and thus distinguishes one epitope from another. Based on the present disclosure, a peptide used in the present invention can be an epitope.

[0095] As used herein, the term "exogenous" refers to any substance that is introduced from or produced outside an organism, cell, tissue or system.

[0096] The term "expand" as used herein refers to an increase in number, such as an increase in the number of T cells. In one embodiment, T cells expanded ex vivo are increased in number compared to the number initially present in the culture. In another embodiment, T cells expanded ex vivo are increased in number compared to other cell types in the culture. The term "ex vivo" as used herein refers to cells removed from a living organism (e.g., a human) and grown outside the organism (e.g., in a culture dish, test tube, or bioreactor).

[0097] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0098] "Expression vector" refers to a vector that contains a recombinant polynucleotide that contains an expression control sequence operably linked to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., Sendai virus, lentivirus, retrovirus, adenovirus and adeno-associated virus) that incorporate recombinant polynucleotides.

[0099] "Identity" as used herein refers to the identity of subunit sequences between two polymer molecules, particularly between two amino acid molecules, e.g., between two polypeptide molecules. If two amino acid sequences have the same residue at the same position, e.g., if each position of the two polypeptide molecules is occupied by arginine, then they are identical at that position. The identity or degree to which two amino acid sequences have the same residue at the same position of alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions, e.g., if half of the positions in the two sequences (e.g., 5 positions in a 10 amino acid long polymer) are identical, the two sequences are 50% identical, and if 90% of the positions (e.g., 9 out of 10) are matching or identical, the two amino acid sequences are 90% identical.

[0100] As used herein, the term "immune response" is defined as a cellular response to an antigen that occurs when lymphocytes identify the antigen molecule as foreign and induce the formation of antibodies and / or activate lymphocytes to eliminate the antigen.

[0101] The term "immunosuppressive" is used herein to refer to decreasing the overall immune response.

[0102] "Isolated" means changed or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein may exist in a substantially purified form, or may exist in a non-native environment, such as, for example, a host cell.

[0103] "Lentivirus" as used herein refers to a genus of the Retroviridae family.Lentiviruses are unique among retroviruses in that they can infect non-dividing cells; they can deliver a significant amount of genetic information to the DNA of host cells, making them one of the most efficient methods of gene delivery vectors.HIV, SIV, and FIV are all examples of lentiviruses.Vectors derived from lentiviruses provide a means to achieve significant levels of gene transfer in vivo.

[0104] The term "modified" as used herein refers to an altered state or structure of a molecule or cell of the invention. Molecules can be modified in many ways, including chemical, structural, and functional ways. Cells can be modified through the introduction of nucleic acids.

[0105] The term "modulate" as used herein means to mediate a detectable increase or decrease in the level of response in a subject, compared to the level of response in a subject in the absence of a treatment or compound, and / or compared to the level of response in an otherwise identical but untreated subject.This term encompasses disrupting and / or affecting a natural signal or response, thereby mediating a beneficial therapeutic response in a subject, preferably a human.

[0106] In the context of the present invention, the following abbreviations for commonly occurring nucleobases are used: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine and "U" refers to uridine.

[0107] The term "oligonucleotide" typically refers to a short polynucleotide. When a nucleotide sequence is represented by a DNA sequence (i.e., A, T, C, G), it will be understood that this also includes an RNA sequence in which "U" replaces "T" (i.e., A, U, C, G).

[0108] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA can also include introns, to the extent that the nucleotide sequence encoding the protein may, in some versions, contain introns.

[0109] "Parenteral" administration of the immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection, or infusion techniques.

[0110] The term "polynucleotide" as used herein is defined as a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Thus, as used herein, "nucleic acid" and "polynucleotide" are interchangeable. Those skilled in the art have the general knowledge that a nucleic acid is a polynucleotide that can be hydrolyzed into monomeric "nucleotides" and contains one or more "nucleotide sequences". The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotide includes, but is not limited to, all nucleic acid sequences (i.e., "nucleotide sequences") obtained by any means available in the art, including, but not limited to, recombinant means, i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques and PCR, etc., as well as synthetic means.

[0111] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to a compound composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that a protein or peptide sequence can contain. A polypeptide includes any peptide or protein that contains two or more amino acids linked together by peptide bonds. As used herein, the term refers to both short chains, also commonly referred to in the art as peptides, oligopeptides, and oligomers, for example, and longer chains, of which there are many varieties, commonly referred to in the art as proteins. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0112] The term "specifically binds" as used herein with respect to an antibody means an antibody that recognizes a particular antigen but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind that antigen from one or more species. However, such species cross-reactivity does not in itself change the classification of the antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to a different allelic form of the antigen. However, such cross-reactivity does not in itself change the classification of the antibody as specific. In some cases, the terms "specific binding" or "specifically binds" may be used with respect to the interaction of an antibody, protein, or peptide with a second chemical species to mean that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) of the chemical species; for example, an antibody recognizes and binds to a particular protein structure, rather than proteins broadly. If an antibody is specific for epitope "A", then in a reaction involving labeled "A" and an antibody, the presence of a molecule containing epitope A (or free unlabeled A) will reduce the amount of labeled A that binds to the antibody.

[0113] The term "stimulation" refers to a primary response induced by the binding of a stimulatory molecule (e.g., the TCR / CD3 complex) with its cognate ligand, thereby mediating a signal transduction event, such as, but not limited to, signal transduction through the TCR / CD3 complex. Stimulation can mediate changes in the expression of certain molecules, such as downregulation of TGF-beta and / or rearrangement of cytoskeletal structure.

[0114] "Stimulatory molecule," as that term is used herein, means a molecule on a T cell that specifically binds to a cognate stimulatory ligand present on an antigen-presenting cell.

[0115] As used herein, "stimulatory ligand" refers to a ligand that, when present on an antigen-presenting cell (e.g., aAPC, dendritic cell, B cell, etc.), can specifically bind to a cognate binding partner (herein referred to as a "stimulatory molecule") on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, etc. Stimulatory ligands are well known in the art and include peptide-loaded MHC class I molecules, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies, among others.

[0116] The term "subject" is intended to include a living organism (e.g., a mammal) in which an immune response can be elicited. A "subject" or "patient" as used herein can be a human or non-human mammal. Non-human mammals include, for example, farm animals and pets, such as ovine, bovine, porcine, canine, feline and murine mammals, as well as monkeys and non-human primate mammals. Preferably, the subject is a human.

[0117] "Target site" or "target sequence" refers to a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule can specifically bind under conditions sufficient for binding to occur. In some embodiments, a target sequence refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule can specifically bind under conditions sufficient for binding to occur.

[0118] As used herein, the term "T cell receptor" or "TCR" refers to a complex of membrane proteins involved in the activation of T cells in response to the presentation of an antigen. TCRs are responsible for the recognition of antigens bound to major histocompatibility complex molecules. TCRs are composed of a heterodimer of alpha (α) and beta (β) chains, although in some cells, TCRs consist of gamma and delta (γ / δ) chains. TCRs can exist in alpha / beta and gamma / delta forms, which are structurally similar but have different anatomical locations and functions. Each chain is composed of two extracellular domains, a variable domain and a constant domain. In some embodiments, TCRs can be engineered on any cell that contains a TCR, including, for example, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, and gamma delta T cells.

[0119] The term "therapeutic" as used herein means treatment and / or prophylaxis. The therapeutic effect is achieved by suppression, amelioration, or eradication of the disease state.

[0120] The terms "transfected" or "transformed" or "transduced" as used herein refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0121] "Treating" a disease, as that term is used herein, means reducing the frequency or severity of at least one sign or symptom of the disease or disorder experienced by a subject.

[0122] A "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the inside of a cell. Numerous vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, etc.

[0123] Ranges: Throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to specifically disclose all possible subranges as well as individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to specifically disclose subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0124] B. Chimeric antigen receptors (CARs) The present invention provides modified immune cells or their precursors (e.g., modified T cells) engineered to express a chimeric antigen receptor (CAR) comprising an intracellular domain comprising the interleukin-9 receptor alpha (IL9Ra) intracellular signaling domain (IL-9Ra ICD). The CAR further comprises an extracellular tumor antigen binding domain, a transmembrane domain, and an intracellular domain. The extracellular tumor antigen binding domain of the CAR is operably linked to another domain of the CAR, such as a hinge domain, a transmembrane domain, or an intracellular domain, each of which is described elsewhere herein.

[0125] The tumor antigen binding domains described herein can be combined with any transmembrane domain described herein, any intracellular or cytoplasmic domain described herein, or any other domain described herein that can be included in a CAR of the invention, such as a hinge domain or spacer sequence.

[0126] The CAR of the present invention may also include a leader sequence as described herein. The CAR of the present invention may also include a hinge domain as described herein. The CAR of the present invention may also include one or more spacer domains or linkers as described herein, which may serve to link one domain of the CAR to the next domain.

[0127] Antigen-binding domain The antigen-binding domain of the CAR is the extracellular region of the CAR for binding to a specific target antigen, including proteins, carbohydrates, and glycolipids. The CAR of the present invention comprises an antigen-binding domain capable of binding a tumor antigen. Suitable tumor antigens are known in the art, including alpha fetoprotein (AFP) / HLA-A2, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA, claudin 18.2, c-Met, DLL3, DR5, EGFR, EGFRvIII, These include, but are not limited to, EpCAM, EphA2, FAP, folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, glycolipid F77, glypican-2 (GPC2), glypican-3 (GPC3), HER2, HLA-A2, ICAM1, IL3Ra, IL13Ra2, LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan-A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, Nectin 4 / FAP, NKG2D ligands (MIC-A, MIC-B, and ULBP 1-6), NY-ESO-1, P16, PD-L1, PSCA, PSMA, ROR1, ROR2, TIM-3, TM4SF1, TnMuc1, VEGFR2, and any combination thereof. In some embodiments, the tumor antigen is selected from mesothelin, GD2, HER2, TnMuc1, GPC2, CD70, PMSA, and EGFRvIII.

[0128] The antigen-binding domain can comprise any domain that binds to an antigen (e.g., a tumor antigen) and can include, but is not limited to, a monoclonal antibody (mAb), a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a non-human antibody, a single domain antibody, a full-length antibody or any antigen-binding fragment thereof, a Fab, and a single chain variable fragment (scFv). In some embodiments, the antigen-binding domain comprises an aglycosylated antibody or a fragment thereof or a scFv thereof. In some embodiments, the tumor antigen-binding domain is a scFv.

[0129] As used herein, the term "single-chain variable fragment" or "scFv" refers to a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulins (e.g., mouse or human) covalently linked to form a VH::VL heterodimer. The variable heavy (VH) and variable light (VL) chains are either directly linked or linked by a peptide linker connecting the N-terminus of the VH to the C-terminus of the VL or the C-terminus of the VH to the N-terminus of the VL. In some embodiments, the antigen-binding domain (e.g., tumor antigen-binding domain) comprises an scFv having a VH-linker-VL configuration from the N-terminus to the C-terminus. In some embodiments, the antigen-binding domain comprises an scFv having a VL-linker-VH or VH-linker-VL configuration from the N-terminus to the C-terminus. One skilled in the art will be able to select the appropriate configuration for use in the present invention.

[0130] The linker is usually glycine-rich for flexibility and serine or threonine-rich for solubility. The linker can link the heavy and light chain variable regions of the extracellular antigen-binding domain. Non-limiting examples of linkers are disclosed in Shen et al., Anal.Chem.80(6):1910-1917(2008) and WO 2014 / 087010, the contents of which are incorporated herein by reference in their entirety. Various linker sequences are known in the art, including, but not limited to, glycine serine (GS) linkers. Those skilled in the art will be able to select appropriate linker sequences for use in the present invention. In one embodiment, the antigen-binding domain of the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), and the VH and VL are separated by a linker sequence.

[0131] Despite the removal of the constant region and the introduction of the linker, the scFv protein retains the specificity of the original immunoglobulin. Single chain Fv polypeptide antibodies can be expressed from nucleic acids containing sequences encoding VH and VL as described by Huston, et al. (Proc.Nat.Acad.Sci.USA, 85:5879-5883, 1988). See also U.S. Patent Nos. 5,091,513, 5,132,405 and 4,956,778, and U.S. Patent Application Publication Nos. 20050196754 and 20050196754. Antagonist scFvs with inhibitory activity have been described (e.g., Zhao et al., Hybridoma(Larchmt)2008 27(6):455-51; Peter et al., J Cachexia Sarcopenia Muscle 2012 August 12; Shieh et al., J Imunol 2009 183(4):2277-85; Giomarelli et al., Thromb Haemost 2007 97(6):955-63; Fife et al., J Clin Invst 2006 116(8):2252-61; Brocks et al., Immunotechnology 1997 3(3):173-84; Moosmayer et al., Ther Immunol 1995 116(8):2252-61). 2(10:31-40). Agonistic scFvs with stimulatory activity have been described (see, e.g., Peter et al., J Bioi Chem 2003 25278(38):36740-7; Xie et al., Nat Biotech 1997 15(8):768-71; Ledbetter et al., Crit Rev Immunol 1997 17(5-6):427-55; Ho et al., BioChim Biophys Acta 2003 1638(3):257-66).

[0132] As used herein, "Fab" refers to the fragment of an antibody structure that binds an antigen but is monovalent and does not have an Fc portion; for example, digestion of an antibody with the enzyme papain produces two Fab fragments and one Fc fragment (e.g., heavy (H) chain constant region; the Fc region that does not bind antigen).

[0133] As used herein, "F(ab')2" refers to an antibody fragment produced by pepsin digestion of a whole IgG antibody, which has two antigen-binding (ab') (bivalent) regions, each (ab') region containing two separate amino acid chains, a portion of a heavy chain and a light (L) chain linked by an S-S bond for binding to the antigen, with the remaining portions of the heavy chain linked together. The "F(ab')2" fragment can be split into two individual Fab' fragments.

[0134] In other embodiments, the antigen binding domain comprises an antibody mimetic protein such as, for example, a designed ankyrin repeat protein (DARPin), an affibody, a monobody, (i.e., an adnectin), an affilin, an affimer, an affitin, an alphabody, an avimer, a Kunitz domain peptide, or anticalin. Constructs with specific binding affinities can be generated using DARPin libraries, for example, as described in Seeger, et al., , Protein Sci., 22:1239-1257(2013).

[0135] In some embodiments, the antigen binding domain can be derived from the same species that the CAR will ultimately be used in. For example, for use in humans, the antigen binding domain of the CAR can comprise a human antibody or a fragment thereof. In some embodiments, the antigen binding domain can be derived from a different species that the CAR will ultimately be used in. For example, for use in humans, the antigen binding domain of the CAR can comprise a mouse antibody or a fragment thereof, or a humanized mouse antibody or a fragment thereof.

[0136] In certain embodiments, the antigen binding domain comprises a heavy chain variable region comprising three heavy chain complementarity determining regions (HCDRs) and a light chain variable region comprising three light chain complementarity determining regions (LCDRs). In certain embodiments, the antigen binding domain comprises a linker.

[0137] Transmembrane domain The CAR of the present invention may comprise a transmembrane domain that connects the antigen-binding domain of the CAR to the intracellular domain of the CAR. The transmembrane domain of the CAR is a region that can span the plasma membrane of a cell (e.g., an immune cell or its precursor). In some embodiments, the transmembrane domain is sandwiched between the antigen-binding domain and the intracellular domain of the CAR.

[0138] In some embodiments, the transmembrane domain is naturally associated with one or more of the domains in the CAR (e.g., a CAR that includes an IL-9R ICD). In some embodiments, the transmembrane domain may be selected to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins, or may be modified by one or more amino acid substitutions, to minimize interactions with other members of the receptor complex.

[0139] The transmembrane domain can be derived from either natural or synthetic sources.When the source is natural, the domain can be derived from any membrane-associated or transmembrane protein, such as type I transmembrane protein.When the source is synthetic, the transmembrane domain can be any artificial sequence, such as an artificial hydrophobic sequence, that facilitates the insertion of the CAR (e.g., a CAR that comprises IL-9R ICD) into the cell membrane. Examples of transmembrane domains that are particularly useful in the present invention include, but are not limited to, transmembrane domains derived from (i.e., comprising at least the transmembrane region of) the alpha, beta or zeta chains of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), ICOS, CD278, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, or transmembrane domains derived from killer immunoglobulin-like receptors (KIR).

[0140] In certain embodiments, the transmembrane domain comprises the transmembrane domain of CD8. In certain embodiments, the transmembrane domain of CD8 is the transmembrane domain of CD8α.

[0141] In some embodiments, the transmembrane domain may be synthetic, in which case it comprises primarily hydrophobic residues such as leucine and valine. Preferably, triplets of phenylalanine, tryptophan and valine are found at each end of the synthetic transmembrane domain.

[0142] The transmembrane domains described herein can be combined with any of the antigen binding domains described herein, any of the intracellular domains described herein, or any of the other domains described herein that can be included in a CAR comprising an IL-9R ICD described herein.

[0143] In some embodiments, the transmembrane domain further comprises a hinge region. The CAR of the present invention may also comprise a hinge region. The hinge region of the CAR is a hydrophilic region located between the antigen binding domain and the transmembrane domain. In some embodiments, this domain promotes proper protein folding of the CAR. The hinge region is an optional component of the CAR. The hinge region may comprise a domain selected from an Fc fragment of an antibody, a hinge region of an antibody, a CH2 region of an antibody, a CH3 region of an antibody, an artificial hinge sequence, or a combination thereof. Examples of hinge regions include, but are not limited to, CD8a hinges, artificial hinges made of polypeptides that may be as small as three glycines (Gly), and the CH1 and CH3 domains of IgG (such as human IgG4).

[0144] In some embodiments, the CAR of the present disclosure comprises a hinge region that connects the antigen binding domain to the transmembrane domain, which in turn connects to the intracellular domain. The hinge region can preferably support the antigen binding domain to recognize and bind to the target antigen on the target cell (see, for example, Hudecek et al., Cancer Immunol.Res.(2015)3(2):125-135). In some embodiments, the hinge region is a flexible domain, which thus allows the antigen binding domain to have a structure that optimally recognizes the specific structure and density of the target antigen on a cell, such as a tumor cell (Hudecek et al. supra). The flexibility of the hinge region allows it to adopt many different conformations.

[0145] In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. In some embodiments, the hinge region is a receptor-derived hinge region polypeptide (e.g., a CD8-derived hinge region).

[0146] The hinge region can have a length of about 4 amino acids to about 50 amino acids, for example, about 4 aa to about 10 aa, about 10 aa to about 15 aa, about 15 aa to about 20 aa, about 20 aa to about 25 aa, about 25 aa to about 30 aa, about 30 aa to about 40 aa, or about 40 aa to about 50 aa. In some embodiments, the hinge region can have a length of more than 5 aa, more than 10 aa, more than 15 aa, more than 20 aa, more than 25 aa, more than 30 aa, more than 35 aa, more than 40 aa, more than 45 aa, more than 50 aa, more than 55 aa, or more.

[0147] A suitable hinge region can be readily selected and can be any of a number of suitable lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 to 15 amino acids, from 3 to 12 amino acids, such as from 4 to 10 amino acids, from 5 to 9 amino acids, from 6 to 8 amino acids, or from 7 to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids. Suitable hinge regions can have a length of more than 20 amino acids (e.g., 30, 40, 50, 60 or more amino acids).

[0148] For example, the hinge region may be a glycine polymer (G) n, glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured and therefore can function as neutral tethers between components. Glycine polymers can be used; glycine has access to significantly more phi-psi space than alanine and is much less restricted than residues with longer side chains (see, e.g., Scheraga, Rev.Computational.Chem.(1992)2:73-142). The hinge region can include the amino acid sequence of the hinge region of human IgG1, IgG2, IgG3, or IgG4 (see, e.g., Yan et al., J.Biol.Chem.(2012)287:5891-5897). In one embodiment, the hinge region can include an amino acid sequence derived from human CD8, or a variant thereof.

[0149] Intracellular signaling domains The CAR of the present invention also comprises an intracellular signaling domain. The terms "intracellular signaling domain" and "intracellular domain" are used interchangeably herein. The intracellular signaling domain of the CAR is responsible for activating at least one effector function of the cell (e.g., immune cell) in which the CAR is expressed. The intracellular signaling domain transmits an effector function signal and instructs the cell (e.g., immune cell) to perform its specialized function, such as damaging and / or destroying a target cell.

[0150] Examples of intracellular domains for use in the present invention include, but are not limited to, the cytoplasmic portion of a surface receptor, a costimulatory molecule, and any molecule that acts in concert to initiate signaling in a T cell, as well as any derivatives or variants of these elements, and any synthetic sequences having the same functional capabilities.

[0151] Examples of intracellular signaling domains include, but are not limited to, the ζ chain of the T cell receptor complex or any of its homologs, such as the η chain, FcsRI γ and β chains, MB1 (Iga) chain, B29 (Ig) chain, human CD3 zeta chain, CD3 polypeptides (Δ, δ and ε), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.), and other molecules involved in T cell transduction such as CD2, CD5 and CD28. In one embodiment, the intracellular signaling domain may comprise an intracellular signaling domain of a protein selected from human CD3 zeta chain, FcyRIII, FcsRI, DAP10, DAP12, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor with an immunoreceptor tyrosine-based activation motif (ITAM), and combinations thereof.

[0152] In one embodiment, the intracellular signaling domain of the CAR comprises any portion of one or more costimulatory molecules, such as CD2, CD3, CD8, CD27, CD28, ICOS, 4-1BB, PD-1, any derivative or variant thereof, such as any synthetic sequence thereof having the same functional capability, and any combination thereof.

[0153] Other examples of intracellular domains include TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc epsilon RIb), CD79a, CD79b, Fc gamma RIIa, DAP10, DAP12, T cell receptor (TCR), CD8, CD27, CD28, 4-1BB (CD137), OX9, OX40, CD30, CD40, PD-1, ICOS, KIR family proteins, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ligand that specifically binds CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, 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, CDlib, ITGAX, CD11c, ITGBl, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT Examples of co-stimulatory molecules include, but are not limited to, AM, 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, NKp44, NKp30, NKp46, NKG2D, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, IL-9R, IL-21R, other co-stimulatory molecules described herein, any derivative, variant or fragment thereof, any synthetic sequence of a co-stimulatory molecule having the same functional capability, and any combination thereof.

[0154] Further examples of intracellular domains include the intracellular signaling domains of several types of various other immune signaling receptors, including, but not limited to, first, second and third generation T cell signaling proteins, including, but not limited to, CD3, B7 family costimulatory molecules and tumor necrosis factor receptor (TNFR) superfamily receptors (see, e.g., Park and Brentjens, J. Clin. Oncol. (2015) 33(6):651-653). Additionally, the intracellular signaling domain can include signaling domains used by NK cells and NKT cells (see, e.g., Hermanson and Kaufman, Front. Immunol. (2015) 6:195), such as the signaling domains of NKp30 (B7-H6) (see, e.g., Zhang et al., J. Immunol. (2012) 189(5):2290-2299), and DAP12 (see, e.g., Topfer et al., J. Immunol. (2015) 194(7):3201-3212), NKG2D, NKp44, NKp46, DAP10, and CD3z.

[0155] Intracellular signaling domains suitable for use in the CAR of the present invention include any desired signaling domain that provides a distinct detectable signal (e.g., increased production of one or more cytokines by the cell; altered transcription of a target gene; altered activity of a protein; altered cell behavior, such as cell death; cell proliferation; cell differentiation; cell survival; modulation of cell signaling responses, etc.) in response to activation of the CAR (i.e., activated by an antigen and a dimerization agent). In some embodiments, the intracellular signaling domain comprises at least one (e.g., 1, 2, 3, 4, 5, 6, etc.) ITAM motif described below. In some embodiments, the intracellular signaling domain comprises a DAP10 / CD28-type signaling chain. In some embodiments, the intracellular signaling domain is not covalently attached to the membrane-bound CAR, but instead is diffused in the cytoplasm.

[0156] Intracellular signaling domains suitable for use in the CARs of the invention include immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptides. In some embodiments, the ITAM motif is repeated twice in the intracellular signaling domain, with the first and second occurrences of the ITAM motif being separated from each other by 6-8 amino acids. In one embodiment, the intracellular signaling domain of the CAR comprises three ITAM motifs.

[0157] In some embodiments, the intracellular signaling domain comprises a signaling domain of a human immunoglobulin receptor containing an immunoreceptor tyrosine-based activation motif (ITAM), such as, but not limited to, Fc gamma RI, Fc gamma RIIA, Fc gamma RIIC, Fc gamma RIIIA, FcRL5 (see, e.g., Gillis et al., Front. Immunol. (2014) 5:254).

[0158] Suitable intracellular signaling domains can be ITAM motif-containing moieties derived from ITAM motif-containing polypeptides. For example, suitable intracellular signaling domains can be ITAM motif-containing domains derived from any ITAM motif-containing protein. Thus, suitable intracellular signaling domains do not need to include the entire sequence of the entire protein from which they are derived. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to, DAP12, FCER1G (Fc epsilon receptor I gamma chain), CD3D (CD3 delta), CD3E (CD3 epsilon), CD3G (CD3 gamma), CD3Z (CD3 zeta), and CD79A (antigen receptor complex-associated protein alpha chain).

[0159] In one embodiment, the intracellular signaling domain is derived from DAP12 (also known as TYROBP; TYRO protein tyrosine kinase binding protein; KARAP; PLOSL; DNAX activating protein 12; KAR associated protein; TYRO protein tyrosine kinase binding protein; killer activating receptor associated protein; killer activating receptor associated protein, etc.). In one embodiment, the intracellular signaling domain is derived from FCER1G (also known as FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma chain; fc epsilon RI gamma; fcR gamma; fceRl gamma; high affinity immunoglobulin epsilon receptor subunit gamma; high affinity gamma chain of immunoglobulin E receptor, etc.). In one aspect, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 delta chain (also known as CD3D; CD3-DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD3d antigen, delta polypeptide (TiT3 complex); OKT3, delta chain; T cell receptor T3 delta chain; T cell surface glycoprotein CD3 delta chain, etc.). In one aspect, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 epsilon chain (also known as CD3e, T cell surface antigen T3 / Leu-4 epsilon chain, T cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3 epsilon, T3e, etc.). In one aspect, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 gamma chain (also known as CD3G, T cell receptor T3 gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3 complex), etc.). In one aspect, the intracellular signaling domain is derived from the T cell surface glycoprotein CD3 zeta chain (also known as CD3Z, T cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.). In one aspect, the intracellular signaling domain is derived from CD79A (also known as B cell antigen receptor complex associated protein alpha chain; CD79a antigen (immunoglobulin-associated alpha); MB-1 membrane glycoprotein; ig alpha; membrane-bound immunoglobulin-associated protein; surface IgM-associated protein, etc.).In one embodiment, the intracellular signaling domain suitable for use in the CAR of the present disclosure comprises an IL-9Ra or IL-21R type signaling chain. In one embodiment, the intracellular signaling domain suitable for use in the CAR of the present disclosure comprises an IL-9Ra intracellular domain as described herein. In some embodiments, the intracellular signaling domain comprises the cytoplasmic signaling domain of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, or CD66d. In one embodiment, the intracellular signaling domain in the CAR comprises the cytoplasmic signaling domain of human CD3 zeta.

[0160] Typically, the entire intracellular signaling domain can be used, but in many cases it is not necessary to use the entire molecule. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain, so long as it transmits an effector function signal. The intracellular signaling domain includes any truncated portion of the intracellular signaling domain sufficient to transmit an effector function signal.

[0161] The intracellular domains described herein can be combined with any of the antigen binding domains described herein, any of the transmembrane domains described herein, or any of the other domains described herein that can be included in a CAR.

[0162] In certain embodiments, the intracellular domain comprises a costimulatory domain of 4-1BB. In certain embodiments, the intracellular domain comprises an intracellular domain of CD3zeta or a variant thereof. In certain embodiments, the intracellular domain comprises a costimulatory domain of 4-1BB and an intracellular domain of CD3zeta.

[0163] CAR containing an ICD that contains the intracellular signaling domain of IL9Ra In one aspect, the present invention provides a CAR that includes an extracellular tumor antigen binding domain, a transmembrane domain, and an intracellular domain that includes an intracellular signaling domain of IL9Ra. The CAR and modified cells (e.g., immune cells) that express the CAR improve CAR cell immunotherapy for treating cancer by (1) utilizing tumor antigens in the tumor to enhance immune stimulatory signals in immune cells (e.g., T cells), (2) changing the phenotype of the immune cells that express the CAR, and (3) enabling IL-9 signaling in the immune cells that express the CAR to improve effector function in situ.

[0164] In some embodiments, the IL9Ra ICD comprises SEQ ID NO:1, SEQ ID NO:4 or SEQ ID NO:8. In some embodiments, the IL9Ra ICD is encoded by SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO:124.

[0165] In various embodiments, the intracellular domain of the CAR may further comprise one or more costimulatory and / or signaling domains described elsewhere herein, such as a CD28 and / or 4-1BB costimulatory domain and / or a CD3z stimulatory domain. In some embodiments, the intracellular domain comprises a CD28 costimulatory domain, an intracellular signaling domain of IL9Ra, and a CD3z stimulatory domain. In some embodiments, the intracellular domain comprises a 4-1BB costimulatory domain, an intracellular signaling domain of IL9Ra, and a CD3z stimulatory domain.

[0166] In various embodiments, the tumor antigen binding domain comprises a domain selected from an anti-mesothelin antigen binding domain, an anti-GD2 antigen binding domain, an anti-HER2 antigen binding domain, an anti-GPC2 antigen binding domain, an anti-CD19 antigen binding domain, an anti-TnMuc1 antigen binding domain, an anti-CD70 antigen binding domain, an anti-PMSA antigen binding domain, and an EGFRvIII antigen binding domain.

[0167] In some embodiments, the CAR comprises an extracellular anti-mesothelin antigen binding domain, an intracellular domain comprising a CD8 hinge, a CD28 TM and a CD28 costimulatory domain, an IL9Ra ICD and a CD3z signaling domain.

[0168] In some embodiments, the CAR comprises an extracellular anti-mesothelin antigen binding domain, a CD8 hinge, a CD8 TM and an intracellular domain comprising a 4-1BB costimulatory domain, an IL9Ra ICD and a CD3z signaling domain.

[0169] Permitted variations in individual CAR domain sequences (leader, antigen binding domain, hinge, transmembrane and / or intracellular domains) will be known to one of skill in the art. For example, in certain embodiments, the CAR domain comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any naturally occurring or known sequence.

[0170] The amino acid and nucleotide sequences for certain embodiments of CAR and its domains are set forth below. TIFF2024534417000001.tif98163TIFF2024534417000002.tif206163TIFF2024534417000003.tif212163TIFF2024 534417000004.tif205163TIFF2024534417000005.tif206163TIFF2024534417000006.tif214163TIFF20245344170 00007.tif208163TIFF2024534417000008.tif207163TIFF2024534417000009.tif215163TIFF2024534417000010.t if208163TIFF2024534417000011.tif212163TIFF2024534417000012.tif213163TIFF2024534417000013.tif213163 TIFF2024534417000014.tif213163TIFF2024534417000015.tif212163TIFF2024534417000016.tif212163TIFF202 4534417000017.tif213163TIFF2024534417000018.tif205166TIFF2024534417000019.tif208165TIFF20245344170 00020.tif214163TIFF2024534417000021.tif212163TIFF2024534417000022.tif199163TIFF2024534417000023.t if213163TIFF2024534417000024.tif212163TIFF2024534417000025.tif213163TIFF2024534417000026.tif205163

[0171] C. Nucleic acids and expression vectors In one aspect, the present invention provides an isolated nucleic acid comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) comprising a tumor antigen binding domain, a transmembrane domain, and an intracellular domain comprising the intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra).

[0172] In certain embodiments, the nucleic acid of the present disclosure comprises a first nucleotide sequence and a second nucleotide sequence. The first and second nucleotide sequences can be separated by a linker. A linker for use in the present disclosure allows multiple proteins to be encoded by the same nucleic acid sequence (e.g., a multicistronic or bicistronic sequence), which are translated as a polyprotein that is dissociated into separate protein components. In certain embodiments, the nucleic acid comprises, from 5' to 3', a first nucleotide sequence, a linker, and a second nucleotide sequence. In certain embodiments, the nucleic acid comprises, from 5' to 3', a second nucleotide sequence, a linker, and a first nucleotide sequence.

[0173] In some embodiments, the linker comprises a nucleic acid sequence encoding an internal ribosome entry site (IRES). As used herein, "internal ribosome entry site" or "IRES" refers to an element that promotes direct internal ribosome entry into a start codon, such as ATG, of a protein coding region, thereby resulting in cap-independent translation of the gene. A variety of internal ribosome entry sites are known to those skilled in the art, including, but not limited to, IRESs obtained from viral or cellular mRNA sources, such as immunoglobulin heavy chain binding protein (BiP); vascular endothelial growth factor (VEGF); fibroblast growth factor 2; insulin-like growth factor; translation initiation factor eIF4G; yeast transcription factors TFIID and HAP4; and IRESs obtained from, for example, cardioviruses, rhinoviruses, aphthoviruses, HCV, Friend murine leukemia virus (FrMLV) and Moloney murine leukemia virus (MoMLV). Those skilled in the art will be able to select the appropriate IRES for use in the present invention.

[0174] In some embodiments, the linker comprises a nucleic acid sequence that encodes a self-cleaving peptide. As used herein, "self-cleaving peptide" or "2A peptide" refers to an oligopeptide that allows multiple proteins to be encoded as a polyprotein that dissociates into component proteins upon translation. The use of the term "self-cleaving" is not intended to imply a proteolytic cleavage reaction. A variety of self-cleaving or 2A peptides are known to those of skill in the art, including, but not limited to, those found in members of the Picornaviridae virus family, such as foot and mouth disease virus (FMDV), equine rhinitis A virus (ERAV0, Thosea asigna virus (TaV), and porcine teschovirus-1 (PTV-1), as well as cardioviruses such as tylovirus and encephalomyocarditis virus. The 2A peptides derived from FMDV, ERAV, PTV-1, and TaV are referred to herein as "F2A," "E2A," "P2A," and "T2A," respectively. One of skill in the art would be able to select an appropriate self-cleaving peptide for use in the present invention.

[0175] In some embodiments, the construct optionally includes a linker further comprising a nucleic acid sequence encoding a furin cleavage site. Furin is a ubiquitously expressed protease that resides in the trans-Golgi and processes protein precursors before their secretion. Furin cleaves at the COOH-terminus of its consensus recognition sequence. A variety of furin consensus recognition sequences (or "furin cleavage sites") are known to those skilled in the art. Those skilled in the art will be able to select a suitable furin cleavage site for use in the present invention.

[0176] In some embodiments, the linker comprises a nucleic acid sequence encoding a combination of a furin cleavage site and a 2A peptide. Examples include, but are not limited to, a linker comprising a nucleic acid sequence encoding a furin cleavage site and F2A, a linker comprising a nucleic acid sequence encoding a furin cleavage site and E2A, a linker comprising a nucleic acid sequence encoding a furin cleavage site and P2A, and a linker comprising a nucleic acid sequence encoding a furin cleavage site and T2A. Those skilled in the art will be able to select a suitable combination for use in the present invention. In such embodiments, the linker may further comprise a spacer sequence between the furin cleavage site and the 2A peptide. In some embodiments, the linker comprises a furin cleavage site 5' to the 2A peptide. In some embodiments, the linker comprises a 2A peptide 5' to the furin cleavage site. A variety of spacer sequences are known in the art, including, but not limited to, a glycine serine (GS) spacer (also known as a GS linker). Those skilled in the art will be able to select a suitable spacer sequence for use in the present invention.

[0177] In some aspects, the nucleotide sequences of the present disclosure may be operably linked to transcriptional control elements, such as promoters and enhancers. Suitable promoter and enhancer elements are known to those of skill in the art.

[0178] In certain embodiments, the promoter is selected from the phosphoglycerate kinase 1 (PGK) promoter, the EF-1a promoter, and the CMV promoter.

[0179] For expression in bacterial cells, suitable promoters include, but are not limited to, lacI, lacZ, T3, T7, gpt, lambda P and trc.For expression in eukaryotic cells, suitable promoters include, but are not limited to, light and / or heavy chain immunoglobulin gene promoter and enhancer elements; cytomegalovirus immediate early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoter; promoters present in long terminal repeat sequences from retroviruses; mouse metallothionein-I promoter; and various tissue-specific promoters known in the art.Suitable reversible promoters, including reversible inducible promoters, are known in the art.Such reversible promoters can be isolated and derived from many organisms, for example eukaryotes and prokaryotes. The modification of a reversible promoter from a first organism for use in a second organism (e.g., a first prokaryote and a second eukaryote, a first eukaryote and a second prokaryote, etc.) is well known in the art.Such reversible promoters, and systems based on such reversible promoters but also comprising additional regulatory proteins, include, but are not limited to, alcohol-regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to alcohol transactivator protein (A1cR), etc.), tetracycline-regulated promoters (e.g., promoter systems including TetActivator, TetON, TetOFF, etc.), steroid-regulated promoters (e.g., rat glucocorticoid receptor promoter system, human estrogen receptor promoter system, retinoid promoter system, thyroid promoter system, ecdysone promoter system, mifepristone promoter system, etc.), metal-regulated promoters (e.g., metallothionein promoter system, etc.), pathogenesis-associated regulated promoters (e.g., salicylic acid-regulated promoters, ethylene-regulated promoters, benzothiadiazole-regulated promoters, etc.), temperature-regulated promoters (e.g., heat shock-inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.), light-regulated promoters, synthetic inducible promoters, etc.

[0180] In some embodiments, the promoter is a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, or a NK cell-specific promoter.For example, the CD4 gene promoter can be used; see, for example, Salmon et al.Proc.Natl.Acad.Sci.USA(1993)90:7739; and Marodon et al.(2003)Blood 101:3416.As another example, the CD8 gene promoter can be used.NK cell-specific expression can be achieved by using the NcrI(p46) promoter; see, for example, Eckelhart et al.Blood(2011)117:1565.

[0181] For expression in yeast cells, suitable promoters are constitutive promoters such as ADH1 promoter, PGK1 promoter, ENO promoter, PYK1 promoter; or regulatable promoters such as GAL1 promoter, GAL10 promoter, ADH2 promoter, PHOS promoter, CUP1 promoter, GALT promoter, MET25 promoter, MET3 promoter, CYC1 promoter, HIS3 promoter, ADH1 promoter, PGK promoter, GAPDH promoter, ADC1 promoter, TRP1 promoter, URA3 promoter, LEU2 promoter, ENO promoter, TP1 promoter, and AOX1 (e.g., for use in Pichia). The selection of suitable vectors and promoters is well within the level of one skilled in the art. Suitable promoters for use in prokaryotic host cells include the bacteriophage T7 RNA polymerase promoter; the trp promoter; the lac operon promoter; hybrid promoters, such as the lac / tac hybrid promoter, the tac / trc hybrid promoter, the trp / lac promoter, the T7 / lac promoter; the trc promoter; the tac promoter, and the like; the araBAD promoter; in vivo regulated promoters (e.g., Dunstan, et al., J. Bacteriol. (1991) 173(1):86-93; Alpuche-Aranda et al., Proc. Natl. Acad. Sci. USA (1992) 89(21):10079-83), such as the nirB promoter (Harborne et al. Mol. Micro. (1992) 6:2805-2813), et al., Infect.Immun.(1999)67:5133-5141; McKelvie et al., Vaccine(2004)22:3243-3255; and Chatfield et al., Biotechnol.(1992) 10:888-892); sigma70 promoters, such as the consensus sigma70 promoter (see, e.g., GenBank Accession Nos. AX798980, AX798961 and AX798183); stationary phase promoters, such as the dps promoter, the spv promoter, and the like; promoters from pathogenicity island SPI-2 (see, e.g., WO 96 / 17951); actA promoters (see, e.g., Shetron-Rama et al., Infect. Immun. (2002) 70:1087-1096); rpsM promoters (see, e.g., Valdivia and Falkow Mol. Microbiol. (1996) 22:367); tet promoters (see, e.g., Hillen, W. and Wissmann, A. (1989) In Saenger, W. and Heinemann, U. (eds), Topics in Molecular and Structural Biology, Protein-Nucleic Acid Interaction. Macmillan, London, UK, Vol. 10, pp. 143-162); SP6 promoter (see, e.g., Melton et al., Nucl. Acids Res.(1984) 12:7035), and the like. Strong promoters suitable for use in prokaryotes, such as Escherichia coli, include, but are not limited to, Trc, Tac, T5, T7, and Plambda. Non-limiting examples of operators for use in bacterial host cells include the lactose promoter operator (the LacI repressor protein changes conformation upon contact with lactose, thereby preventing the Lad repressor protein from binding to the operator), the tryptophan promoter operator (when complexed with tryptophan, the TrpR repressor protein has a conformation that binds to the operator; in the absence of tryptophan, the TrpR repressor protein has a conformation that does not bind to the operator), and the tac promoter operator (e.g., deBoer et al., Proc. Natl. Acad. Sci. USA (1983) 80:21-25).

[0182] Other examples of suitable promoters include the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can drive high levels of expression of any polynucleotide sequence operably linked to it. Other constitutive promoter sequences can also be used, including, but not limited to, the Simian Virus 40 (SV40) early promoter, the mouse mammary tumor virus (MMTV) or human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, the MoMuLV promoter, the avian leukosis virus promoter, the Epstein-Barr virus immediate early promoter, the Rous sarcoma virus promoter, the EF-1 alpha promoter, and human gene promoters, including, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on the expression of the operably linked polynucleotide sequence when such expression is desired, or turn off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to, a metallothionein promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0183] In some embodiments, the locus or construct or transgene containing a suitable promoter is irreversibly switched by the induction of an inducible system. Suitable systems for the induction of irreversible switches are well known in the art, for example, the induction of irreversible switches can utilize Cre-lox mediated recombination (see, for example, Fuhrmann-Benzakein, et al., Proc.Natl.Acad.Sci.USA (2000) 28:e99, the disclosure of which is incorporated herein by reference). Any suitable combination of recombinases, endonucleases, ligases, recombination sites, etc., known in the art can be used to create irreversibly switchable promoters. The methods, mechanisms and requirements for performing site-specific recombination described elsewhere herein are utilized in creating irreversibly switched promoters and are well known in the art, see, e.g., Grindley et al. Annual Review of Biochemistry (2006) 567-605; and Tropp, Molecular Biology (2012) (Jones & Bartlett Publishers, Sudbury, Mass.), the disclosures of which are incorporated herein by reference.

[0184] In some embodiments, the nucleic acid of the present disclosure further comprises a nucleic acid sequence encoding a CAR-inducible expression cassette. In one embodiment, the CAR-inducible expression cassette is used for the production of a transgenic polypeptide product released upon CAR signaling. See, e.g., Chmielewski and Abken, Expert Opin.Biol.Ther.(2015)15(8):1145-1154; and Abken, Immunotherapy(2015)7(5):535-544. In some embodiments, the nucleic acid of the present disclosure further comprises a nucleic acid sequence encoding a cytokine operably linked to a T cell activation responsive promoter. In some embodiments, the cytokine operably linked to the T cell activation responsive promoter is present on a separate nucleic acid sequence. In one embodiment, the cytokine is IL-12.

[0185] The nucleic acids of the present disclosure may be present in an expression vector and / or a cloning vector. Expression vectors may include a selection marker, an origin of replication, and other features that provide for replication and / or maintenance of the vector. Suitable expression vectors include, for example, plasmids, viral vectors, and the like. Numerous suitable vectors and promoters are known to those of skill in the art; many are commercially available for making recombinant constructs of interest. The following vectors are provided by way of example and should not be construed as limiting in any way: Bacteria: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).

[0186] Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. A selectable marker functional in the expression host may also be present. Suitable expression vectors include viral vectors (e.g., vaccinia virus; poliovirus; adenovirus-based viral vectors (e.g., Li et al., Invest. Opthalmol. Vis. Sci. (1994) 35:2543-2549; Borras et al., Gene Ther. (1999) 6:515-524; Li and Davidson, Proc. Natl. Acad. Sci. USA (1995) 92:7700-7704; Sakamoto et al., H. Gene Ther. (1999) 5:1088-1097; WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; see WO 95 / 11984 and WO 95 / 00655); adeno-associated virus (e.g., Ali et al., Hum. Gene Ther. (1998) 9:81-86, Flannery et al., Proc. Natl. Acad. Sci. USA (1997) 94:6916-6921; Bennett et al., Invest. Opthalmol. Vis. Sci. (1997) 38:2857-2863; Jomary et al., Gene Ther. (1997) 4:683 690, Rolling et al., Hum. Gene Ther. (1999) 10:641-648; Ali et al., Hum. Mol. Genet. (1996) 5:591-594; Srivastava in WO 93 / 09239, Samulski et al., J. Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988) 166:154-165; and Flotte et al., Proc. Natl. Acad. Sci.USA (1993) 90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., Proc. Natl. Acad. Sci. USA (1997) 94:10319-23; Takahashi et al., J. Virol. (1999) 73:7812-7816); retroviral vectors (e.g., vectors derived from murine leukemia virus, spleen necrosis virus, and retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus).

[0187] Further suitable expression vectors for use include, but are not limited to, lentivirus vectors, gamma retrovirus vectors, foamy virus vectors, adeno-associated virus vectors, adenovirus vectors, poxvirus vectors, herpes virus vectors, engineered hybrid virus vectors, transposon-mediated vectors, etc. Viral vector technology is well known in the art and 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 manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.

[0188] Generally, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No. 6,326,193).

[0189] In some embodiments, an expression vector (e.g., lentiviral vector) may be used to introduce a nucleic acid into an immune cell or its precursor (e.g., T cell). Thus, an expression vector (e.g., lentiviral vector) of the present invention may comprise a nucleic acid of the present invention comprising one or more nucleotide sequences encoding a CAR of the present invention. In some embodiments, an expression vector (e.g., lentiviral vector) comprises additional elements that aid in the functional expression of a receptor encoded therein. In some embodiments, an expression vector comprising a nucleic acid of the present invention further comprises an elongation factor 1 alpha promoter (EF-1α promoter). Use of the EF-1α promoter may increase the efficiency of expression of a downstream transgene (e.g., a nucleotide sequence encoding a CAR). Physiological promoters (e.g., EF-1α promoter) are less likely to induce integration-mediated genotoxicity and may negate the ability of a retroviral vector to transform stem cells. Other physiological promoters suitable for use in vectors (e.g., lentiviral vectors) are known to those of skill in the art and may be incorporated into the vectors of the present invention. In some embodiments, a vector (e.g., lentiviral vector) further comprises non-essential cis-acting sequences that may improve titer and gene expression. One non-limiting example of a non-essential cis-acting sequence is the central polypurine tract and central termination sequence (cPPT / CTS), which is important for efficient reverse transcription and nuclear import. Other non-essential cis-acting sequences are known to those skilled in the art and can be incorporated into the vectors of the present invention (e.g., lentiviral vectors). In some embodiments, the vector further comprises a post-transcriptional regulatory element. The post-transcriptional regulatory element can improve RNA translation, improve transgene expression, and stabilize RNA transcripts. One example of a post-transcriptional regulatory element is the Woodchuck Hepatitis Virus post-transcriptional regulatory element (WPRE). Thus, in some embodiments, the vector for the present invention further comprises a WPRE sequence. A variety of post-transcriptional regulatory elements are known to those skilled in the art and can be incorporated into the vectors of the present invention (e.g., lentiviral vectors).The vector of the present invention may further comprise additional elements such as a rev response element (RRE) for RNA transport, a packaging sequence, and 5' and 3' long terminal repeats (LTR). The term "long terminal repeat" or "LTR" refers to a domain of base pairs located at the end of retroviral DNA, including the U3, R and U5 regions. LTRs generally provide functions necessary for retroviral gene expression (e.g., promoting, initiating and polyadenylation of gene transcripts) and viral replication. In one embodiment, the vector of the present invention (e.g., lentiviral vector) comprises a 3'U3 deleted LTR. Thus, the vector of the present invention (e.g., lentiviral vector) may comprise any combination of elements described herein to increase the efficiency of functional expression of the transgene. For example, the vector of the present invention (e.g., lentiviral vector) may comprise a WPRE sequence, a cPPT sequence, an RRE sequence, a 5'LTR, a 3'U3 deleted LTR' in addition to the nucleic acid encoding the CAR of the present invention.

[0190] The vector of the present invention can be a self-inactivating vector. As used herein, the term "self-inactivating vector" refers to a vector in which the 3'LTR enhancer promoter region (U3 region) is modified (e.g., by deletion or substitution). A self-inactivating vector can prevent viral transcription beyond the first round of viral replication. As a result, a self-inactivating vector can infect and then integrate into a host genome (e.g., a mammalian genome) only once, and cannot pass further. Therefore, a self-inactivating vector can greatly reduce the risk of generating a replicative virus.

[0191] In some embodiments, the nucleic acid of the present invention can be RNA, for example, in vitro synthesized RNA. Methods for in vitro synthesis of RNA are known to those skilled in the art; any known method can be used to synthesize RNA comprising a sequence encoding a CAR of the present disclosure. Methods for introducing RNA into a host cell are known in the art. See, for example, Zhao et al.Cancer Res.(2010)15:9053. Introducing RNA comprising a nucleotide sequence encoding a CAR of the present disclosure into a host cell can be performed in vitro, ex vivo, or in vivo. For example, host cells (e.g., NK cells, cytotoxic T lymphocytes, etc.) can be electroporated with RNA comprising a nucleotide sequence encoding a CAR of the present disclosure in vitro or ex vivo.

[0192] To evaluate the expression of a polypeptide or a part thereof, the expression vector introduced into the cell can also include either a selectable marker gene or a reporter gene or both, to facilitate the identification and selection of expressing cells from the population of cells to be transfected or infected via viral vector.In some embodiments, the selectable marker can be carried on a separate piece of DNA and used in a co-transfection procedure.Both the selectable marker and the reporter gene can be adjacent to the appropriate regulatory sequence that allows expression in the host cell.Useful selectable markers include, but are not limited to, antibiotic resistance genes.

[0193] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences.Generally, reporter genes are genes that code for polypeptides that are not present in or expressed by recipient organisms or tissues, and whose expression is manifested by some easily detectable characteristic, such as enzymatic activity.The expression of reporter genes is evaluated at a suitable time after DNA is introduced into recipient cells.Suitable reporter genes may include, but are not limited to, genes that code for luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82).

[0194] In some aspects, the nucleic acids of the disclosure provide for the production of a CAR described herein, e.g., in a mammalian cell. In some aspects, the nucleic acids of the disclosure provide for the amplification of a nucleic acid encoding a CAR.

[0195] D. Engineered immune cells The present invention further provides a modified cell comprising: The cell is an immune cell or a precursor thereof, and The cells are engineered to express a chimeric antigen receptor (CAR) comprising a tumor antigen binding domain, a transmembrane domain, and an intracellular domain comprising the intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra), Modified cells are provided.

[0196] In some embodiments, the modified immune cell or a precursor thereof is selected from a T cell, a natural killer T (NKT) cell, a gamma delta T cell, a natural killer (NK) cell, and a macrophage.

[0197] In some embodiments, the modified cells are autologous cells. In some embodiments, the modified cells are autologous cells. In some embodiments, the cells are human cells obtained from a human subject. In some embodiments, the modified cells are T cells.

[0198] E. Treatment methods In another aspect, the present invention provides a method of treating cancer in a subject in need thereof, comprising administering to said subject a population of modified cells, wherein said cells are immune cells or precursor cells thereof, and said cells have been engineered to express a chimeric antigen receptor (CAR) comprising a tumor antigen binding domain, a transmembrane domain, and an intracellular domain comprising the intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra).

[0199] The modified cells (e.g., T cells) described herein can be included in a composition for immunotherapy. The composition can include a pharmaceutical composition and can further include a pharma- ceutically acceptable carrier. A therapeutically effective amount of the pharmaceutical composition containing the modified T cells can be administered.

[0200] In one aspect, the invention includes a method for adoptive cell transfer therapy comprising administering to a subject in need thereof a population of modified cells of the invention, the cells being immune cells or precursor cells thereof (e.g., T cells).

[0201] The administration method of immune cells for adoptive cell therapy is known and can be used in combination with the provided methods and compositions.For example, the method of adoptive immune cell therapy is described in, for example, U.S. Patent Application Publication No. 2003 / 0170238 to Gruenberg et al.; U.S. Patent No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol.8(10):577-85). For example, Themeli et al.(2013)Nat Biotechnol.31(10):928-933; Tsukahara et al.(2013)Biochem Biophys Res Commun 438(1):84-9; Davila et al.(2013)PLoS ONE 8(4):e61338; Lee et al., Int J.Mol Sci.(2021)22(9):4590; Banerjee et al., JCO Clin Cancer Inform.(2021)5:668-678; Robbins et al., Stem Cell Res Ther.(2021)12(1):350; Wrona et al., Int J Mol Sci.(2021)22(11):5899; Atrash and Moyo, Onco Targets See Ther.(2021)14:2185-2201; Martinez Bedoya et al., Front Immunol.(2021)12:640082; Morgan et al., Front Immunol.(2020)11:1965; Chicaybam et al., Cancers(Basel)(2020)12(9):2360; and Rafiq et al., Nat Rev Clin Oncol.(2020)17(3):147-167. In some embodiments, cell therapy, e.g., adoptive T cell therapy, is performed by autologous transplantation, where cells are isolated and / or otherwise prepared from a subject who is to receive cell therapy, or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from a subject, e.g., a patient, in need of treatment, and after isolation and processing, the cells are administered to the same subject.

[0202] In some embodiments, cell therapy, such as adoptive T cell therapy, is carried out by allogeneic transplantation, in which cells are isolated and / or otherwise prepared from a subject other than the subject that is to receive cell therapy or the subject that will ultimately receive cell therapy, such as a first subject.In such embodiments, cells are then administered to a different subject of the same species, such as a second subject.In some embodiments, the first subject and the second subject are genetically identical.In some embodiments, the first subject and the second subject are genetically similar.In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.

[0203] In some embodiments, the subject is treated with a therapeutic agent that targets a disease or condition, such as a tumor, prior to administration of the cell or cell-containing composition.In some aspects, the subject is refractory or non-responsive to other therapeutic agents.In some embodiments, the subject has persistent or recurrent disease after treatment with another therapeutic intervention, including, for example, chemotherapy, radiation, and / or hematopoietic stem cell transplantation (HSCT), such as allogeneic HSCT.In some embodiments, administration effectively treats the subject even though the subject has become resistant to another therapy.

[0204] In some embodiments, the subject is responsive to another therapeutic agent, and treatment with the therapeutic agent reduces disease burden. In some aspects, the subject is initially responsive to the therapeutic agent, but shows recurrence of disease or condition over time. In some embodiments, the subject has not relapsed. In some such embodiments, the subject is determined to be at risk of relapse, e.g., at high risk of relapse, and thus the cells are administered prophylactically, e.g., to reduce the likelihood of relapse or prevent relapse. In some aspects, the subject has not been previously treated with another therapeutic agent.

[0205] In some embodiments, the subject has persistent or recurrent disease after treatment with another therapeutic intervention, including, for example, chemotherapy, radiation, and / or hematopoietic stem cell transplantation (HSCT), e.g., allogeneic HSCT. In some embodiments, administration effectively treats the subject despite the subject having become refractory to another therapy.

[0206] The modified immune cells of the present invention can be administered to animals, preferably mammals, and even more preferably humans, to treat cancer. Furthermore, the cells of the present invention can be used for the treatment of any condition related to cancer, particularly cell-mediated immune response against tumor cell(s), when it is desired to treat or alleviate the disease. The types of cancer that can be treated with the modified cells or pharmaceutical compositions of the present invention include certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignant tumors, such as sarcomas, carcinomas, and melanomas. Exemplary cancers include, but are not limited to, B-cell malignancies (such as B-cell lymphomas and leukemias), lung cancer, non-small cell lung cancer, small cell lung cancer, Merkel cell carcinoma, melanoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, urothelial carcinoma, gastric cancer, cervical cancer, cutaneous squamous cell carcinoma, renal cell carcinoma, breast cancer, triple-negative breast cancer, colon cancer, esophageal cancer, gastric cancer, liver cancer, kidney cancer, pancreatic cancer, prostate cancer, brain cancer, lung adenocarcinoma, glioblastoma, hepatocellular carcinoma, gallbladder cancer, cervical cancer, cervical squamous cell carcinoma, colorectal cancer, ovarian cancer, and renal cancer. The cancer may be a non-solid tumor (such as a hematological tumor) or a solid tumor. Adult tumors / cancers and pediatric tumors / cancers are also included. In one embodiment, the cancer is a solid tumor or a hematological tumor.

[0207] The administered cells can be autologous to the subject undergoing treatment.

[0208] Administration of the cells of the present invention can be carried out in any convenient manner known to those skilled in the art. The cells of the present invention can be administered to a subject by aerosol inhalation, injection, oral ingestion, transfusion, implantation or transplantation. The compositions described herein can be administered to a patient intraarterially, subcutaneously, intradermally, intratumorally, intranodally, intramuscularly, by intravenous (iv) injection, or via intraperitoneal route. In other examples, the cells of the present invention are directly injected into the subject's inflammation site, the subject's local disease site, lymph nodes, organs, tumors, etc.

[0209] In some embodiments, cells are administered at a desired dosage, which in some aspects includes a desired dose or number of cells or cell types and / or a desired ratio of cell types.Thus, the dosage of cells in some embodiments is based on the total number of cells (or number per kg body weight) and the desired ratio of individual populations or subtypes, such as CD4+ to CD8+ ratio.In some embodiments, the dosage of cells is based on the desired total number of cells (or number per kg body weight) in each population or each cell type.In some embodiments, the dosage is based on a combination of such characteristics, such as the desired number of total cells, the desired ratio, and the desired total number of cells in each population.

[0210] In some embodiments, CD8 + and CD4 + A population or subtype of cells, such as T cells, is administered at or within a tolerance of a desired dose of total cells, such as a desired dose of T cells. In some aspects, the desired dose is a desired number of cells or a desired number of cells per unit body weight of the subject to whom the cells are administered, e.g., cells / kg. In some aspects, the desired dose is above a minimum or minimum number of cells, or above a minimum or minimum number of cells per unit body weight. In some aspects, among the total cells administered at a desired dose, individual populations or subtypes are administered at a desired production ratio (CD4 + vs. CD8 + ratio, etc.) or a ratio approaching that ratio, e.g., within a particular tolerance or error of such ratio.

[0211] In some embodiments, the cells are administered at or within a tolerance of the desired dose of one or more of the individual populations or subtypes of cells, such as a desired dose of CD4+ cells and / or a desired dose of CD8+ cells. In some aspects, the desired dose is the desired number of cells of a subtype or population, or the desired number of such cells per unit body weight of the subject to whom the cells are administered, e.g., cells / kg. In some aspects, the desired dose is above the minimum or minimum number of cells of the population or subtype, or above the minimum or minimum number of cells of the population or subtype per unit body weight. Thus, in some embodiments, the dosage is based on a desired fixed dose and a desired ratio of total cells, and / or based on one or more, e.g., a desired fixed dose of each, of individual subtypes or subpopulations. Thus, in some embodiments, the dosage is based on a desired fixed or minimum dose of T cells and a desired fixed or minimum dose of CD4+ cells. + vs. CD8 + Based on the desired ratio of cells and / or CD4 + and / or CD8 + Based on a desired fixed or minimum dose of cells.

[0212] In certain embodiments, individual populations of cells, or subtypes of cells, are in the range of about 1 million to about 100 billion cells, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), e.g., 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, or a range defined by any two of the foregoing values). cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases, about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells), or any value between these ranges.

[0213] In some embodiments, the dose of total cells and / or the dose of individual subpopulations of cells is greater than or equal to 1×10 5 cells / kg ~ approx. 1×10 11 cells / kg 10 4 or about 1 x 10 5 cells / kg ~ approx. 1×10 11 cells / kg 10 4 From, 10 11 or about 10 11 cells / kg body weight, e.g., 10 5 ~10 6 In the range of cells / kg body weight, e.g., 1 x 10 5 cells / kg, 1.5×10 5 cells / kg, 2×10 5 Cells / kg or 1×10 6 cells / kg, or approximately 1 x 10 5 cells / kg, approximately 1.5×10 5 cells / kg, approximately 2×10 5Cells / kg or approximately 1 x 10 6 For example, in some embodiments, the cells are at 10 4 or about 10 4 From, 10 9 or about 10 9 T cells / kilogram (kg) body weight, e.g., 10 5 ~10 6 For example, 1 x 10 T cells / kg body weight 5 T cells / kg, 1.5×10 5 T cells / kg, 2×10 5 T cells / kg or 1×10 6 T cells / kg body weight, or approximately 1 × 10 5 T cells / kg, approximately 1.5×10 5 T cells / kg, approximately 2×10 5 T cells / kg or approximately 1 × 10 6 In another exemplary embodiment, a suitable dosage range of modified cells for use in the methods of the present disclosure is, but is not limited to, about 1×10 5 cells / kg ~ approx. 1×10 6 cells / kg, approximately 1×10 6 cells / kg ~ approx. 1×10 7 cells / kg, approximately 1×10 7 cells / kg ~ approx. 1×10 8 cells / kg, approximately 1×10 8 cells / kg ~ approx. 1×10 9 cells / kg, approximately 1×10 9 cells / kg ~ approx. 1×10 10 cells / kg, approximately 1×10 10 cells / kg ~ approx. 1×10 11 In an exemplary embodiment, a suitable dosage for use in the methods of the present disclosure is about 1×10 8 In an exemplary embodiment, a suitable dosage for use in the methods of the present disclosure is about 1×10 7 In other embodiments, a suitable dosage is about 1×10 cells / kg. 7 Total cells ~ approx. 5 x 10 7 In some embodiments, a suitable dose is about 1×10 whole cells. 8 Total cells ~ approx. 5 x 108 In some embodiments, a suitable dose is about 1.4×10 whole cells. 7 Total cells ~ approx. 1.1 x 10 9 In an exemplary embodiment, a suitable dosage for use in the methods of the present disclosure is about 7×10 9 All cells in the organism.

[0214] In some embodiments, the cells are 4 or about 10 4 From, 10 9 or about 10 9 CD4 + and / or CD8 + cells / kilogram (kg) body weight, e.g. 10 5 ~10 6 CD4 + and / or CD8 + For example, 1 x 10 cells / kg body weight 5 CD4 + and / or CD8 + cells / kg, 1.5×10 5 CD4 + and / or CD8 + cells / kg, 2×10 5 CD4 + and / or CD8 + cells / kg, or 1 x 10 6 CD4 + and / or CD8 + cells / kg body weight, or approximately 1 x 10 5 CD4 + and / or CD8 + cells / kg, approximately 1.5×10 5 CD4 + and / or CD8 + cells / kg, approximately 2×10 5 CD4 + and / or CD8 + cells / kg body weight, or approximately 1 x 10 6 CD4 + and / or CD8 +In some embodiments, the cells are administered at about 1×10 cells / kg body weight, or within a certain margin of error thereof. 6 Super, about 2.5×10 6 Super, about 5×10 6 Super, about 7.5×10 6 Over or about 9 x 10 6 Super CD4 + cells, and / or at least about 1 x 10 6 , about 2.5×10 6 , about 5×10 6 , about 7.5×10 6 Or about 9 x 10 6 CD4 + cells, and / or at least about 1 x 10 6 , about 2.5×10 6 , about 5×10 6 , about 7.5×10 6 Or about 9 x 10 6 CD8+ cells, and / or at least about 1 x 10 6 , about 2.5×10 6 , about 5×10 6 , about 7.5×10 6 Or about 9 x 10 6 In some embodiments, the cells are administered at about 10 T cells or within a certain margin of error thereof. 8 ~10 12 Or about 10 10 ~10 11 T cells, approximately 10 8 ~10 12 Or about 10 10 ~10 11 CD4 + cells, and / or about 10 8 ~10 12 Or about 10 10 ~10 11 CD8 + Administered in cells or within their specified margin of error.

[0215] In some embodiments, the cells are administered at a desired production ratio, or within a tolerance range, of multiple cell populations or subtypes, such as CD4+ and CD8+ cells or subtypes. In some aspects, the desired ratio can be a specific ratio or can be a range of ratios, e.g., in some embodiments, the desired ratio (e.g., CD4 + vs. CD8 + The ratio of cells is from 5:1 or about 5:1 to 5:1 or about 5:1 (or from about 1:5 to about 5:1), or from 1:3 or about 1:3 to 3:1 or about 3:1 (or from about 1:3 to about 3:1), such as from 2:1 or about 2:1 to 1:5 or about 1:5 (or from about 1:5 to about 2:1), such as 5:1, 4.5:1, 4:1, 3.5:1, 3:1, 2.5:1, 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1 :1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 or 1:5, or about 5:1, about 4.5:1, about 4:1, about 3.5:1, about 3:1, about 2.5:1, about 2:1, about 1.9:1, about 1.8:1, about 1.7:1, about 1.6:1, about 1.5:1 , about 1.4:1, about 1.3:1, about 1.2:1, about 1.1:1, about 1:1, about 1:1.1, about 1:1.2, about 1:1.3, about 1:1.4, about 1:1.5, about 1:1.6, about 1:1.7, about 1:1.8, about 1:1.9, about 1:2, about 1:2.5, about 1:3, about 1:3.5, about 1:4, about 1:4.5, or about 1:5. In some aspects, the tolerance is within about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50% of the desired ratio, including any value between these ranges.

[0216] In some embodiments, the dose of modified cells is administered to the subject in need thereof in a single dose or multiple doses.In some embodiments, the dose of modified cells is administered in multiple doses, for example, once a week or every 7 days, once every 2 weeks or every 14 days, once every 3 weeks or every 21 days, once every 4 weeks or every 28 days.In exemplary embodiments, a single dose of modified cells is administered to the subject in need thereof.In exemplary embodiments, a single dose of modified cells is administered to the subject in need thereof by rapid intravenous infusion.

[0217] For the prevention or treatment of disease, the appropriate dosage will depend on the type of disease being treated, the type of cells or recombinant receptor, the severity and course of the disease, whether the cells are being administered for prophylactic or therapeutic purposes, previous treatments, the subject's medical history and response to the cells, and the discretion of the attending physician. The compositions and cells are in some embodiments suitably administered to the subject at one time or over a series of treatments.

[0218] In some embodiments, the cells are administered as part of a combination therapy, e.g., simultaneously with another therapeutic intervention, e.g., an antibody or engineered cell or receptor or agent, e.g., a cytotoxic or therapeutic agent, or sequentially in any order. The cells in some embodiments are co-administered simultaneously or sequentially in any order with one or more additional therapeutic agents or in combination with another therapeutic intervention. In some situations, the cells are co-administered with another therapy close enough in time that the cell population enhances the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the cells are administered before one or more additional therapeutic agents. In some embodiments, the cells are administered after one or more additional therapeutic agents. In some embodiments, the one or more additional agents include a cytokine, such as IL-2, e.g., to enhance persistence. In some embodiments, the method includes administration of a chemotherapeutic agent.

[0219] In certain embodiments, the modified cells of the present invention (e.g., modified cells comprising a CAR) may be administered to a subject in combination with an immune checkpoint antibody (e.g., an anti-PD1, anti-CTLA-4, or anti-PDL1 antibody). For example, the modified cells may be administered in combination with an antibody or antibody fragment that targets, for example, PD-1 (programmed death 1 protein). Examples of anti-PD-1 antibodies include, but are not limited to, pembrolizumab (KEYTRUDA®, formerly known as lambrolizumab, MK-3475), and nivolumab (BMS-936558, MDX-1106, ONO-4538, OPDIVA®), or an antigen-binding fragment thereof. In certain embodiments, the modified cells may be administered in combination with an anti-PD-L1 antibody, or an antigen-binding fragment thereof. Examples of anti-PD-L1 antibodies include, but are not limited to, BMS-936559, MPDL3280A (TECENTRIQ®, atezolizumab), and MEDI4736 (durvalumab, Imfinzi). In certain embodiments, the modified cells may be administered in combination with an anti-CTLA-4 antibody or an antigen-binding fragment thereof. Examples of anti-CTLA-4 antibodies include, but are not limited to, ipilimumab (trade name Yervoy). Other types of immune checkpoint modulators may also be used, including, but not limited to, small molecules, siRNA, miRNA, and CRISPR systems. The immune checkpoint modulator may be administered before, after, or simultaneously with the modified cells comprising the CAR. In certain embodiments, a combination therapy comprising an immune checkpoint modulator may increase the therapeutic effect of a therapy comprising the modified cells of the present invention.

[0220] After administration of the cells, in some embodiments the biological activity of the engineered cell population is measured, for example, by any of several known methods. Parameters to be evaluated include specific binding of engineered or natural T cells or other immune cells to antigens in vivo, for example, by imaging, or ex vivo, for example, by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described, for example, in Kochenderfer et al., J.Immunotherapy, 32(7):689-702(2009); Herman et al.J.Immunological Methods, 285(1):25-40(2004); Kiesgen et al., Nat Protoc.(2021)16(3):1331-1342; and Maldini et al., J Immunol Methods(2020)484-485:112830. In certain embodiments, the biological activity of the cells is measured by assaying expression and / or secretion of one or more cytokines, such as CD107a, IFNγ, IL-2, and TNF. In some aspects, the biological activity is measured by assessing a clinical outcome, such as reduction in tumor burden or tumor burden.

[0221] In certain embodiments, the subject is provided with a second-line treatment, including but not limited to chemotherapy, radiation, surgery, and drug therapy.

[0222] In some embodiments, the subject may be administered a conditioning therapy before CAR T cell therapy. In some embodiments, the conditioning therapy comprises administering an effective amount of cyclophosphamide to the subject. In some embodiments, the conditioning therapy comprises administering an effective amount of fludarabine to the subject. In a preferred embodiment, the conditioning therapy comprises administering an effective amount of a combination of cyclophosphamide and fludarabine to the subject. The administration of a conditioning therapy before CAR T cell therapy can enhance the efficacy of CAR T cell therapy. Methods of conditioning patients for T cell therapy are described in U.S. Patent No. 9,855,298, the entirety of which is incorporated herein by reference.

[0223] In some embodiments, certain dosing regimens of the present disclosure include a lymphodepletion step prior to administration of the modified T cells. In exemplary embodiments, the lymphodepletion step includes administration of cyclophosphamide and / or fludarabine.

[0224] In some embodiments, the lymphodepletion step comprises administering about 200 mg / m 2 / day~about 2000mg / m 2 / day (e.g., 200 mg / m 2 / day, 300mg / m 2 / day, or 500 mg / m 2 / day). In an exemplary embodiment, the dose of cyclophosphamide is about 300 mg / m 2 In some embodiments, the lymphodepletion step is at about 20 mg / m 2 / day~about 900mg / m 2 / day (e.g., 20 mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, or 60 mg / m 2 / day). In an exemplary embodiment, the dose of fludarabine is about 30 mg / m 2 / day.

[0225] In some embodiments, the lymphodepletion step comprises administering about 200 mg / m 2 / day~about 2000mg / m 2 / day (e.g., 200 mg / m 2 / day, 300mg / m 2 / day, or 500 mg / m 2 / day), and cyclophosphamide at a dose of approximately 20 mg / m 2 / day~about 900mg / m 2 / day (e.g., 20 mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, or 60 mg / m 2 / day). In an exemplary embodiment, the lymphodepletion step comprises administration of fludarabine at a dose of about 300 mg / m 2 / day dose of cyclophosphamide and approximately 30 mg / m 2 The treatment involves administration of fludarabine at a dose of 100 mg / day.

[0226] In an exemplary embodiment, the dose of cyclophosphamide is 300 mg / m for 3 days. 2 / day and the fludarabine dose was 30 mg / m for 3 days. 2 / day.

[0227] Administration of lymphodepleting chemotherapy may be scheduled from day -6 to day -4 (-1 day window, i.e., administered from day -7 to day -5) relative to the infusion of T cells (e.g., CAR-T, TCR-T, modified T cells, etc.) on day 0.

[0228] In an exemplary embodiment, for subjects with cancer, the subject is administered 300 mg / m 200 mg / ml by intravenous infusion 3 days prior to administration of the modified T cells. 2 In an exemplary embodiment, for subjects with cancer, the subject receives lymphodepleting chemotherapy including cyclophosphamide at 300 mg / m by intravenous infusion for 3 days prior to administration of the modified T cells. 2 will undergo lymphodepleting chemotherapy, including cyclophosphamide.

[0229] In an exemplary embodiment, for a subject with cancer, the subject receives about 20 mg / m 2 / day~about 900mg / m 2 / day (e.g., 20 mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, or 60 mg / m 2 / day). In an exemplary embodiment, for a subject with cancer, the subject receives lymphodepleting chemotherapy including fludarabine at a dose of 30 mg / m 2 Patients will receive lymphodepleting chemotherapy containing a dose of fludarabine for three days.

[0230] In an exemplary embodiment, for a subject with cancer, the subject receives about 200 mg / m 2 / day~about 2000mg / m 2 / day (e.g., 200 mg / m 2 / day, 300mg / m 2 / day, or 500 mg / m 2 / day), and cyclophosphamide at a dose of approximately 20 mg / m 2 / day~about 900mg / m 2 / day (e.g., 20 mg / m 2 / day, 25mg / m 2 / day, 30mg / m 2 / day, or 60 mg / m 2 / day). In an exemplary embodiment, for a subject with cancer, the subject receives lymphodepleting chemotherapy including fludarabine at a dose of about 300 mg / m 2 / day dose of cyclophosphamide and 30 mg / m 2 Patients will receive lymphodepleting chemotherapy containing a dose of fludarabine for three days.

[0231] The cells of the present invention may be administered at dosages and routes and times determined in appropriate preclinical and clinical experiments and trials. The cell compositions may be administered multiple times at dosages within these ranges. Administration of the cells of the present invention may be combined with other methods useful for treating the desired disease or condition as determined by one of skill in the art.

[0232] It is known in the art that one of the adverse effects following infusion of CAR T cells is the development of immune activation known as cytokine release syndrome (CRS). CRS is immune activation resulting in elevated inflammatory cytokines. CRS is a known on-target toxicity, and its occurrence likely correlates with efficacy. Clinical and laboratory measures range from mild CRS (systemic symptoms and / or grade 2 organ toxicity) to severe CRS (sCRS; grade ≥ 3 organ toxicity, aggressive clinical intervention, and / or potentially life-threatening). Clinical features include high fever, malaise, fatigue, myalgia, nausea, anorexia, tachycardia / hypotension, capillary leak, cardiac dysfunction, renal impairment, liver failure, and disseminated intravascular coagulation. Dramatic increases in cytokines including interferon-gamma, granulocyte-macrophage colony-stimulating factor, IL-10, and IL-6 have been shown following CAR T cell infusion. One CRS signature is elevated cytokines, including IL-6 (severely elevated), IFN-gamma, TNF-alpha (moderate), and IL-2 (mild). Elevations of clinically available inflammatory markers, including ferritin and C-reactive protein (CRP), have also been observed to correlate with CRS syndrome. The presence of CRS generally correlates with the expansion of adoptively transferred cells and progressive immune activation. It has been demonstrated that the degree of CRS severity is determined by the disease burden at the time of infusion, as patients with high tumor burden experience more sCRS.

[0233] Therefore, the present invention provides a suitable CRS management strategy to alleviate the physiological symptoms of uncontrolled inflammation after the diagnosis of CRS without compromising the anti-tumor effect of engineered cells (e.g., CAR T cells).CRS management strategies are known in the art.For example, systemic corticosteroids can be administered to rapidly ameliorate the symptoms of sCRS (e.g., grade 3 CRS) without compromising initial anti-tumor response.

[0234] In some embodiments, anti-IL-6R antibody can be administered. One example of anti-IL-6R antibody is the monoclonal antibody tocilizumab, also known as atlizumab (commercially available as Actemra or RoActemra), approved by the Food and Drug Administration. Tocilizumab is a humanized monoclonal antibody against interleukin-6 receptor (IL-6R). Administration of tocilizumab has demonstrated almost immediate reversal of CRS.

[0235] CRS is generally managed based on the severity of the syndrome observed, and interventions are tailored accordingly. CRS management decisions can be based on clinical signs and symptoms and response to interventions, rather than solely on laboratory values.

[0236] Mild to moderate cases are generally treated with symptom management with fluid therapy, nonsteroidal anti-inflammatory drugs (NSAIDs) and antihistamines as needed for adequate symptom relief. More severe cases include patients with any degree of hemodynamic instability, where administration of tocilizumab is recommended. First-line management of CRS may be, in some embodiments, tocilizumab at a labelled dose of 8 mg / kg IV over 60 minutes (not to exceed 800 mg / dose); tocilizumab may be repeated every Q8 hours. In the event of a suboptimal response to the first dose of tocilizumab, additional doses of tocilizumab may be considered. Tocilizumab may be administered alone or in combination with corticosteroid therapy. Patients with persistent or progressive CRS symptoms, inadequate clinical improvement in 12-18 hours, or inadequate response to tocilizumab may be treated with high-dose corticosteroid therapy, typically hydrocortisone 100 mg IV or methylprednisolone 1-2 mg / kg. In patients with more severe hemodynamic instability or more severe respiratory symptoms, patients may be placed on high-dose corticosteroid therapy early in the course of CRS. CRS management guidance may be based on published standards (Lee et al. (2019) Biol Blood Marrow Transplant, doi.org / 10.1016 / j.bbmt.2018.12.758; Neelapu et al. (2018) Nat Rev Clin Oncology, 15:47; Teachey et al. (2016) Cancer Discov, 6(6):664-679).

[0237] Features consistent with macrophage activation syndrome (MAS) or hemophagocytic lymphohistiocytosis (HLH) have been observed in patients treated with CAR-T therapy, in line with the clinical presentation of CRS (Henter, 2007). MAS appears to be a response to immune activation resulting from CRS and should therefore be considered a manifestation of CRS. MAS is similar to HLH (also a response to immune stimulation). The clinical syndrome of MAS is characterized by high-grade non-remitting fever, cytopenias affecting at least two of the three lineages, and hepatosplenomegaly. This is associated with high serum ferritin, soluble interleukin-2 receptor and triglycerides, and reduced circulating natural killer (NK) activity.

[0238] In one aspect, the invention includes a method of treating cancer in a subject in need thereof comprising administering to the subject any one of the modified immune cells or progenitor cells disclosed herein. Yet another aspect of the invention includes a method of treating cancer in a subject in need thereof comprising administering to the subject modified immune cells or progenitor cells produced by any one of the methods disclosed herein.

[0239] F. Source of immune cells In certain embodiments, the source of immune cells (e.g., T cells) is obtained from a subject for ex vivo manipulation and / or in vivo transduction. The source of target cells for ex vivo manipulation can also include, for example, autologous or heterologous donor blood, umbilical cord blood or bone marrow. For example, the source of immune cells can be derived from the subject that is treated with the modified immune cells of the present invention, for example, the subject's blood, the subject's umbilical cord blood or the subject's bone marrow. Non-limiting examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. Preferably, the subject is a human. Methods for in vivo transduction of immune cells for CAR expression have been described, for example, in Pfeiffer et al., EMBO Mol Med.(2018)10(11):e9158; Weidner et al., Nat Protoc.(2021)16(7):3210-3240; Frank et al., Blood Advances(2020)4(22):5702-5715; Nawaz et al., Blood Cancer J.(2021)11(6):119.

[0240] Immune cells can be obtained from many sources, including blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, lymph, or lymphoid organs. Immune cells are cells of the immune system, such as cells of innate or adaptive immunity, for example, lymphoid cells, including bone marrow cells or lymphocytes, typically T cells and / or NK cells. Other exemplary cells include stem cells, such as multipotent and multipotent stem cells, including induced pluripotent stem cells (iPSCs). In some aspects, the cells are human cells. With respect to the subject to be treated, the cells can be allogeneic and / or autologous. The cells are typically primary cells, such as those directly isolated from the subject and / or those isolated from the subject and frozen.

[0241] In certain embodiments, the immune cell is a T cell, e.g., a CD8+ T cell (e.g., a CD8+ naive T cell, a central memory T cell, or an effector memory T cell), a CD4+ T cell, a natural killer T cell (NKT cell), a regulatory T cell (Treg), a stem cell memory T cell, a lymphoid progenitor cell, a hematopoietic stem cell, a natural killer cell (NK cell), a macrophage, or a dendritic cell. In some embodiments, the cell is a monocyte or a granulocyte, e.g., a myeloid cell, a macrophage, a neutrophil, a dendritic cell, a mast cell, an eosinophil, and / or a basophil. In one embodiment, the target cell is an induced pluripotent stem (iPS) cell or a cell derived from an iPS cell, e.g., an iPS cell generated from a subject and engineered to modify (e.g., induce mutations) or manipulate expression of one or more target genes, e.g., differentiated into a T cell, e.g., a CD8+ T cell (e.g., a CD8+ naive T cell, a central memory T cell or an effector memory T cell), a CD4+ T cell, a stem cell memory T cell, a lymphoid progenitor cell, or a hematopoietic stem cell.

[0242] In some embodiments, the cells include one or more subsets of T cells or other cell types, such as the total T cell population, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, differentiation potential, expansion, recirculation, localization, and / or persistence capacity, antigen specificity, antigen receptor type, presence in specific organs or compartments, marker or cytokine secretion profile, and / or degree of differentiation. Among the subtypes and subpopulations of T cells and / or CD4+ and / or CD8+ T cells are naive T (TN) cells, effector T cells (TEFF), memory T cells and their subtypes, such as stem cell memory T (TSCM) cells, central memory T (TCM) cells, effector memory T (TEM) cells, or terminally differentiated effector memory T cells, tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, intrinsic and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and delta / gamma T cells. In certain embodiments, any number of T cell lines available in the art may be used.

[0243] In some embodiments, the method includes steps of isolating immune cells from a subject, preparing, treating, culturing, and / or manipulating them. In some embodiments, the preparation of the engineered cells includes one or more culturing and / or preparation steps. Cells for manipulation as described can be isolated from a sample, e.g., a biological sample, e.g., a sample obtained or derived from a subject. In some embodiments, the subject from which the cells are isolated has a disease or condition or is in need of cell therapy or is a subject to whom cell therapy is administered. The subject in some embodiments is a human in need of a particular therapeutic intervention, e.g., adoptive cell therapy, for which the cells are isolated, treated, and / or manipulated. Thus, the cells in some embodiments are primary cells, e.g., primary human cells. Samples include tissues, body fluids, and other samples taken directly from a subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic manipulation (e.g., transduction with a viral vector), washing, and / or incubation. Biological samples can be samples obtained directly from a biological source or samples that have been processed. Biological samples include, but are not limited to, body fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived from tissue and organ samples.

[0244] In some aspects, the sample from which the cells are derived or isolated is blood or blood-derived sample, or is or is derived from apheresis or leukapheresis product.Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMC), white blood cells, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil, or other organ, and / or cells derived therefrom.Samples include samples from autologous and allogeneic sources in the context of cell therapy, such as adoptive cell therapy.

[0245] In some embodiments, the cells are derived from a cell line, e.g., a T cell line. In some embodiments, the cells are obtained from heterologous sources, e.g., from mice, rats, non-human primates, and pigs. In some embodiments, the isolation of cells includes one or more preparative and / or non-affinity-based cell separation steps. In some examples, the cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, e.g., to remove undesirable components, to enrich for desired components, to lyse or remove cells that are sensitive to a particular reagent. In some examples, the cells are separated based on one or more characteristics, such as density, adhesive properties, size, sensitivity, and / or resistance to a particular component.

[0246] In some examples, cells from the subject's circulating blood are obtained, for example, by apheresis or leukapheresis. The sample includes lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and / or platelets, and in some aspects includes cells other than red blood cells and platelets. In some embodiments, blood cells collected from a subject are washed, for example, to remove the plasma fraction and place the cells in a suitable buffer or medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some aspects, the washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended in various biocompatible buffers after washing. In certain embodiments, components of the blood cell sample are removed and the cells are resuspended directly in culture medium. In some embodiments, the method includes preparation of white blood cells from peripheral blood by lysing red blood cells and density-based cell separation methods such as centrifugation through Percoll or Ficoll gradients.

[0247] In one embodiment, immune cells obtained from the circulating blood of an individual are obtained by apheresis or leukapheresis. Apheresis products typically include lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. Cells collected by apheresis may be washed to remove the plasma fraction and place the cells in a suitable buffer or medium, such as phosphate-buffered saline (PBS), or the washing solution may be calcium-free, magnesium-free, or many, if not all, divalent cations, for subsequent processing steps. After washing, the cells may be resuspended in various biocompatible buffers, such as Ca-free, Mg-free PBS. Alternatively, the undesired components of the apheresis sample may be removed and the cells resuspended directly in culture medium.

[0248] In some embodiments, the isolation method comprises the separation of different cell types based on the expression or presence in cells of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acids. In some embodiments, any known method for such marker-based separation can be used. In some embodiments, the separation is affinity or immunoaffinity-based separation. For example, in some aspects, the isolation comprises the separation of cells and cell populations based on the expression or expression level of one or more markers, typically cell surface markers, by incubation with an antibody or binding partner that specifically binds to such marker, followed generally by a washing step and the separation of the cells that bind to the antibody or binding partner from the cells that do not bind to the antibody or binding partner.

[0249] Such separation steps can be based on positive selection, where cells that bound to the reagent are retained for further use, and / or negative selection, where cells that did not bind to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful when antibodies that specifically identify cell types in a heterogeneous population are not available, so separation is best performed based on markers expressed by cells other than the desired population. Separation does not need to result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection or enrichment of a particular type of cell, such as cells expressing a marker, refers to increasing the number or percentage of such cells, but does not need to result in the complete absence of cells that do not express the marker. Similarly, negative selection, removal, or depletion of a particular type of cell, such as cells expressing a marker, refers to decreasing the number or percentage of such cells, but does not need to result in the complete removal of all such cells.

[0250] In some examples, multiple separation steps are performed, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, cells expressing multiple markers simultaneously can be depleted in a single separation step, such as by incubating cells with multiple antibodies or binding partners, each specific to a marker that is the target of negative selection. Similarly, multiple cell types can be positively selected simultaneously by incubating cells with multiple antibodies or binding partners expressed on different cell types.

[0251] In some embodiments, one or more of the T cell populations are positive for (marker+) or express high levels of one or more particular markers, such as surface markers (marker hlgh ) or cells that are negative (marker-) or express relatively low levels (marker low) cells are enriched or depleted. For example, in some aspects, a particular subpopulation of T cells, such as cells that are positive or express high levels of one or more surface markers, such as CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+ T cells, are isolated by positive or negative selection techniques. In some cases, such markers are markers that are absent or expressed at relatively low levels on certain populations of T cells (such as non-memory cells) but present or expressed at relatively high levels on certain other populations of T cells (such as memory cells). In one embodiment, the cells (such as CD8+ cells or T cells, e.g., CD3+ cells) are enriched for cells that are positive for or express high surface levels of CD45RO, CCR7, CD28, CD27, CD44, CD127, and / or CD62L (i.e., positively selected), and / or depleted for cells that are positive for or express high surface levels of CD45RA (e.g., negatively selected). In some embodiments, the cells are enriched or depleted for cells that are positive for or express high surface levels of CD122, CD95, CD25, CD27, and / or IL7-Ra (CD127). In some examples, the CD8+ T cells are enriched for cells that are positive for CD45RO (or negative for CD45RA) and positive for CD62L. For example, CD3+,CD28+ T cells can be positively selected using CD3 / CD28-conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander).

[0252] In some embodiments, T cells are separated from the PBMC sample by negative selection of markers expressed on non-T cells such as B cells, monocytes, or other white blood cells, e.g., CD14. In some aspects, a CD4+ or CD8+ selection step is used to separate CD4+ helper T cells and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further classified into subpopulations by positive or negative selection for markers expressed or expressed to a relatively high degree on one or more naive T cell, memory T cell, and / or effector T cell subpopulations. In some embodiments, CD8+ cells are further enriched or depleted for naive, central memory, effector memory, and / or central memory stem cells, such as by positive or negative selection based on surface antigens associated with each subpopulation. In some embodiments, enrichment of central memory T (TCM) cells is performed to enhance efficacy, e.g., to improve long-term survival, expansion, and / or engraftment after administration, which in some aspects is particularly robust in such subpopulations. In some embodiments, combining TCM-enriched CD8+ T cells with CD4+ T cells further enhances efficacy.

[0253] In some embodiments, memory T cells are present in both CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMCs can be enriched or depleted for CD62L-CD8+ and / or CD62L+CD8+ fractions using, for example, anti-CD8 and anti-CD62L antibodies. In some embodiments, CD4+ T cell populations and CD8+ T cell subpopulations, such as subpopulations enriched for central memory (TCM) cells. In some embodiments, enrichment of central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD127: in some aspects, this is based on negative selection of cells that express or highly express CD45RA and / or granzyme B. In some aspects, the isolation of CD8+ population enriched in TCM cells is performed by depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment of cells expressing CD62L. In one aspect, the enrichment of central memory T (TCM) cells is performed by starting with the negative fraction of cells selected based on CD4 expression, and subjecting it to negative selection based on CD14 and CD45RA expression, and positive selection based on CD62L. In some aspects, such selections are performed simultaneously, and in other aspects, sequentially in either order. In some aspects, the same selection step based on CD4 expression used to prepare the CD8+ cell population or subpopulation is also used to generate the CD4+ cell population or subpopulation, so that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the method, optionally after one or more additional positive or negative selection steps.

[0254] CD4+ T helper cells are sorted into naive cells, central memory cells, and effector cells by identifying cell populations with cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+, CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ cells are CD62L- and CD45RO. In one example, to enrich for CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CDllb, CD16, HLA-DR, and CD8. In some embodiments, the antibodies or binding partners are bound to a solid support or matrix, such as magnetic or paramagnetic beads, to allow for the separation of cells for positive and / or negative selection.

[0255] In some embodiments, the cells are incubated and / or cultured prior to or in combination with the genetic manipulation. The incubation step may include culturing, stimulating, activating, and / or expanding. In some embodiments, the composition or cells are incubated in the presence of stimulatory conditions or agents. Such conditions include those designed to induce proliferation, expansion, activation, and / or survival of the cells in the population, to mimic antigen exposure, and / or to prime the cells for genetic manipulation, such as the introduction of a recombinant antigen receptor. The conditions may include one or more of a particular medium, temperature, oxygen content, carbon dioxide content, time, agents, such as nutrients, amino acids, antibiotics, ions, and / or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells. In some embodiments, the stimulatory conditions or agents include one or more agents, such as ligands, that can activate the intracellular signaling domain of the TCR complex. In some aspects, the agents activate or initiate the TCR / CD3 intracellular signaling cascade in the T cells. Such agents may include antibodies, such as those specific for TCR components and / or costimulatory receptors, e.g., anti-CD3, anti-CD28, and / or one or more cytokines, bound to a solid support, such as beads. Optionally, the expansion method may further include adding anti-CD3 and / or anti-CD28 antibodies to the medium (e.g., at a concentration of at least about 0.5 ng / ml). In some embodiments, the stimulatory agent includes IL-2 and / or IL-15, e.g., an IL-2 concentration of at least about 10 units / mL.

[0256] In another embodiment, T cells are isolated from peripheral blood by lysing red blood cells and depleting monocytes, for example by centrifugation through a PERCOLL™ gradient. Alternatively, T cells can be isolated from umbilical cord. In either case, specific subpopulations of T cells can be further isolated by positive or negative selection techniques.

[0257] The cord blood mononuclear cells so isolated can be depleted of cells expressing specific antigens, including but not limited to CD34, CD8, CD14, CD19 and CD56. Depletion of these cells can be accomplished using isolated antibodies, biological samples containing antibodies such as ascites fluid, antibodies bound to physical supports, and antibodies bound to cells.

[0258] Enrichment of a T cell population by negative selection can be achieved using a combination of antibodies against surface markers unique to the negatively selected cells. A preferred method is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on the negatively selected cells. For example, negative selection can be used to enrich for CD4 + To enrich for cells, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

[0259] For isolation of a desired population of cells by positive or negative selection, the concentration of cells and surfaces (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly reduce the volume in which beads and cells are mixed together (i.e., 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 is 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 higher concentrations can result in increased cell yield, cell activation, and cell expansion.

[0260] T cells can also be frozen after the washing step, which does not require a monocyte removal step. Without wishing to be bound by theory, the freezing and subsequent thawing steps provide a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing step to remove 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 regard, in a non-limiting example, one method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or other suitable cell freezing medium. The cells are then frozen to -80°C at a rate of 1°C per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing, as well as immediate uncontrolled freezing at -20°C or in liquid nitrogen, may be used.

[0261] In one embodiment, the population of T cells is comprised within cells such as peripheral blood mononuclear cells, umbilical cord blood cells, purified populations of T cells, and T cell lines. In another embodiment, peripheral blood mononuclear cells comprise the population of T cells. In yet another embodiment, purified T cells comprise the population of T cells.

[0262] In certain embodiments, regulatory T cells (Tregs) can be isolated from a sample. Samples can include, but are not limited to, umbilical cord blood or peripheral blood. In certain embodiments, Tregs are isolated by flow cytometry sorting. Samples can be enriched for Tregs before isolation by any means known in the art. Isolated Tregs can be cryopreserved and / or expanded before use. Methods for isolating Tregs are described in U.S. Patent Nos. 7,754,482, 8,722,400 and 9,555,105, and U.S. Patent Application No. 13 / 639,927, the contents of which are incorporated herein in their entirety.

[0263] G. Expansion of immune cells Whether before or after modification of the cells to express the CAR of the invention, the cells may be modified using the methods described in, for example, U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and US Patent Publication No. 20060121005. For example, the T cells of the present invention can be expanded by contact with a surface to which is attached an agent that stimulates CD3 / TCR complex-associated signals and a ligand that stimulates costimulatory molecules on the surface of the T cells. In particular, the T cell population can be stimulated by contact with an anti-CD3 antibody or an antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on the surface, or by contact with a protein kinase C activator (e.g., bryostatin) in combination with a calcium ionophore. For costimulation of accessory molecules on the surface of the T cells, a ligand that binds to the accessory molecule is used. For example, the T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions appropriate to stimulate proliferation of the T cells. Exemplary anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France), which can be used in the present invention, as well as other methods and reagents known in the art (see, e.g., ten Berge et al., Transplant Proc. (1998) 30(8):3975-3977; Haanen et al., J. Exp. Med. (1999) 190(9):1319-1328; and Garland et al., J. Immunol. Methods (1999) 227(1-2):53-63).

[0264] T cells undergoing expansion by the methods disclosed herein can be expanded by about 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 200x, 300x, 400x, 500x, 600x, 700x, 800x, 900x, 1000x, 2000x, 3000x, 4000x, 5000x, 6000x, 7000x, 8000x, 9000x, 10,000x, 100,000x, 1,000,000x, 10,000,000x, or more, and any and all whole or partial integers therebetween. In one embodiment, T cells are expanded in the range of about 20x to about 50x.

[0265] After culturing, the T cells can be incubated in the cell medium in the culture device for a period of time or until the cells reach confluence or high cell density for optimal passaging before sending the cells to another culture device. The culture device can be any culture device commonly used for culturing cells in vitro. Preferably, the level of confluence is 70% or more before sending the cells to another culture device. More preferably, the level of confluence is 90% or more. The period can be any time suitable for culturing cells in vitro. The T cell medium can be replaced at any time during the culture of the T cells. Preferably, the T cell medium is replaced about every 2-3 days. The T cells are then recovered from the culture device, after which the T cells can be used immediately or cryopreserved and stored for later use. In one embodiment, the present invention includes a step of cryopreserving the expanded T cells. The cryopreserved T cells are thawed before introducing a nucleic acid into the T cells.

[0266] In another embodiment, the method includes isolating T cells and expanding the T cells. In another embodiment, the invention further includes cryopreserving the T cells prior to expansion. In yet another embodiment, the cryopreserved T cells are thawed for electroporation with RNA encoding the chimeric membrane protein.

[0267] Another technique for ex vivo expansion cells is described in U.S. Patent No. 5,199,942 (incorporated herein by reference). Expansion as described in U.S. Patent No. 5,199,942 may be an alternative or an addition to other expansion methods described herein. Briefly, ex vivo culture and expansion of T cells includes the addition of cell growth factors as described in U.S. Patent No. 5,199,942, or other factors such as flt3-L, IL-1, IL-3, and c-kit ligand. In one embodiment, expanding T cells includes culturing T cells with factors selected from the group consisting of flt3-L, IL-1, IL-3, and c-kit ligand.

[0268] The culturing step described herein (following contact with an agent described herein or electroporation) can be very short, e.g., less than 24 hours, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours. The culturing step described further herein (contact with an agent described herein) can be longer, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more.

[0269] Various terms are used to describe cells in culture. Cell culture generally refers to cells taken from a living organism and grown under controlled conditions. Primary cell cultures are cultures of cells, tissues, or organs taken directly from an organism and prior to the first subculture. Cells are expanded in culture when placed in a growth medium under conditions that promote cell growth and / or division, resulting in a larger population of cells. When cells are expanded in culture, the rate of cell growth is typically measured by the amount of time required for the cells to double in number, otherwise known as the doubling time.

[0270] Each time of subculture is called a passage. When cells are subcultured, they are said to be passaged. A particular cell population or cell line may be called or characterized by the number of times it has been passaged. For example, a cultured cell population that has been passaged 10 times may be called a P10 culture. A primary culture, i.e., the first culture after isolating cells from tissue, is designated as P0. After the first subculture, the cells are designated as a secondary culture (P1 or passage 1). After the second subculture, the cells become a tertiary culture (P2 or passage 2), and so on. It will be understood by those skilled in the art that there may be many population doublings during a period of passage; therefore, the number of population doublings of a culture is greater than the number of passages. The expansion of cells during the period between passages (i.e., the number of population doublings) depends on many factors, including but not limited to seeding density, substrate, medium, and time between passages.

[0271] In one embodiment, cells may be cultured for a few hours (about 3 hours) to about 14 days or any integer value of time units therebetween. Suitable conditions for T cell culture include a suitable medium (e.g., Minimum Essential Medium or RPMI Medium 1640 or X-vivo 15 (Lonza)) that may contain factors necessary for growth and survival, including serum (e.g., fetal bovine serum or human fetal serum), interleukin 2 (IL-2), insulin, IFN-gamma, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF-beta and TNF-α or any other additive for cell growth known to those of skill in the art. Other additives for cell growth include, but are not limited to, detergents, plasmanate, and reducing agents such as N-acetyl-cysteine ​​and 2-mercaptoethanol. Culture media may include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, which are supplemented with amino acids, sodium pyruvate, and vitamins, and are serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or cytokines in sufficient amounts for T cell growth and expansion. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and not in cultures of cells injected into subjects. Target cells are maintained under conditions necessary to support growth, such as at an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air+5% CO2).

[0272] The medium used to culture T cells can include an agent that can costimulate T cells. For example, an agent that can stimulate CD3 is an antibody to CD3, and an agent that can stimulate CD28 is an antibody to CD28. The cells isolated by the methods disclosed herein can be expanded about 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 200x, 300x, 400x, 500x, 600x, 700x, 800x, 900x, 1000x, 2000x, 3000x, 4000x, 5000x, 6000x, 7000x, 8000x, 9000x, 10,000x, 100,000x, 1,000,000x, 10,000,000x, or more. In one embodiment, T cells are expanded from about 20-fold to about 50-fold or more. In one embodiment, human regulatory T cells are expanded via anti-CD3 antibody-coated KT64.86 artificial antigen presenting cells (aAPCs). Methods for expanding and activating T cells can be found in U.S. Patent Nos. 7,754,482, 8,722,400 and 9,555,105, the contents of which are incorporated herein in their entireties.

[0273] In one embodiment, the method of expanding T cells may further comprise isolating the expanded T cells for further use. In another embodiment, the method of expanding may further comprise subsequent electroporation of the expanded T cells followed by culturing. Subsequent electroporation may comprise introducing a nucleic acid encoding an agent into the expanded population of T cells, such as transducing the expanded T cells, transfecting the expanded T cells, or electroporating the expanded T cells with a nucleic acid, and the agent may further stimulate the T cells. The agent may stimulate the T cells, such as by stimulating further expansion, effector function, or another T cell function.

[0274] H. Pharmaceutical Compositions and Formulations Also provided are populations of immune cells of the present invention, compositions containing such cells and / or enriched with such cells, for example compositions in which cells expressing CAR (e.g., IL-containing CAR) constitute at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the total cells in the composition or a particular type of cells, such as T cells or CD8+ or CD4+ cells. Among the compositions are pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy. Also provided are therapeutic methods for administering the cells and compositions to a subject, e.g., a patient.

[0275] Also provided are compositions containing cells for administration, including pharmaceutical compositions and formulations, such as compositions in unit dose form that contain the number of cells for administration at a given dose or fraction thereof.Pharmaceutical compositions and formulations generally include one or more optional pharmaceutically acceptable carriers or excipients.In some embodiments, the composition includes at least one additional therapeutic agent.

[0276] The term "pharmaceutical formulation" or "pharmaceutical composition" refers to a preparation that is in a form that allows the biological activity of the active ingredient contained therein to be effective and does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered. A "pharmaceutical acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than the active ingredient, that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives. In some aspects, the choice of carrier is determined, in part, by the particular cell and / or by the method of administration. Thus, there are a variety of suitable formulations. For example, the pharmaceutical composition may include a preservative. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, for example, in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to, the following: buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; A sugar such as sucrose, mannitol, trehalose or sorbitol; a salt-forming counterion such as sodium; a metal complex (e.g., a Zn-protein complex); and / or a non-ionic surfactant such as polyethylene glycol (PEG).

[0277] In some aspects, a buffering agent is included in the composition. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixture is typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail, for example, in Remington: The Science and Practice of Pharmacy, Lippincott Williams&Wilkins; 21st ed.(May 1, 2005).

[0278] The formulation may comprise an aqueous solution. The formulation or composition may also contain multiple active ingredients, preferably those with complementary activities, useful for the particular indication, disease, or condition treated with the cells, provided that the respective activities do not adversely affect each other. Such active ingredients are suitably present in combination in amounts effective for the intended purpose. Thus, in some embodiments, the pharmaceutical composition further comprises other pharma- ceutical active agents or drugs, such as chemotherapeutic agents, such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and / or vincristine. The pharmaceutical composition in some embodiments contains the cells in an amount effective to treat or prevent the disease or condition, such as a therapeutically or prophylactically effective amount. The therapeutic or prophylactic effectiveness in some embodiments is monitored by periodic evaluation of the treated subject. The desired dosage can be delivered by a single bolus of cells, by multiple boluses of cells, or by continuous infusion of cells.

[0279] Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the cell population is administered parenterally. The term "parenteral" as used herein includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the cells are administered to the subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection. The compositions in some embodiments are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which in some aspects may be buffered to a selected pH. Liquid preparations are usually easier to prepare than gels, other viscous compositions, and solid compositions. Furthermore, liquid compositions are somewhat more convenient to administer, especially by injection. On the other hand, viscous compositions can be formulated within an appropriate viscosity range to provide longer contact periods with specific tissues. A liquid or viscous composition may contain a carrier, which may be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.

[0280] Sterile injectable solutions can be prepared by incorporating the cells in a solvent, for example, by mixing with a suitable carrier, diluent, or excipient, such as sterile water, physiological saline, glucose, dextrose, etc. The composition can contain auxiliary substances, such as wetting agents, dispersing or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or thickening additives, preservatives, flavoring agents, and / or coloring agents, depending on the desired route of administration and preparation. In some aspects, standard textbooks can be consulted to prepare suitable preparations.

[0281] Various additives can be added to enhance the stability and sterility of the composition, including antibacterial preservatives, antioxidants, chelating agents, and buffers.Prevention of microbial action can be ensured by various antibacterial and antifungal agents, such as paraben, chlorobutanol, phenol, and sorbic acid.Prolonged absorption of injectable pharmaceutical forms can be achieved by the use of agents that delay absorption, such as aluminum monostearate and gelatin.

[0282] Formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, for example, by filtration through sterile filtration membranes.

[0283] The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein are incorporated herein by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from such articles, patents, patent applications, or other physical and electronic documents.

[0284] Although the present invention has been described with reference to specific embodiments thereof, it should be understood by those skilled in the art that various modifications may be made and equivalents may be substituted without departing from the true spirit and scope of the present invention. It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein may be made using appropriate equivalents without departing from the scope of the embodiments disclosed herein. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the appended claims. Although specific embodiments have now been described in detail, this will be more clearly understood by reference to the following examples, which are included for illustrative purposes only and are not intended to be limiting. EXAMPLES

[0285] Experimental Example The invention will now be described with reference to the following examples, which are provided for illustrative purposes only, and the invention is not limited to these examples, but rather includes all variations that become apparent as a result of the teachings provided herein.

[0286] material and method Retroviruses encoding chimeric antigen receptors were generated using Plat-E packaging cells (Cell Biolabs) by transfection with pMSGV vectors using Lipofectamine 2000 (ThermoFisher). Culture medium was replaced after 24 hours, and the medium was harvested after a further 24 hours, clarified by centrifugation, passed through a 0.45 um filter, and stored at -80°C.

[0287] Retroviral transduction of mouse CAR T cells was achieved by enriching donor CD45.1+ mouse bulk splenocytes for CD3+ cells by magnetic bead separation (Stemcell Technologies). T cells were activated with mouse CD3 / CD28 Dynabeads (ThermoFisher) for 48 h in the presence of 50 U / ml recombinant human IL-2 (Peprotech) prior to spinfection (1 h) with retroviral supernatant on Retronectin-coated (Takara Bio) plates. Cells were harvested for flow cytometry 2 days after spinfection.

[0288] Retrovirally transduced mouse T cells were incubated with antibodies specific for the CAR for 20 min at room temperature in the dark, followed by flow cytometric detection of the chimeric antigen receptor by acquiring at least 10,000 events on an LSR II flow cytometer (BD Biosciences). Data were analyzed with FlowJo software (BD Biosciences).

[0289] The results of the experiment will now be described.

[0290] Example 1: Chimeric antigen receptor containing IL9Ra ICD Adoptively transferred genetically engineered T cell therapy has demonstrated significant antitumor activity in patients with hematopoietic malignancies, but has been of limited benefit in patients with solid tumors (Rosenberg and Restifo, Science (2015) 348:62-68). One major limitation is that adoptively transferred T cells expand and persist poorly in vivo, necessitating lymphodepleting conditioning chemotherapy, a toxic regimen that limits patient eligibility (Goff et al., J Clin Oncol. (2016) 34:2389-2397; Dudley et al., J Clin Oncol. (2008) 26:5233-5239; Dutcher et al., Journal for ImmunoTherapy of Cancer (2014) 2:26). Even T cells that proliferate and persist eventually become differentiated and dysfunctional (Philip et al., Nature, (2017) 545: 452-456; Schietinger et al., Immunity (2016) 45: 389-401). T cells with stem-like phenotypes can overcome these limitations and exhibit superior antitumor activity in mouse models and humans (Gattinoni et al., Nature Reviews Cancer (2012) 12: 671-684; Krishna et al., Science (2020) 370: 1328-1334), but therapeutic manipulations to select or expand stem-like T cells are limited to the cell manufacturing stage and cannot be performed in vivo.

[0291] The IL-9 receptor (CD129) is a less studied member of the γc cytokine receptor family that binds IL-9 and forms a heterodimeric receptor signaling complex with γc that can lead to activation of pSTAT1, pSTAT3 and pSTAT5 (Demoulin et al., Mol Cell Biol. (1996) 16:4710-4716; Knoops et al., Growth Factors (2004) 22:207-215; Bauer et al., J Biol Chem. (1998) 273:9255-9260). IL-9R is naturally expressed by mast cells, memory B cells, innate lymphoid cells and hematopoietic progenitor cells (Knoops et al., Growth Factors (2004) 22:207-215; Takatsuka et al., Nature Immunology (2018) 19:1025-1034; Townsend et al., Immunity (2000) 13:573-583; Williams et al., Blood (1990) 76:906-911; Turner et al., J Exp Med (2013) 210:2951-2965).Although T cell subsets that produce IL-9 have been described (Lu et al., J Clin Invest (2012) 122:4160-4171; Lu et al., Proc Natl Acad Sci USA (2014) 111:2265; Purwar et al., Nat Med (2012) 18:1248-1253), the effects of IL-9R signaling on T cells are not well characterized (Elyaman et al., Proc Natl Acad Sci USA (2009) 106:12885-12890; Nowak et al., J Exp Med (2009) 206:1653-1660; Li et al., Eur J Immunol (2011) 41:2197-2206; Houssiau et al., J Immunol (2011) 41:2197-2206; Immunol (1993) 150:2634-2640; Louahed et al., J Immunol (1995) 154:5061-5070; Lehrnbecher et al., Cytokine (1994) 6:279-284). For example, it is well documented that naive T cells are insensitive to IL-9 and T cell development is not impaired in IL-9-deficient mice, suggesting that IL-9 is not a critical natural cytokine in T cell biology (Townsend et al., Immunity (2000) 13:573-583; Houssiau et al., J Immunol (1993) 150:2634-2640). More recently, common gamma chain (γc) cytokine receptor signaling accompanied by STAT1, STAT3 and STAT5 activation was observed in T cells engineered to express orthogonal chimeric cytokine receptors (WO 2021 / 050752; Kalbasi, et al., 2022, Nature, 607(7918):360-365). These T cells take on characteristics of stem cell memory and effector T cells.

[0292] As an alternative approach to transduce T cells with common gamma chain (γc) cytokine receptor signaling, we engineered a chimeric antigen receptor (CAR) containing the IL9Ra ICD (Figure 1A). Human and mouse versions of this CAR were engineered as outlined in Table 1.

[0293] Table 1. Chimeric antigen receptors containing IL9Ra ICD TIFF2024534417000027.tif86166

[0294] Expression of the mouse CAR (containing anti-MSLN scFv, CD8 hinge and TM, 41BB ICD, IL9Ra ICD, and CD3z stimulatory domains) from the lentiviral construct was tested and demonstrated in transduced mouse T cells (Figure 1B). An important advantage of the CAR of the present invention compared to orthogonal cytokine receptor systems is that the CAR of the present invention does not require administration of orthogonal cytokines.

[0295] Expression of these CARs containing the IL9Ra ICD on transduced T cells is predicted to activate STAT1, STAT3 and STAT5 in T cells, thereby resulting in T cells with stem cell memory characteristics (Tscm) with improved trafficking and effector function, and improved anti-tumor activity against difficult-to-treat solid tumors in patients.

[0296] Example 2: Co-expression of WT IL9Ra and CAR in T cells It is envisioned in this example that co-expressing the IL9Ra receptor or a chimeric cytokine receptor comprising IL9Ra ICD on transduced T cells together with a CAR targeting a tumor antigen will similarly activate STAT1, STAT3 and STAT5 in T cells in vivo, thereby resulting in T cells with stem cell memory (Tscm) characteristics with improved trafficking and effector function, thereby improving anti-tumor activity against difficult-to-treat solid tumors in patients (as previously shown using an orthogonal chimeric cytokine receptor comprising IL9Ra ICD).

[0297] To demonstrate this and to further explore the mechanisms driving the unique phenotype and superior antitumor efficacy of CAR T cells engineered to receive IL-9 signals, human and mouse lentiviral constructs for expression of WT IL9Ra and anti-mesothelin CAR were designed and generated. Human and mouse T cells were transduced with the respective constructs and co-expression of CAR and IL9Ra was demonstrated by flow cytometry after transduction. Figure 3A shows the data for human T cells.

[0298] Co-expression of the mouse IL9Ra cytokine receptor and anti-mesothelin CAR on transduced mouse T cells was tested and demonstrated (Figure 2A and Figure 3B). Furthermore, these cells demonstrated a stem cell memory (Tscm) phenotype as indicated by the expression of surface markers CD44, CD62L and Fas (CD95) 24 hours after stimulation with 100 ng / mL wild-type mIL9 or wild-type mIL2 (Figure 2B and Figure 4). Total RNA was then extracted from transduced T cells cultured for 24 hours in the presence of mIL-2 or mIL-9. The RNA was analyzed with the Nanostring nCounter Mouse Immunology Panel (562 genes) and plotted using nSolver 4.0 software to obtain a global gene expression profile (Figure 2C). These data further demonstrate the Tscm phenotype of the transduced cells.

[0299] Transduced T cells co-expressing IL9Ra and CAR containing tumor antigen binding domain will be used with oncolytic adenoviral vector expressing IL9 to treat cancer in subjects. Therefore, we tested and demonstrated the expression of IL9 from Ad5 vector in mouse pancreatic cancer cell line (Figure 2D). Mouse pancreatic cancer cell line PDA7940b (10,000 cells / well) was infected with 100 virus particles / cell of Ad-mIL9, and cell culture supernatant was analyzed for mIL-9 by ELISA at the indicated time points.

[0300] Next, mouse T cells were starved of IL-2 for 24 h and then incubated in increasing concentrations of IL-9 for 20 min. Cells were fixed, permeabilized, stained, and analyzed by phosphorescent flow cytometry. Cells co-expressing IL9Ra and CAR showed a significant increase in phosphorylation of STAT1, STAT3, and STAT5 compared to cells expressing only CAR (A03) or untransduced cells (Figure 5). After incubating cells with IL9 for 48 h, cytokine secretion was analyzed by Luminex assay. Co-expression of CAR and IL9Ra significantly increased the secretion of IFNγ, TNFα, IL-10, IL-18, IL-1β, IL-6, IL-17A, IL-9, IL-22, IL-23, IL-4, IL-5, IL-13, IL-12p70, and IL-27 (Figure 6, right side of each panel). For T cells expressing CAR only (without co-expression of IL9Ra), no significant increases were observed for IFNγ, TNFα, and IL-10, and the increases in IL-4 and IL-13 were less significant than those observed for cells co-expressing CAR and IL9Ra (Figure 6, left side of each panel).

[0301] Tumor cell killing was then assessed by seeding 5000 PDA7940b (pancreatic tumor) cells. Primary mouse T cells were preincubated with IL-9 (100 nM) (or, as a control, no IL9) for 48 h and added at T cell:tumor cell ratios of 3:1, 1:1 and 1:3. Tumor cell killing was significantly enhanced for T cells expressing both IL9Ra and anti-mesothelin CAR (A03) in the presence of IL9 (Figure 7).

[0302] Gene expression profiles were determined using the Nanostring nCounter Mouse Immunology Panel for mouse T cells expressing CAR and IL9Ra preincubated with IL9 (or IL2 as a control) (Figure 8A). To compare with the orthogonal chimeric cytokine receptor system (see WO 2021 / 050752; Kalbasi, et al., Nature, 2022, 607:360-365), gene expression profiles were also determined for T cells expressing CAR and an orthogonal chimeric cytokine receptor (ortho-IL2Rβ-IL9Ra ("o9R") preincubated with ortho-IL2 (or IL2 as a control) (Figure 8B). The top 20 up- and down-regulated genes for each are shown (p-adj<0.05) (Figures 8A-B). The gene expression profiles for the two were similar. The top common up- and down-regulated genes are shown in Figure 8C.

[0303] We next performed gene set mutation analysis (GSVA) ​​on T cells expressing anti-meso CAR and IL9Ra preincubated with either IL9 or IL2, as well as on T cells expressing anti-meso CAR and ortho-IL2Rβ-IL9Ra chimeric cytokine receptor (o9R) preincubated with ortho-IL2 or IL-2. Pathways significantly enriched in CAR T cells stimulated with IL9 versus IL2 are shown in Figure 9A-C. In particular, the interferon gamma and interferon alpha pathways are significantly enriched in CAR T cells stimulated with IL-9 (Figure 9D-F).

[0304] Next, we established an in vivo syngeneic mouse model of PDA (Figure 10A) and titrated an Ad vector expressing IL-9 (Ad-mIL9) (Figure 10B). Transduction efficiency data is shown in Figure 10C. In vivo tumor growth was determined at various doses of Ad-mIL9 in mice injected with T cells expressing CAR and IL9Ra, along with control conditions as indicated (Figure 10D).

[0305] Enumerated Aspects The following enumerated aspects are provided, the numbering of which should not be construed as designating a level of importance. Aspect 1 provides: A chimeric antigen receptor (CAR) comprising a tumor antigen-binding domain, a transmembrane domain, and an intracellular domain comprising the intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra). Aspect 2 provides: The tumor antigen is alpha fetoprotein (AFP) / HLA-A2, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA, claudin 18.2, c-Met, DLL3, DR5, EGFR, EGFRvIII, EpCAM, Ep 2. The CAR of embodiment 1, wherein the CAR is selected from the group consisting of hA2, FAP, folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, glycolipid F77, glypican-2 (GPC2), glypican-3 (GPC3), HER2, HLA-A2, ICAM1, IL3Ra, IL13Ra2, LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, MelanA, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, Nectin4 / FAP, NKG2D ligands (MIC-A, MIC-B, and ULBP 1-6), NY-ESO-1, P16, PD-L1, PSCA, PSMA, ROR1, ROR2, TIM-3, TM4SF1, TnMuc1, VEGFR2, and any combination thereof. Aspect 3 provides: The CAR of embodiment 1 or embodiment 2, wherein said tumor antigen is selected from mesothelin, GD2, HER2, TnMuc1, CD70, PMSA, and EGFRvIII. Aspect 4 provides: 4. The CAR of any one of embodiments 1-3, wherein said tumor antigen binding domain is selected from the group consisting of a full length antibody or antigen-binding fragment thereof, a monospecific antibody, a bispecific antibody, a Fab, a Fab', a F(ab')2, an Fv, a single chain variable fragment (scFv), a linear antibody, a single domain antibody (sdAb) and an antibody mimetic (e.g., a designed ankyrin repeat protein (DARPin), an affibody, a monobody (adnectin), an affilin, an affimer, an affitin, an alphabody, an avimer, a Kunitz domain peptide, anticalin, and a syntherin). Aspect 5 provides the following: The CAR according to any one of aspects 1 to 4, wherein the tumor antigen-binding domain is a single chain variable fragment (scFv). Aspect 6 provides the following: the tumor antigen-binding domain is (a) an anti-mesothelin scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from SEQ ID NO:49 and SEQ ID NO:65; (b) an anti-GD2 scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:108; (c) an anti-HER2 scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:110 or SEQ ID NO:112; (d) an anti-TnMuc1 scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:114; (e) an anti-CD70 scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:116; (f) an anti-PMSA scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:120; and (g) an anti-EGFRvIII scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:122. The CAR according to any one of embodiments 1 to 5, wherein the CAR is selected from the group consisting of Aspect 7 provides the following: 7. The CAR of any one of embodiments 1-6, wherein said intracellular domain of said CAR further comprises a costimulatory domain of a protein selected from the group consisting of the TNFR superfamily of proteins, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3 (CD276), or a variant thereof, or an intracellular domain derived from a killer immunoglobulin-like receptor (KIR). Aspect 8 provides the following: 8. The CAR of any one of embodiments 1-7, wherein said intracellular domain of said CAR further comprises an intracellular signaling domain of a protein selected from the group consisting of CD3 zeta chain (CD3ζ), FcγRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor with an immunoreceptor tyrosine-based activation motif (ITAM), TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or a variant thereof. Aspect 9 provides the following: The CAR of any one of embodiments 1-8, wherein said intracellular domain of said CAR further comprises a costimulatory domain of CD28, a costimulatory domain of 4-1BB, an intracellular signaling domain of CD3 zeta, or any combination thereof. Aspect 10 provides the following: The CAR of any one of embodiments 1-9, further comprising a hinge domain. Aspect 11 provides the following: (a) anti-human mesothelin scFv, human CD8 hinge domain, human CD28 transmembrane domain, human CD28 costimulatory domain, human IL9Ra intracellular signaling domain and human CD3z signaling domain; (b) anti-human mesothelin scFv, human CD8 hinge domain, human CD8 transmembrane domain, human 4-1BB costimulatory domain, human IL9Ra intracellular signaling domain and human CD3z signaling domain; (c) anti-mouse mesothelin scFv, mouse CD8 hinge domain, mouse CD28 transmembrane domain, mouse CD28 costimulatory domain, mouse IL9Ra intracellular signaling domain and mouse CD3z signaling domain; or (d) anti-mouse mesothelin scFv, mouse CD8 hinge domain, mouse CD8 transmembrane domain, mouse 4-1BB costimulatory domain, mouse IL9Ra intracellular signaling domain, and mouse CD3z signaling domain 11. The CAR of any one of embodiments 1 to 10, comprising: Aspect 12 provides the following: 12. The CAR of any one of embodiments 1-11, comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from SEQ ID NOs: 81, 83, 85, and 87. Aspect 13 provides the following: 13. The CAR of any one of embodiments 1-12, encoded by a nucleic acid comprising a nucleotide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from SEQ ID NOs: 82, 84, 86, and 88. Aspect 14 provides the following: 1. An isolated nucleic acid comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) comprising a tumor antigen binding domain, a transmembrane domain, and an intracellular domain comprising the intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra). Aspect 15 provides the following: The tumor antigen is alpha fetoprotein (AFP) / HLA-A2, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA, claudin 18.2, c-Met, DLL3, DR5, EGFR, EGFRvIII, EpCAM, Ep 15. The isolated nucleic acid of embodiment 14, wherein the nucleic acid is selected from the group consisting of hA2, FAP, folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, glycolipid F77, glypican-2 (GPC2), glypican-3 (GPC3), HER2, HLA-A2, ICAM1, IL3Ra, IL13Ra2, LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan-A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, Nectin4 / FAP, NKG2D ligands (MIC-A, MIC-B, and ULBP 1-6), NY-ESO-1, P16, PD-L1, PSCA, PSMA, ROR1, ROR2, TIM-3, TM4SF1, TnMuc1, VEGFR2, and any combination thereof. Aspect 16 provides the following: The isolated nucleic acid of embodiment 14 or embodiment 15, wherein said tumor antigen is selected from mesothelin, GD2, HER2, TnMuc1, CD70, PMSA, and EGFRvIII. Aspect 17 provides the following: 17. The isolated nucleic acid of any one of embodiments 14-16, wherein the tumor antigen-binding domain is selected from the group consisting of a full length antibody or antigen-binding fragment thereof, a monospecific antibody, a bispecific antibody, a Fab, a Fab', a F(ab')2, a Fv, a single chain variable fragment (scFv), a linear antibody, a single domain antibody (sdAb) and an antibody mimetic (e.g., a designed ankyrin repeat protein (DARPin), an affibody, a monobody (adnectin), an affilin, an affimer, an affitin, an alphabody, an avimer, a Kunitz domain peptide, anticalin, and a syntherin). Aspect 18 provides the following: 18. The isolated nucleic acid according to any one of aspects 14 to 17, wherein the tumor antigen-binding domain is a single-chain variable fragment (scFv). Aspect 19 provides the following: the tumor antigen-binding domain is (a) an anti-mesothelin scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from SEQ ID NO:49 and SEQ ID NO:65; (b) an anti-GD2 scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:108; (c) an anti-HER2 scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:110 or SEQ ID NO:112; (d) an anti-TnMuc1 scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:114; (e) an anti-CD70 scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:116; (f) an anti-PMSA scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:120; and (g) an anti-EGFRvIII scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:122. 19. The isolated nucleic acid according to any one of embodiments 14 to 18, selected from: Aspect 20 provides the following: 20. The isolated nucleic acid of any one of embodiments 14-19, wherein said intracellular domain of said CAR further comprises a costimulatory domain of a protein selected from the group consisting of the TNFR superfamily of proteins, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3 (CD276), or a variant thereof, or an intracellular domain derived from a killer immunoglobulin-like receptor (KIR). Aspect 21 provides the following: 21. The isolated nucleic acid of any one of embodiments 14-20, wherein said intracellular domain of said CAR further comprises an intracellular signaling domain of a protein selected from the group consisting of CD3 zeta chain (CD3ζ), FcγRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor with an immunoreceptor tyrosine-based activation motif (ITAM), TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or a variant thereof. Aspect 22 provides the following: 22. The isolated nucleic acid of any one of embodiments 14-21, wherein said intracellular domain of said CAR further comprises a costimulatory domain of CD28, a costimulatory domain of 4-1BB, an intracellular signaling domain of CD3 zeta, or any combination thereof. Aspect 23 provides the following: 23. The isolated nucleic acid of any one of embodiments 14 to 22, further comprising a hinge domain. Aspect 24 provides the following: The CAR, (a) anti-human mesothelin scFv, human CD8 hinge domain, human CD28 transmembrane domain, human CD28 costimulatory domain, human IL9Ra intracellular signaling domain and human CD3z signaling domain; (b) anti-human mesothelin scFv, human CD8 hinge domain, human CD8 transmembrane domain, human 4-1BB costimulatory domain, human IL9Ra intracellular signaling domain and human CD3z signaling domain; (c) anti-mouse mesothelin scFv, mouse CD8 hinge domain, mouse CD28 transmembrane domain, mouse CD28 costimulatory domain, mouse IL9Ra intracellular signaling domain and mouse CD3z signaling domain; or (d) anti-mouse mesothelin scFv, mouse CD8 hinge domain, mouse CD8 transmembrane domain, mouse 4-1BB costimulatory domain, mouse IL9Ra intracellular signaling domain, and mouse CD3z signaling domain 24. The isolated nucleic acid of any one of embodiments 14 to 23, comprising: Aspect 25 provides the following: 25. The isolated nucleic acid of any one of embodiments 14-24, wherein the CAR comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from SEQ ID NOs: 81, 83, 85, and 87. Aspect 26 provides the following: 26. The isolated nucleic acid of any one of embodiments 14-25, wherein the CAR is encoded by a nucleic acid comprising a nucleotide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from SEQ ID NOs: 82, 84, 86, and 88. Aspect 27 provides the following: A vector comprising the isolated nucleic acid according to any one of embodiments 14 to 26. Aspect 28 provides the following: 28. The vector of embodiment 27, which is a retroviral vector or a lentiviral vector. Aspect 29 provides the following: A modified cell comprising: The cell is an immune cell or a precursor thereof, and The cells are engineered to express a chimeric antigen receptor (CAR) comprising a tumor antigen binding domain, a transmembrane domain, and an intracellular domain comprising the intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra), The modified cell. Aspect 30 provides the following: The tumor antigen is alpha fetoprotein (AFP) / HLA-A2, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA, claudin 18.2, c-Met, DLL3, DR5, EGFR, EGFRvIII, EpCAM, Ep 30. The modified cell of embodiment 29, wherein the polypeptide is selected from the group consisting of hA2, FAP, folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, glycolipid F77, glypican-2 (GPC2), glypican-3 (GPC3), HER2, HLA-A2, ICAM1, IL3Ra, IL13Ra2, LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan-A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, Nectin4 / FAP, NKG2D ligands (MIC-A, MIC-B, and ULBP 1-6), NY-ESO-1, P16, PD-L1, PSCA, PSMA, ROR1, ROR2, TIM-3, TM4SF1, TnMuc1, VEGFR2, and any combination thereof. Aspect 31 provides the following: The modified cell of embodiment 29 or embodiment 30, wherein said tumor antigen is selected from mesothelin, GD2, HER2, TnMuc1, CD70, PMSA, and EGFRvIII. Aspect 32 provides the following: 32. The modified cell of any one of embodiments 29-31, wherein the tumor antigen-binding domain is selected from the group consisting of a full length antibody or antigen-binding fragment thereof, a monospecific antibody, a bispecific antibody, a Fab, a Fab', a F(ab')2, an Fv, a single chain variable fragment (scFv), a linear antibody, a single domain antibody (sdAb) and an antibody mimetic (e.g., a designed ankyrin repeat protein (DARPin), an affibody, a monobody (adnectin), an affilin, an affimer, an affitin, an alphabody, an avimer, a Kunitz domain peptide, anticalin, and a syntherin). Aspect 33 provides the following: 33. The modified cell of any one of aspects 29 to 32, wherein the tumor antigen-binding domain is a single-chain variable fragment (scFv). Aspect 34 provides the following: the tumor antigen-binding domain is (a) an anti-mesothelin scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from SEQ ID NO:49 and SEQ ID NO:65; (b) an anti-GD2 scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:108; (c) an anti-HER2 scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:110 or SEQ ID NO:112; (d) an anti-TnMuc1 scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:114; (e) an anti-CD70 scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:116; (f) an anti-PMSA scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:120; and (g) an anti-EGFRvIII scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:122. The modified cell of any one of embodiments 29 to 33, selected from the group consisting of Embodiment 35 provides the following: 35. The modified cell of any one of embodiments 29-34, wherein said intracellular domain of said CAR further comprises a costimulatory domain of a protein selected from the group consisting of proteins of the TNFR superfamily, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3 (CD276), or a variant thereof, or an intracellular domain derived from a killer immunoglobulin-like receptor (KIR). Aspect 36 provides the following: 36. The modified cell of any one of embodiments 29-35, wherein said intracellular domain of said CAR further comprises an intracellular signaling domain of a protein selected from the group consisting of CD3 zeta chain (CD3ζ), FcγRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor with an immunoreceptor tyrosine-based activation motif (ITAM), TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or a variant thereof. Aspect 37 provides the following: 37. The modified cell of any one of embodiments 29-36, wherein said intracellular domain of said CAR further comprises a costimulatory domain of CD28, a costimulatory domain of 4-1BB, an intracellular signaling domain of CD3 zeta, or any combination thereof. Embodiment 38 provides the following: The modified cell of any one of embodiments 29 to 37, further comprising a hinge domain. Aspect 39 provides the following: The CAR, (a) anti-human mesothelin scFv, human CD8 hinge domain, human CD28 transmembrane domain, human CD28 costimulatory domain, human IL9Ra intracellular signaling domain and human CD3z signaling domain; (b) anti-human mesothelin scFv, human CD8 hinge domain, human CD8 transmembrane domain, human 4-1BB costimulatory domain, human IL9Ra intracellular signaling domain and human CD3z signaling domain; (c) anti-mouse mesothelin scFv, mouse CD8 hinge domain, mouse CD28 transmembrane domain, mouse CD28 costimulatory domain, mouse IL9Ra intracellular signaling domain and mouse CD3z signaling domain; or (d) anti-mouse mesothelin scFv, mouse CD8 hinge domain, mouse CD8 transmembrane domain, mouse 4-1BB costimulatory domain, mouse IL9Ra intracellular signaling domain, and mouse CD3z signaling domain 39. The modified cell of any one of embodiments 29 to 38, comprising: Aspect 40 provides the following: 40. The modified cell of any one of embodiments 29-39, wherein the CAR comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from SEQ ID NOs: 81, 83, 85, and 87. Aspect 41 provides the following: 41. The modified cell of any one of embodiments 29-40, wherein the CAR is encoded by a nucleic acid comprising a nucleotide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from SEQ ID NOs: 82, 84, 86, and 88. Aspect 42 provides the following: 42. The modified cell of any one of embodiments 29 to 41, wherein the cell is a T cell, an autologous cell, a human cell, or any combination thereof. Aspect 43 provides the following: 43. The modified cell of any one of embodiments 29 to 42, wherein the cell is capable of activating STAT1, STAT3, STAT5, or any combination thereof. Aspect 44 provides the following: A pharmaceutical composition comprising a population of modified cells according to any one of aspects 29 to 43 and at least one pharma- ceutically acceptable carrier. Aspect 45 provides the following: 1. A method of treating cancer in a subject in need thereof, comprising administering to the subject a population of modified cells, wherein the cells are immune cells or precursor cells thereof, and the cells have been engineered to express a chimeric antigen receptor (CAR) comprising a tumor antigen binding domain, a transmembrane domain, and an intracellular domain comprising the intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra). Aspect 46 provides the following: The tumor antigen is alpha fetoprotein (AFP) / HLA-A2, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA, claudin 18.2, c-Met, DLL3, DR5, EGFR, EGFRvIII, EpCAM, Ep 46. ​​The method of embodiment 45, wherein the IL-13R is selected from the group consisting of hA2, FAP, folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, glycolipid F77, glypican-2 (GPC2), glypican-3 (GPC3), HER2, HLA-A2, ICAM1, IL3Ra, IL13Ra2, LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan-A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, Nectin4 / FAP, NKG2D ligands (MIC-A, MIC-B, and ULBP 1-6), NY-ESO-1, P16, PD-L1, PSCA, PSMA, ROR1, ROR2, TIM-3, TM4SF1, TnMuc1, VEGFR2, and any combination thereof. Aspect 47 provides the following: The method of embodiment 45 or embodiment 46, wherein said tumor antigen is selected from mesothelin, GD2, HER2, TnMuc1, CD70, PMSA, and EGFRvIII. Aspect 48 provides the following: 48. The method of any one of embodiments 45-47, wherein the tumor antigen-binding domain is selected from the group consisting of a full-length antibody or antigen-binding fragment thereof, a monospecific antibody, a bispecific antibody, a Fab, a Fab', a F(ab')2, an Fv, a single-chain variable fragment (scFv), a linear antibody, a single domain antibody (sdAb) and an antibody mimetic (e.g., a designed ankyrin repeat protein (DARPin), an affibody, a monobody (adnectin), an affilin, an affimer, an affitin, an alphabody, an avimer, a Kunitz domain peptide, anticalin, and a syntherin). Aspect 49 provides the following: The method according to any one of aspects 45 to 48, wherein the tumor antigen-binding domain is a single-chain variable fragment (scFv). Embodiment 50 provides the following: the tumor antigen-binding domain is (a) an anti-mesothelin scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from SEQ ID NO:49 and SEQ ID NO:65; (b) an anti-GD2 scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:108; (c) an anti-HER2 scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:110 or SEQ ID NO:112; (d) an anti-TnMuc1 scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:114; (e) an anti-CD70 scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:116; (f) an anti-PMSA scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:120; and (g) an anti-EGFRvIII scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to SEQ ID NO:122. 50. The method of any one of embodiments 45 to 49, selected from the group consisting of Aspect 51 provides the following: 51. The method of any one of embodiments 45-50, wherein said intracellular domain of said CAR further comprises a costimulatory domain of a protein selected from the group consisting of the TNFR superfamily of proteins, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3 (CD276), or a variant thereof, or an intracellular domain derived from a killer immunoglobulin-like receptor (KIR). Embodiment 52 provides the following: 52. The method of any one of embodiments 45-51, wherein said intracellular domain of said CAR further comprises an intracellular signaling domain of a protein selected from the group consisting of CD3 zeta chain (CD3ζ), FcγRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor with an immunoreceptor tyrosine-based activation motif (ITAM), TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or a variant thereof. Embodiment 53 provides the following: 53. The method of any one of embodiments 45-52, wherein said intracellular domain of said CAR further comprises a costimulatory domain of CD28, a costimulatory domain of 4-1BB, an intracellular signaling domain of CD3 zeta, or any combination thereof. Embodiment 54 provides the following: The method of any one of embodiments 45 to 53, further comprising a hinge domain. Embodiment 55 provides the following: The CAR, (a) anti-human mesothelin scFv, human CD8 hinge domain, human CD28 transmembrane domain, human CD28 costimulatory domain, human IL9Ra intracellular signaling domain and human CD3z signaling domain; (b) anti-human mesothelin scFv, human CD8 hinge domain, human CD8 transmembrane domain, human 4-1BB costimulatory domain, human IL9Ra intracellular signaling domain and human CD3z signaling domain; (c) anti-mouse mesothelin scFv, mouse CD8 hinge domain, mouse CD28 transmembrane domain, mouse CD28 costimulatory domain, mouse IL9Ra intracellular signaling domain and mouse CD3z signaling domain; or (d) anti-mouse mesothelin scFv, mouse CD8 hinge domain, mouse CD8 transmembrane domain, mouse 4-1BB costimulatory domain, mouse IL9Ra intracellular signaling domain, and mouse CD3z signaling domain 55. The method of any one of embodiments 45 to 54, comprising: Embodiment 56 provides the following: 56. The method of any one of embodiments 45-55, wherein the CAR comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from SEQ ID NOs:81, 83, 85, and 87. Embodiment 57 provides the following: 57. The method of any one of embodiments 45-56, wherein the CAR is encoded by a nucleic acid comprising a nucleotide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a sequence selected from SEQ ID NOs:82, 84, 86, and 88. Embodiment 58 provides the following: 58. The method of any one of embodiments 45-57, wherein said population of cells comprises T cells, autologous cells, human cells, or any combination thereof. Embodiment 59 provides the following: The method of any one of embodiments 45-58, wherein said population of cells is capable of activating STAT1, STAT3, STAT5, or any combination thereof. Embodiment 60 provides the following: 60. The method of any one of aspects 45 to 59, wherein the subject is a human. Aspect 61 provides the following: 61. The method of any one of aspects 45 to 60, wherein the cancer is selected from B-cell malignancies (such as B-cell lymphoma or leukemia), lung cancer, non-small cell lung cancer, small cell lung cancer, Merkel cell carcinoma, melanoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, urothelial carcinoma, gastric cancer, cervical cancer, cutaneous squamous cell carcinoma, renal cell carcinoma, breast cancer, triple-negative breast cancer, colon cancer, esophageal cancer, stomach cancer, liver cancer, kidney cancer, pancreatic cancer, prostate cancer, brain cancer, lung adenocarcinoma, glioblastoma, hepatocellular carcinoma, gallbladder cancer, cervical cancer, cervical squamous cell carcinoma, colorectal cancer, ovarian cancer, and renal cancer.

[0306] Other Aspects The disclosures of any and all patents, patent applications, and publications cited herein are incorporated herein by reference in their entirety. Although the present invention has been disclosed with reference to certain embodiments, it is clear that other embodiments and modifications of the present invention may be devised by those skilled in the art without departing from the true spirit and scope of the present invention. It is intended that the appended claims be construed to include all such embodiments and equivalent modifications.

Claims

1. From the amino terminus to the carboxy terminus, (a) a tumor antigen-binding domain; (b) a transmembrane domain; (c) a costimulatory domain; (d) the intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra); (e) an intracellular signaling domain of a protein selected from the group consisting of CD3 zeta chain (CD3ζ), FcγRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor with an immunoreceptor tyrosine-based activation motif (ITAM), TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or a variant thereof; and capable of activating STAT1, STAT3, STAT5, or any combination thereof.

2. the tumor antigen-binding domain is (a) Alpha-fetoprotein (AFP) / HLA-A2, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA, claudin 18.2, c-Met, DLL3, DR5, EGFR, EGFRvIII, EpCAM, EphA 2, FAP, folate receptor alpha (FRa) / folate binding protein (FBP), GD-2, glycolipid F77, glypican-2 (GPC2), glypican-3 (GPC3), HER2, HLA-A2, ICAM1, IL3Ra, IL13Ra2, LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan-A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, nectin-4 / FAP, NKG2D ligands (MIC-A, MIC-B, and ULBP 1-6), NY-ESO-1, P16, PD-L1, PSCA, PSMA, ROR1, ROR2, TIM-3, TM4SF1, TnMuc1, VEGFR2, and any combination thereof; or (b) Mesothelin, GD2, HER2, TnMuc1, CD70, PMSA, and EGFRvIII The CAR of claim 1, wherein the CAR binds to a tumor antigen selected from the group consisting of: (a) the tumor antigen-binding domain is selected from the group consisting of a full-length antibody or antigen-binding fragment thereof, a monospecific antibody, a bispecific antibody, Fab, Fab', F(ab')2, Fv, a single-chain variable fragment (scFv), a linear antibody, a single-domain antibody (sdAb), and an antibody mimetic (e.g., a designed ankyrin repeat protein (DARPin), an affibody, a monobody (adnectin), an affilin, an affimer, an affitin, an alphabody, an avimer, a Kunitz domain peptide, anticalin, and a syntheline); or (b) the tumor antigen-binding domain is a single-chain variable fragment (scFv); 10. The CAR of claim 1.

4. the tumor antigen-binding domain is (a) an anti-mesothelin scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:49 or SEQ ID NO:65; (b) an anti-GD2 scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:108; (c) an anti-HER2 scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:110 or SEQ ID NO:112; (d) an anti-TnMuc1 scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:114; (e) an anti-CD70 scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:116; (f) an anti-PMSA scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:120; or (g) an anti-EGFRvIII scFv comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

122.

2. The CAR of claim 1, wherein the CAR is selected from: (a) the costimulatory domain is a costimulatory domain of a protein selected from the group consisting of proteins of the TNFR superfamily, CD28, 4-1BB (CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3 (CD276), or a variant thereof, or an intracellular domain derived from a killer immunoglobulin-like receptor (KIR); and / or (b) the intracellular signaling domain is the intracellular signaling domain of CD3 zeta; and / or (c) the CAR further comprises a hinge domain; 10. The CAR of claim 1.

6. The CAR of claim 1, wherein the CAR comprises the costimulatory domain of CD28 or the costimulatory domain of 4-1BB, and the intracellular signaling domain of CD3 zeta, or any combination thereof.

7. From the amino terminus to the carboxy terminus, (a) Anti-human mesothelin scFv, human CD8 hinge domain, human CD28 transmembrane domain, human CD28 costimulatory domain, human IL9Ra intracellular signaling domain, and human CD3z signaling domain; (b) anti-human mesothelin scFv, human CD8 hinge domain, human CD8 transmembrane domain, human 4-1BB costimulatory domain, human IL9Ra intracellular signaling domain, and human CD3z signaling domain; (c) anti-mouse mesothelin scFv, mouse CD8 hinge domain, mouse CD28 transmembrane domain, mouse CD28 costimulatory domain, mouse IL9Ra intracellular signaling domain and mouse CD3z signaling domain; or (d) Anti-mouse mesothelin scFv, mouse CD8 hinge domain, mouse CD8 transmembrane domain, mouse 4-1BB costimulatory domain, mouse IL9Ra intracellular signaling domain, and mouse CD3z signaling domain 2. The CAR of claim 1, comprising:

8. The CAR of claim 1, comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 81, 83, 85, and 87.

9. An isolated nucleic acid comprising a chimeric antigen receptor (CAR) described in any one of claims 1 to 8.

10. 10. The isolated nucleic acid of claim 9, wherein the CAR is encoded by a nucleic acid comprising a nucleotide sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 82, 84, 86, and 88.

11. A vector comprising the isolated nucleic acid of claim 9.

12. 12. The vector of claim 11, which is a retroviral vector or a lentiviral vector.

13. (a) engineered to express a chimeric antigen receptor (CAR) according to any one of claims 1 to 8; or (b) comprising the isolated nucleic acid of claim 9; or (c) comprising the vector of claim 11; Modified immune cells or their precursor cells.

14. The modified immune cell or precursor thereof of claim 13, which is a T cell, an autologous cell, a human cell, or any combination thereof.

15. The modified immune cell or precursor thereof of claim 13, which is capable of activating STAT1, STAT3, STAT5, or any combination thereof.

16. 14. A pharmaceutical composition comprising a population of modified immune cells or progenitor cells thereof according to claim 13 and at least one pharmaceutically acceptable carrier.

17. A pharmaceutical composition for treating cancer in a subject in need thereof, comprising a population of modified immune cells or their precursor cells as described in claim 13. (a) the population of modified immune cells or their precursor cells comprises T cells, autologous cells, human cells, or any combination thereof; and / or (b) the population of modified immune cells or their precursor cells is capable of activating STAT1, STAT3, STAT5, or any combination thereof; 18. The pharmaceutical composition of claim 17.

19. 18. The pharmaceutical composition of claim 17, wherein the subject is a human.

20. The cancer is (a) selected from the group consisting of B-cell malignancies, lung cancer, non-small cell lung cancer, small cell lung cancer, Merkel cell carcinoma, melanoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, urothelial carcinoma, gastric cancer, cervical cancer, cutaneous squamous cell carcinoma, renal cell carcinoma, breast cancer, triple-negative breast cancer, colon cancer, esophageal cancer, stomach cancer, liver cancer, kidney cancer, pancreatic cancer, prostate cancer, brain cancer, lung adenocarcinoma, glioblastoma, hepatocellular carcinoma, gallbladder cancer, cervical cancer, cervical squamous cell carcinoma, colorectal cancer, ovarian cancer, and renal cancer; or (b) B-cell lymphoma or leukemia; 18. The pharmaceutical composition of claim 17.

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