Interleukin-9 signaling in chimeric antigen receptor (CAR) immune cells

Abstract

The present disclosure provides a chimeric cytokine receptor comprising an intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra). The present disclosure also provides modified cells, i.e., immune cells or precursor cells thereof, engineered to express a) interleukin-9 receptor alpha (IL9Ra) or a chimeric cytokine receptor disclosed herein, and b) a chimeric antigen receptor (CAR). The present disclosure further provides vectors (e.g., oncolytic adenoviral vectors) comprising a nucleic acid sequence encoding a cytokine, and methods of using the modified cells and vectors to treat cancer in a subject in need thereof. Also provided are modified immune cells or precursor cells thereof engineered to express a chimeric antigen receptor (CAR), wherein expression of cullin 5 in the cells is reduced and / or eliminated. Also provided are methods and uses of the modified cells, e.g., 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.C. 119(e) to 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] Background of the Invention Current advances in immunotherapy 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 in cancer treatment is solid tumors. CAR-T cells have lacked effectiveness in combating solid tumors due to numerous challenges, including the lack of tumor-specific antigens, treatment resistance, tumor heterogeneity, poor proliferation and persistence, and overcoming the extrinsic functional and physical barriers to T cell infiltration posed by the dense immunosuppressive tumor microenvironment (TME). One major limitation is the poor in vivo proliferation and persistence of adoptively transferred T cells, necessitating lymphodepleting conditioning chemotherapy, a toxic regimen that limits patient eligibility. Even T cells that proliferate and persist eventually differentiate and become dysfunctional. T cells with stem-like phenotypes can overcome these limitations and exhibit excellent anti-tumor 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 cytokine receptor comprising: (a) an extracellular domain containing the ligand binding domain (LBD) of a receptor selected from interleukin-13 receptor alpha type 2 (IL13Ra2), interleukin-2 receptor beta (IL2Rb), interleukin-18 receptor alpha (IL18Ra), and interleukin-18 receptor beta (IL18Rb); (b) transmembrane domain and; (c) an intracellular domain containing the intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra); The chimeric cytokine receptor comprises:

[0004] In some embodiments, the transmembrane domain is an IL9Ra transmembrane domain.

[0005] In some embodiments, the chimeric cytokine receptor is (a) Human IL13Ra2 LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (b) human IL2Rb LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (c) human IL18Ra LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (d) human IL18Rb LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (e) mouse IL13Ra2 LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; (f) mouse IL2Rb LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; (g) a mouse IL18Ra LBD, a mouse IL9Ra transmembrane domain, and a mouse IL9Ra intracellular signaling domain; or (h) Mouse IL18Rb LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain Includes:

[0006] In some embodiments, the chimeric cytokine receptor 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: 15, 17, 19, 21, 23, 51, 53, 55, 57, and 59.

[0007] In some embodiments, the chimeric cytokine receptor 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: 16, 18, 20, 22, 24, 52, 54, 56, 58, and 60.

[0008] In some embodiments, an isolated nucleic acid is provided comprising a nucleotide sequence encoding the chimeric cytokine receptor of any one of the preceding embodiments.

[0009] In some embodiments, a vector comprising the isolated nucleic acid of embodiment 6 is provided.

[0010] In some embodiments, the vector is a retroviral or lentiviral vector.

[0011] In some aspects, a) a first nucleotide sequence encoding a chimeric cytokine receptor comprising: (i) an extracellular domain comprising the ligand binding domain (LBD) of a receptor selected from interleukin-13 receptor alpha type 2 (IL13Ra2), interleukin-2 receptor beta (IL2Rb), interleukin-18 receptor alpha (IL18Ra), and interleukin-18 receptor beta (IL18Rb), (ii) a first transmembrane domain, and (iii) an intracellular domain comprising the intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra); and b) a second nucleotide sequence encoding a chimeric antigen receptor (CAR) comprising an extracellular tumor antigen binding domain, a second transmembrane domain, and a second intracellular domain; An isolated nucleic acid is provided, comprising:

[0012] In some embodiments, the transmembrane domain is an IL9Ra transmembrane domain.

[0013] In some embodiments, the chimeric cytokine receptor is (a) Human IL13Ra2 LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (b) human IL2Rb LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (c) human IL18Ra LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (d) human IL18Rb LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (e) mouse IL13Ra2 LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; (f) mouse IL2Rb LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; (g) a mouse IL18Ra LBD, a mouse IL9Ra transmembrane domain, and a mouse IL9Ra intracellular signaling domain; or (h) Mouse IL18Rb LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain Includes:

[0014] In some embodiments, the chimeric cytokine receptor 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: 15, 17, 19, 21, 23, 51, 53, 55, 57, and 59.

[0015] In some embodiments of the isolated nucleic acid of any one of the foregoing embodiments, the first nucleotide sequence is a 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: 16, 18, 20, 22, 24, 52, 54, 56, 58, and 60.

[0016] In some embodiments of the isolated nucleic acid of any one of the preceding aspects, the tumor antigen is alphafetoprotein (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, EG The antibody is selected from the group consisting of FRvIII, 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.

[0017] In some embodiments, the tumor antigen is selected from mesothelin, GD2, HER2, TnMuc1, CD70, PMSA, and EGFRvIII.

[0018] 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'), 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 syntheline).

[0019] In some embodiments, the tumor antigen-binding domain is a single-chain variable fragment (scFv).

[0020] 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: 79 and SEQ ID NO: 95; (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: 117; (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: 119 or SEQ ID NO: 121; (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: 123; (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: 125; (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: 129; 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: 131. is selected from.

[0021] In some embodiments, the intracellular domain of the CAR 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).

[0022] In some embodiments, the intracellular domain of the CAR comprises the 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.

[0023] In some embodiments, the intracellular domain of the CAR comprises the costimulatory domain of CD28, the costimulatory domain of 4-1BB, the intracellular signaling domain of CD3 zeta, or any combination thereof.

[0024] In some aspects, a vector is provided that includes the isolated nucleic acid of any one of the preceding embodiments.

[0025] In some embodiments, the vector is a retroviral or lentiviral vector.

[0026] In some aspects, modified cells are provided that comprise a chimeric cytokine receptor of any one of the chimeric cytokine receptor-comprising embodiments, the isolated nucleic acid-comprising embodiments, and / or the vector-comprising embodiments, wherein the cell is an immune cell or a precursor cell thereof.

[0027] In some embodiments, the cells are T cells, autologous cells, human cells, or any combination thereof.

[0028] In some embodiments, the cell is an immune cell or a progenitor thereof, and the cell comprises: a) interleukin-9 receptor alpha (IL9Ra), or A chimeric cytokine receptor comprising: (i) an extracellular domain comprising the ligand binding domain (LBD) of a receptor selected from interleukin-13 receptor alpha type 2 (IL13Ra2), interleukin-2 receptor beta (IL2Rb), interleukin-18 receptor alpha (IL18Ra), and interleukin-18 receptor beta (IL18Rb), (ii) a first transmembrane domain, and (iii) an intracellular domain comprising the intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra); and b) a chimeric antigen receptor (CAR) comprising an extracellular tumor antigen-binding domain, a second transmembrane domain, and a second intracellular domain; It has been engineered to express

[0029] In some embodiments, the transmembrane domain is an IL9Ra transmembrane domain.

[0030] In some embodiments, the chimeric cytokine receptor is (a) Human IL13Ra2 LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (b) human IL2Rb LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (c) human IL18Ra LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (d) human IL18Rb LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (e) mouse IL13Ra2 LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; (f) mouse IL2Rb LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; (g) a mouse IL18Ra LBD, a mouse IL9Ra transmembrane domain, and a mouse IL9Ra intracellular signaling domain; or (h) Mouse IL18Rb LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain Includes:

[0031] In some embodiments, the chimeric cytokine receptor 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: 15, 17, 19, 21, 23, 51, 53, 55, 57, and 59.

[0032] In some embodiments, the first nucleotide sequence is a 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: 16, 18, 20, 22, 24, 52, 54, 56, 58, and 60.

[0033] In some embodiments of the modified cells, the tumor antigen is alphafetoprotein (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, 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.

[0034] In some embodiments, the tumor antigen is selected from mesothelin, GD2, HER2, TnMuc1, CD70, PMSA, and EGFRvIII.

[0035] 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'), 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 syntheline).

[0036] In some embodiments, the tumor antigen-binding domain is a single-chain variable fragment (scFv).

[0037] 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: 79 and SEQ ID NO: 95; (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: 117; (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: 119 or SEQ ID NO: 121; (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: 123; (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: 125; (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: 129; 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: 131. is selected from.

[0038] In some embodiments, the intracellular domain of the CAR 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).

[0039] In some embodiments, the intracellular domain of the CAR comprises the 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.

[0040] In some embodiments, the intracellular domain of the CAR comprises the costimulatory domain of CD28, the costimulatory domain of 4-1BB, the intracellular signaling domain of CD3 zeta, or any combination thereof.

[0041] In some embodiments, the cells are T cells, autologous cells, human cells, or any combination thereof.

[0042] In some embodiments, the IL9Ra or chimeric cytokine receptor is capable of activating STAT1, STAT3, STAT5, or any combination thereof in a cell.

[0043] In another aspect, a pharmaceutical composition is provided comprising a population of modified cells of any one of the embodiments comprising the modified cells and at least one pharmaceutically acceptable carrier.

[0044] In another aspect, there is provided a system for enabling IL9 signaling in a cell, comprising: (a)(i) interleukin-9 receptor alpha (IL9Ra), or a chimeric cytokine receptor comprising an extracellular domain comprising the ligand binding domain (LBD) of a receptor selected from interleukin-13 receptor alpha type 2 (IL13Ra2), interleukin-2 receptor beta (IL2Rb), interleukin-18 receptor alpha (IL18Ra), and interleukin-18 receptor beta (IL18Rb), and an intracellular domain comprising a first transmembrane domain and the intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra); and (ii) a chimeric antigen receptor (CAR) comprising an extracellular tumor antigen-binding domain, a second transmembrane domain, and a second intracellular domain; modified immune cells that have been engineered to express the (b) a vector comprising a nucleotide sequence encoding a cytokine selected from IL9, IL13, IL2, and IL18; The system includes:

[0045] In some embodiments, the vector is an adenoviral vector.

[0046] In some embodiments, the vector is a serotype 5 adenoviral vector.

[0047] In some embodiments, the vector is an oncolytic adenoviral vector.

[0048] In some embodiments, the transmembrane domain is an IL9Ra transmembrane domain.

[0049] In some embodiments, the chimeric cytokine receptor is (a) comprising a human IL13Ra2 LBD, a human IL9Ra transmembrane domain, and a human IL9Ra intracellular signaling domain, and the cytokine is IL13; (b) comprising a human IL2Rb LBD, a human IL9Ra transmembrane domain, and a human IL9Ra intracellular signaling domain, and the cytokine is IL2; (c) comprising a human IL18Ra LBD, a human IL9Ra transmembrane domain, and a human IL9Ra intracellular signaling domain, and the cytokine is IL18; (d) comprising a human IL18Rb LBD, a human IL9Ra transmembrane domain, and a human IL9Ra intracellular signaling domain, and the cytokine is IL18; (e) comprising a mouse IL13Ra2 LBD, a mouse IL9Ra transmembrane domain, and a mouse IL9Ra intracellular signaling domain, and the cytokine is IL13; (f) comprising a mouse IL2Rb LBD, a mouse IL9Ra transmembrane domain, and a mouse IL9Ra intracellular signaling domain, and the cytokine is IL2; (g) comprising a murine IL18Ra LBD, a murine IL9Ra transmembrane domain, and a murine IL9Ra intracellular signaling domain, and the cytokine is IL18; or (h) A polypeptide comprising a mouse IL18Rb LBD, a mouse IL9Ra transmembrane domain, and a mouse IL9Ra intracellular signaling domain, wherein the cytokine is IL18.

[0050] In some embodiments, the chimeric cytokine receptor 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: 15, 17, 19, 21, 23, 51, 53, 55, 57, and 59.

[0051] In some embodiments, the first nucleotide sequence is a 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: 16, 18, 20, 22, 24, 52, 54, 56, 58, and 60.

[0052] 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 The protein is selected from the group consisting of 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, 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.

[0053] In some embodiments, the tumor antigen is selected from mesothelin, GD2, HER2, TnMuc1, CD70, PMSA, and EGFRvIII.

[0054] 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'), 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 syntheline).

[0055] In some embodiments, the tumor antigen-binding domain is a single-chain variable fragment (scFv).

[0056] 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: 79 and SEQ ID NO: 95; (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: 117; (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: 119 or SEQ ID NO: 121; (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: 123; (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: 125; (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: 129; 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: 131. is selected from.

[0057] In some embodiments, the intracellular domain of the CAR 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).

[0058] In some embodiments, the intracellular domain of the CAR comprises the 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.

[0059] In some embodiments, the intracellular domain of the CAR comprises the costimulatory domain of CD28, the costimulatory domain of 4-1BB, the intracellular signaling domain of CD3 zeta, or any combination thereof.

[0060] In some embodiments, (a) IL9 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 NO: 25 and SEQ ID NO: 61; (b) IL13 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 NO: 27 and SEQ ID NO: 63; (c) the IL13 is an IL13-TQM variant 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: 29; (d) IL2 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 NO: 31 and SEQ ID NO: 67; (e) the IL2 is an IL2 F42A variant 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: 33; or (f) IL18 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 NO: 35 and SEQ ID NO: 71.

[0061] In some embodiments, the cells are T cells, autologous cells, human cells, or any combination thereof.

[0062] In some embodiments, the IL9Ra or chimeric cytokine receptor is capable of activating STAT1, STAT3, STAT5, or any combination thereof in a cell.

[0063] In another aspect, provided is a method of treating cancer in a subject in need thereof, comprising the steps of: (a) a population of modified cells, wherein the cells are immune cells or precursor cells thereof, and the cells comprise: (i) interleukin-9 receptor alpha (IL9Ra), or a chimeric cytokine receptor comprising an extracellular domain comprising the ligand binding domain (LBD) of a receptor selected from interleukin-13 receptor alpha type 2 (IL13Ra2), interleukin-2 receptor beta (IL2Rb), interleukin-18 receptor alpha (IL18Ra), and interleukin-18 receptor beta (IL18Rb), and an intracellular domain comprising a first transmembrane domain and the intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra); and (ii) a chimeric antigen receptor (CAR) comprising an extracellular tumor antigen-binding domain, a second transmembrane domain, and a second intracellular domain; a population of the modified cells that have been engineered to express (b) a vector comprising a nucleotide sequence encoding a cytokine selected from IL9, IL13, IL2, and IL18; to said subject.

[0064] In some embodiments, the vector is an adenoviral vector.

[0065] In some embodiments, the vector is a serotype 5 adenoviral vector.

[0066] In some embodiments, the vector is an oncolytic adenoviral vector.

[0067] In some embodiments, the transmembrane domain is an IL9Ra transmembrane domain.

[0068] In some embodiments, the chimeric cytokine receptor is (a) comprising a human IL13Ra2 LBD, a human IL9Ra transmembrane domain, and a human IL9Ra intracellular signaling domain, and the cytokine is IL13; (b) comprising a human IL2Rb LBD, a human IL9Ra transmembrane domain, and a human IL9Ra intracellular signaling domain, and the cytokine is IL2; (c) comprising a human IL18Ra LBD, a human IL9Ra transmembrane domain, and a human IL9Ra intracellular signaling domain, and the cytokine is IL18; (d) comprising a human IL18Rb LBD, a human IL9Ra transmembrane domain, and a human IL9Ra intracellular signaling domain, and the cytokine is IL18; (e) comprising a mouse IL13Ra2 LBD, a mouse IL9Ra transmembrane domain, and a mouse IL9Ra intracellular signaling domain, and the cytokine is IL13; (f) comprising a mouse IL2Rb LBD, a mouse IL9Ra transmembrane domain, and a mouse IL9Ra intracellular signaling domain, and the cytokine is IL2; (g) comprising a murine IL18Ra LBD, a murine IL9Ra transmembrane domain, and a murine IL9Ra intracellular signaling domain, and the cytokine is IL18; or (h) A polypeptide comprising a mouse IL18Rb LBD, a mouse IL9Ra transmembrane domain, and a mouse IL9Ra intracellular signaling domain, wherein the cytokine is IL18.

[0069] In some embodiments, the chimeric cytokine receptor 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: 15, 17, 19, 21, 23, 51, 53, 55, 57, and 59.

[0070] In some embodiments, the first nucleotide sequence is a 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: 16, 18, 20, 22, 24, 52, 54, 56, 58, and 60.

[0071] 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 The protein is selected from the group consisting of 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, 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.

[0072] In some embodiments, the tumor antigen is selected from mesothelin, GD2, HER2, TnMuc1, CD70, PMSA, and EGFRvIII.

[0073] 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'), 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 syntheline).

[0074] In some embodiments, the tumor antigen-binding domain is a single-chain variable fragment (scFv).

[0075] 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: 79 and SEQ ID NO: 95; (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: 117; (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: 119 or SEQ ID NO: 121; (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: 123; (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: 125; (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: 129; 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: 131. is selected from.

[0076] In some embodiments, the intracellular domain of the CAR 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).

[0077] In some embodiments, the intracellular domain of the CAR comprises the 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.

[0078] In some embodiments, the intracellular domain of the CAR comprises the costimulatory domain of CD28, the costimulatory domain of 4-1BB, the intracellular signaling domain of CD3 zeta, or any combination thereof.

[0079] In some embodiments, (a) IL9 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 NO: 25 and SEQ ID NO: 61; (b) IL13 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 NO: 27 and SEQ ID NO: 63; (c) the IL13 is an IL13-TQM variant 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: 29; (d) IL2 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 NO: 31 and SEQ ID NO: 67; (e) the IL2 is an IL2 F42A variant 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: 33; or (f) IL18 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 NO: 35 and SEQ ID NO: 71.

[0080] In some embodiments, the population of cells comprises T cells, autologous cells, human cells, or any combination thereof.

[0081] In some embodiments, the population of cells is capable of activating STAT1, STAT3, STAT5, or any combination thereof.

[0082] In some embodiments, the subject is a human.

[0083] 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'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.

[0084] In another aspect, there is provided a modified cell, wherein the cell is an immune cell or a precursor cell thereof, and the cell has been engineered to express a chimeric antigen receptor (CAR) comprising a tumor antigen-binding domain, a transmembrane domain, and an intracellular domain, and further wherein expression of Cullin 5 in the cell has been reduced and / or eliminated via genetic engineering techniques or by introduction of an inhibitory RNA.

[0085] In some embodiments, the genetic engineering techniques include zinc finger nucleases, transcription activator-like effector nucleases (TALENs), or clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 systems.

[0086] In some embodiments, the inhibitory RNA comprises an siRNA or an shRNA.

[0087] 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 The protein is selected from the group consisting of 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, 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.

[0088] In some embodiments, the tumor antigen is selected from mesothelin, GD2, HER2, TnMuc1, CD70, PMSA, and EGFRvIII.

[0089] 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'), 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 syntheline).

[0090] In some embodiments, the tumor antigen-binding domain is a single-chain variable fragment (scFv).

[0091] 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: 79 and SEQ ID NO: 95; (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: 117; (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: 119 or SEQ ID NO: 121; (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: 123; (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: 125; (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: 129; 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: 131. is selected from.

[0092] In some embodiments, the intracellular domain of the CAR 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).

[0093] In some embodiments, the intracellular domain of the CAR comprises the 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.

[0094] In some embodiments, the intracellular domain of the CAR comprises the costimulatory domain of CD28, the costimulatory domain of 4-1BB, the intracellular signaling domain of CD3 zeta, or any combination thereof.

[0095] In some embodiments, the cells are T cells, autologous cells, human cells, or any combination thereof.

[0096] In some embodiments, STAT1, STAT3, STAT5, or any combination thereof, is activated in the cell.

[0097] In another aspect, a pharmaceutical composition is provided that includes a population of modified cells of the above embodiment and at least one pharmaceutically acceptable carrier.

[0098] In another aspect, there is provided a method of treating cancer in a subject in need thereof, comprising the step of administering to the subject a population of modified cells, wherein the cells are immune cells or precursor cells thereof and have been engineered to express a chimeric antigen receptor (CAR) comprising a tumor antigen-binding domain, a transmembrane domain, and an intracellular domain, and further wherein expression of Cullin 5 in the cells has been reduced and / or eliminated via genetic engineering techniques or by introduction of an inhibitory RNA.

[0099] In some embodiments, the genetic engineering techniques include zinc finger nucleases, transcription activator-like effector nucleases (TALENs), or clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 systems.

[0100] In some embodiments, the inhibitory RNA comprises an siRNA or an shRNA.

[0101] 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 The protein is selected from the group consisting of 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, 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.

[0102] In some embodiments, the tumor antigen is selected from mesothelin, GD2, HER2, TnMuc1, CD70, PMSA, and EGFRvIII.

[0103] 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'), 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 syntheline).

[0104] In some embodiments, the tumor antigen-binding domain is a single-chain variable fragment (scFv).

[0105] 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: 79 and SEQ ID NO: 95; (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: 117; (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: 119 or SEQ ID NO: 121; (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: 123; (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: 125; (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: 129; 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: 131. is selected from.

[0106] In some embodiments, the intracellular domain of the CAR 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).

[0107] In some embodiments, the intracellular domain of the CAR comprises the 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.

[0108] In some embodiments, the intracellular domain of the CAR comprises the costimulatory domain of CD28, the costimulatory domain of 4-1BB, the intracellular signaling domain of CD3 zeta, or any combination thereof.

[0109] In some embodiments, the population of cells comprises T cells, autologous cells, human cells, or any combination thereof.

[0110] In some embodiments, STAT1, STAT3, STAT5, or any combination thereof, is activated in the population of cells.

[0111] In some embodiments, the subject is a human.

[0112] 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'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.

[0113] In another aspect, a chimeric cytokine receptor is provided that comprises an extracellular domain comprising the ligand binding domain (LBD) of an inhibitory immunoreceptor or an anti-checkpoint inhibitor antigen binding domain, a transmembrane domain, and an intracellular domain comprising the intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra).

[0114] In some embodiments, the inhibitory immunoreceptor is selected from programmed cell death protein 1 (PD1), transforming growth factor beta receptor I (TGFbRI), transforming growth factor beta receptor II (TGFbRII), T cell immunoreceptor with Ig and ITIM domains (TIGIT), and T cell immunoglobulin and mucin domain containing 3 (TIM3), and further said checkpoint inhibitor is selected from cytotoxic T lymphocyte-associated protein 4 (CTLA4), programmed cell death protein 1 (PD1), and programmed death-ligand-1 (PD-L1).

[0115] In some embodiments, the transmembrane domain is an IL9Ra transmembrane domain.

[0116] In some embodiments, the chimeric cytokine receptor is (a) Human PD1 LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (b) human TGFbRI LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (c) human TGFbRII LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (d) human TIGIT LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (e) human TIM3 LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (f) mouse PD1 LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; (g) mouse TGFbRI LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; (h) mouse TGFbRII LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; (i) mouse TIGIT LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; (j) mouse TIM3 LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; (k) anti-human CTLA4 antigen-binding domain, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (l) anti-human PD1 antigen-binding domain, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (m) anti-human PD-L1 antigen-binding domain, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (n) anti-mouse CTLA4 antigen-binding domain, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; (o) an anti-mouse PD1 antigen-binding domain, a mouse IL9Ra transmembrane domain, and a mouse IL9Ra intracellular signaling domain; or (p) Anti-mouse PD-L1 antigen-binding domain, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain Includes:

[0117] In some embodiments, the chimeric cytokine receptor 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: 215, 217, 219, 221, 223, 239, 241, 243, 245, 247, and 300.

[0118] In some embodiments, the chimeric cytokine receptor 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: 216, 218, 220, 222, 224, 240, 242, 244, 246, 248, and 301.

[0119] In another aspect, there is provided an isolated nucleic acid comprising a nucleotide sequence encoding the chimeric cytokine receptor of any one of the preceding embodiments.

[0120] In another aspect, a vector comprising an isolated nucleic acid of the invention is provided.

[0121] In some embodiments, the vector is a retroviral or lentiviral vector.

[0122] In some aspects, a) a first nucleotide sequence encoding a chimeric cytokine receptor comprising an extracellular domain comprising a ligand-binding domain of an inhibitory immunoreceptor or an anti-checkpoint inhibitor antigen-binding domain, a first transmembrane domain, and an intracellular domain comprising the intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra); and b) a second nucleotide sequence encoding a chimeric antigen receptor (CAR) comprising an extracellular tumor antigen binding domain, a second transmembrane domain, and a second intracellular domain; An isolated nucleic acid is provided, comprising:

[0123] In some embodiments, the inhibitory immunoreceptor is selected from programmed death protein 1 (PD1), transforming growth factor beta receptor I (TGFbRI), transforming growth factor beta receptor II (TGFbRII), T cell immunoreceptor with Ig and ITIM domains (TIGIT), and T cell immunoglobulin and mucin domain-containing 3 (TIM3), and further said checkpoint inhibitor is selected from cytotoxic T lymphocyte-associated protein 4 (CTLA4), programmed death protein 1 (PD1), and programmed death-ligand-1 (PD-L1).

[0124] In some embodiments, the transmembrane domain is an IL9Ra transmembrane domain.

[0125] In some embodiments, the chimeric cytokine receptor is (a) Human PD1 LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (b) human TGFbRI LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (c) human TGFbRII LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (d) human TIGIT LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (e) human TIM3 LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (f) mouse PD1 LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; (g) mouse TGFbRI LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; (h) mouse TGFbRII LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; (i) mouse TIGIT LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; (j) Mouse TIM3 LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain (k) anti-human CTLA4 antigen-binding domain, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (l) anti-human PD1 antigen-binding domain, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (m) anti-human PD-L1 antigen-binding domain, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (n) anti-mouse CTLA4 antigen-binding domain, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; (o) an anti-mouse PD1 antigen-binding domain, a mouse IL9Ra transmembrane domain, and a mouse IL9Ra intracellular signaling domain; or (p) Anti-mouse PD-L1 antigen-binding domain, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain Includes:

[0126] In some embodiments, the chimeric cytokine receptor 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: 215, 217, 219, 221, 223, 239, 241, 243, 245, 247, and 300.

[0127] In some embodiments, the first nucleotide sequence is a 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: 216, 218, 220, 222, 224, 240, 242, 244, 246, 248, and 301.

[0128] 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 The protein is selected from the group consisting of 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, 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.

[0129] In some embodiments, the tumor antigen is selected from mesothelin, GD2, HER2, TnMuc1, CD70, PMSA, and EGFRvIII.

[0130] 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'), 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 syntheline).

[0131] In some embodiments, the tumor antigen-binding domain is a single-chain variable fragment (scFv).

[0132] 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: 79 and SEQ ID NO: 95; (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: 117; (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: 119 or SEQ ID NO: 121; (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: 123; (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: 125; (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: 129; 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: 131. is selected from.

[0133] In some embodiments, the intracellular domain of the CAR 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).

[0134] In some embodiments, the intracellular domain of the CAR comprises the 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.

[0135] In some embodiments, the intracellular domain of the CAR comprises the costimulatory domain of CD28, the costimulatory domain of 4-1BB, the intracellular signaling domain of CD3 zeta, or any combination thereof.

[0136] In some aspects, a vector is provided that includes the isolated nucleic acid of the above embodiment.

[0137] In some embodiments, the vector is a retroviral or lentiviral vector.

[0138] In other aspects, a modified cell is provided that comprises the chimeric cytokine receptor of the previous embodiment, the isolated nucleic acid of the previous embodiment, and / or the vector of the previous embodiment, wherein the cell is an immune cell or a precursor cell thereof.

[0139] In some embodiments, the cells are T cells, autologous cells, human cells, or any combination thereof.

[0140] In some embodiments, the cell is an immune cell or a progenitor thereof, and the cell is a) a chimeric cytokine receptor comprising an extracellular domain comprising the ligand-binding domain of an inhibitory immunoreceptor or an anti-checkpoint inhibitor antigen-binding domain, a first transmembrane domain, and an intracellular domain comprising the intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra); and b) a chimeric antigen receptor (CAR) comprising an extracellular tumor antigen-binding domain, a second transmembrane domain, and a second intracellular domain; It has been engineered to express

[0141] In some embodiments, the inhibitory immunoreceptor is selected from programmed death protein 1 (PD1), transforming growth factor beta receptor I (TGFbRI), transforming growth factor beta receptor II (TGFbRII), T cell immunoreceptor with Ig and ITIM domains (TIGIT), and T cell immunoglobulin and mucin domain-containing 3 (TIM3), and further said checkpoint inhibitor is selected from cytotoxic T lymphocyte-associated protein 4 (CTLA4), programmed death protein 1 (PD1), and programmed death-ligand-1 (PD-L1).

[0142] In some embodiments, the transmembrane domain is an IL9Ra transmembrane domain.

[0143] In some embodiments, the chimeric cytokine receptor is (a) Human PD1 LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (b) human TGFbRI LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (c) human TGFbRII LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (d) human TIGIT LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (e) human TIM3 LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (f) mouse PD1 LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; (g) mouse TGFbRI LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; (h) mouse TGFbRII LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; (i) mouse TIGIT LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; (j) mouse TIM3 LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; (k) anti-human CTLA4 antigen-binding domain, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (l) anti-human PD1 antigen-binding domain, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (m) anti-human PD-L1 antigen-binding domain, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (n) anti-mouse CTLA4 antigen-binding domain, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; (o) an anti-mouse PD1 antigen-binding domain, a mouse IL9Ra transmembrane domain, and a mouse IL9Ra intracellular signaling domain; or (p) Anti-mouse PD-L1 antigen-binding domain, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain Includes:

[0144] In some embodiments, the chimeric cytokine receptor 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: 215, 217, 219, 221, 223, 239, 241, 243, 245, 247, and 300.

[0145] In some embodiments, the first nucleotide sequence is a 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: 216, 218, 220, 222, 224, 240, 242, 244, 246, 248, and 301.

[0146] 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 The protein is selected from the group consisting of 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, 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.

[0147] In some embodiments, the tumor antigen is selected from mesothelin, GD2, HER2, TnMuc1, CD70, PMSA, and EGFRvIII.

[0148] 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'), 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 syntheline).

[0149] In some embodiments, the tumor antigen-binding domain is a single-chain variable fragment (scFv).

[0150] 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: 79 and SEQ ID NO: 95; (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: 117; (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: 119 or SEQ ID NO: 121; (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: 123; (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: 125; (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: 129; 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: 131. is selected from.

[0151] In some embodiments, the intracellular domain of the CAR 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).

[0152] In some embodiments, the intracellular domain of the CAR comprises the 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.

[0153] In some embodiments, the intracellular domain of the CAR comprises the costimulatory domain of CD28, the costimulatory domain of 4-1BB, the intracellular signaling domain of CD3 zeta, or any combination thereof.

[0154] In some embodiments, the cells are T cells, autologous cells, human cells, or any combination thereof.

[0155] In some embodiments, the chimeric cytokine receptor is capable of activating STAT1, STAT3, STAT5, or any combination thereof in a cell.

[0156] In another aspect, a pharmaceutical composition is provided that includes a population of modified cells of the above embodiment and at least one pharmaceutically acceptable carrier.

[0157] 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: a) a chimeric cytokine receptor comprising an extracellular domain comprising the ligand-binding domain of an inhibitory immunoreceptor or an anti-checkpoint inhibitor antigen-binding domain, a first transmembrane domain, and an intracellular domain comprising the intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra); and b) a chimeric antigen receptor (CAR) comprising an extracellular tumor antigen-binding domain, a second transmembrane domain, and a second intracellular domain; The method is provided, wherein the vector is engineered to express

[0158] In some embodiments, the inhibitory immunoreceptor is selected from programmed death protein 1 (PD1), transforming growth factor beta receptor I (TGFbRI), transforming growth factor beta receptor II (TGFbRII), T cell immunoreceptor with Ig and ITIM domains (TIGIT), and T cell immunoglobulin and mucin domain-containing 3 (TIM3), and further said checkpoint inhibitor is selected from cytotoxic T lymphocyte-associated protein 4 (CTLA4), programmed death protein 1 (PD1), and programmed death-ligand-1 (PD-L1).

[0159] In some embodiments, the transmembrane domain is an IL9Ra transmembrane domain.

[0160] In some embodiments, the chimeric cytokine receptor is (a) Human PD1 LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (b) human TGFbRI LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (c) human TGFbRII LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (d) human TIGIT LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (e) human TIM3 LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (f) mouse PD1 LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; (g) mouse TGFbRI LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; (h) mouse TGFbRII LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; (i) mouse TIGIT LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; (j) mouse TIM3 LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; (k) anti-human CTLA4 antigen-binding domain, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (l) anti-human PD1 antigen-binding domain, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (m) anti-human PD-L1 antigen-binding domain, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (n) anti-mouse CTLA4 antigen-binding domain, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; (o) an anti-mouse PD1 antigen-binding domain, a mouse IL9Ra transmembrane domain, and a mouse IL9Ra intracellular signaling domain; or (p) Anti-mouse PD-L1 antigen-binding domain, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain Includes:

[0161] In some embodiments, the chimeric cytokine receptor 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: 215, 217, 219, 221, 223, 239, 241, 243, 245, 247, and 300.

[0162] In some embodiments, the first nucleotide sequence is a 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: 216, 218, 220, 222, 224, 240, 242, 244, 246, 248, and 301.

[0163] 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 The protein is selected from the group consisting of 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, 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.

[0164] In some embodiments, the tumor antigen is selected from mesothelin, GD2, HER2, TnMuc1, CD70, PMSA, and EGFRvIII.

[0165] 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'), 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 syntheline).

[0166] In some embodiments, the tumor antigen-binding domain is a single-chain variable fragment (scFv).

[0167] 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: 79 and SEQ ID NO: 95; (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: 117; (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: 119 or SEQ ID NO: 121; (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: 123; (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: 125; (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: 129; 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: 131. is selected from.

[0168] In some embodiments, the intracellular domain of the CAR 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).

[0169] In some embodiments, the intracellular domain of the CAR comprises the 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.

[0170] In some embodiments, the intracellular domain of the CAR comprises the costimulatory domain of CD28, the costimulatory domain of 4-1BB, the intracellular signaling domain of CD3 zeta, or any combination thereof.

[0171] In some embodiments, the population of cells comprises T cells, autologous cells, human cells, or any combination thereof.

[0172] In some embodiments, the chimeric cytokine receptor is capable of activating STAT1, STAT3, STAT5, or any combination thereof in a cell.

[0173] In some embodiments, the subject is a human.

[0174] 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'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. [Brief explanation of the drawings]

[0175] These 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.

[0176] [Figure 1] Figures 1A-1D provide schematic diagrams illustrating various embodiments of the cytokine receptors of the present invention. Figure 1A is a schematic diagram illustrating a wild-type IL9Ra cytokine receptor co-expressed with an exemplary CAR. Figure 1B is a schematic diagram illustrating an IL13Ra2-IL9Ra chimeric cytokine receptor co-expressed with an exemplary CAR. Figure 1C is a schematic diagram illustrating an IL2Rb-IL9Ra chimeric cytokine receptor co-expressed with an exemplary CAR. Figure 1D is a schematic diagram illustrating an IL18R-IL9Ra chimeric cytokine receptor co-expressed with an exemplary CAR. [Figure 2]Figures 2A-2F provide schematic diagrams illustrating various exemplary expression constructs for cytokine receptors, CARs, and ligands disclosed herein. Figure 2A is a schematic diagram of two lentiviral constructs for the expression of human IL9Ra and human CAR. The top and bottom constructs show that the nucleotide sequence encoding the cytokine receptor and the CAR are linked by a nucleotide sequence encoding either a 2A self-cleaving peptide (2A) or an internal ribosome entry site (IRES), respectively. Figure 2B is a schematic diagram of an Ad5 adenoviral construct for expressing human IL-9 under the control of a CMV promoter. Figure 2C is a schematic diagram of an Ad5 adenoviral construct for expressing murine IL-9 under the control of a CMV promoter. Figure 2D is a schematic diagram of an Ad5 adenoviral construct for expressing the IL-13 TQM mutant under the control of a CMV promoter. Figure 2E is a schematic diagram of an Ad5 adenoviral construct for expressing the IL-2 F42A mutant under the control of a CMV promoter. FIG. 2F is a schematic diagram of the Ad5 adenoviral construct for expressing IL-18 under the control of the CMV promoter. [Figure 3A]Figures 3A-3D provide data on the wild-type murine IL9Ra cytokine receptor coexpressed with an exemplary CAR on transduced murine CD3+ T cells. Figure 3A provides data showing the coexpression of murine IL9Ra and murine CAR on transduced murine CD3+ T cells compared to untransduced (UTD) cells. Figure 3B 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 3C provides global gene expression profile data in transduced murine 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 using the Nanostring nCounter Mouse Immunology Panel (562 genes) and plotted using nSolver 4.0 software. Figure 3D provides a graph showing in vitro expression of mIL-9 via the adenoviral vector construct Ad-mIL-9 shown in Figure 2C. The mouse pancreatic cancer cell line PDA7940b (10,000 cells / well) was infected with Ad-mIL-9 at 100 viral particles / cell, and cell culture supernatants were analyzed for mIL-9 by ELISA at the indicated time points. [Figure 3B-1] See legend to Figure 3A. [Figure 3B-2] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 3D] See legend to Figure 3A. [Figure 4A]Figures 4A-4B provide data on the human IL13Ra2-IL9Ra chimeric cytokine receptor. Figure 4A provides flow cytometry data showing the expression of the human IL13Ra2-IL9Ra chimeric cytokine receptor on lentivirally transduced human T cells compared to untransduced cells (UTD). Figure 4B provides Western blots from four donors showing pSTAT1 / pSTAT3 / pSTAT5 expression in hIL13Ra2-IL9Ra-expressing T cells. 10 x 106 lentivirally transduced T cells were starved overnight in RPMI containing 0.1% FBS and either left untreated or treated with hIL-13 (100 ng / mL) for 30 minutes. Western blot detection of phosphorylated STAT1, STAT3, and STAT5 using GAPDH as a loading control. [Figure 4B] See legend to Figure 4A. [Figure 5] Figure 5 provides flow cytometry data showing expression of the chimeric hIL2Rb-IL9Ra receptor on lentivirally transduced human T cells. UTD, untransduced. [Figure 6-1] Figures 6A-6D provide schematic diagrams illustrating chimeric IL-9R cytokine receptors of the present invention. Figure 6A is a schematic diagram illustrating a PD1-IL9Ra chimeric cytokine receptor co-expressed with an exemplary CAR. Figure 6B is a schematic diagram illustrating a TGFbRII-IL9Ra chimeric cytokine receptor co-expressed with an exemplary CAR. Figure 6C is a schematic diagram illustrating a TIGIT-IL9Ra chimeric cytokine receptor co-expressed with an exemplary CAR. Figure 6D is a schematic diagram illustrating a TIM3-IL9Ra chimeric cytokine receptor co-expressed with an exemplary CAR. [Figure 6-2]Figure 6E is a schematic diagram illustrating a chimeric cytokine receptor comprising an anti-CTLA4(H+L) antigen-binding domain and an IL9Ra ICD co-expressed with an exemplary CAR. Figure 6F is a schematic diagram illustrating a chimeric cytokine receptor comprising an anti-CTLA4 scFv antigen-binding domain and an IL9Ra ICD co-expressed with an exemplary CAR. [Figure 7] Figure 7 provides flow cytometry data showing co-expression of the murine PD1-IL9Ra chimeric cytokine receptor and the murine anti-mesothelin CAR containing the A03 scFv on transduced murine T cells compared to untransduced (UTD) cells. [Figure 8] FIG. 8 provides flow cytometry data showing expression of the murine TGFbRII-IL9Ra chimeric cytokine receptor on transduced murine T cells compared to untransduced (UTD) cells. [Figure 9A] Figure 9A provides a schematic diagram 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 9B] Figure 9B 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 10] FIG. 10 provides flow cytometry data illustrating the finding that IL9Ra signaling in T cells leads to a Tscm phenotype. [Figure 11] Figure 11 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 12-1]Figure 12 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 12-2] See the description of Figure 12-1. [Figure 12-3] See the description of Figure 12-1. [Figure 13] FIG. 13 shows the finding that IL9Ra signaling in mouse T cells enhances tumor cell killing. [Figure 14A] Figures 14A-14C illustrate the finding that IL9a signaling induces similar gene expression profiles in T cells engineered to express an anti-mesoCAR and IL9Ra or an anti-mesoCAR and an orthogonal chimeric cytokine receptor (ortho-IL2Rβ-IL9Ra chimeric cytokine receptor (o9R)). Figure 14A shows the top 20 up- and down-regulated genes for T cells expressing an anti-mesoCAR and IL9Ra preincubated with either IL9 or IL2. Figure 14B shows the top 20 up- and down-regulated genes for T cells expressing an anti-mesoCAR and o9R preincubated with ortho-IL2 or IL-2. Figure 14C shows the common up- and down-regulated genes. [Figure 14B] See the legend to Figure 14A. [Figure 14C] See the legend to Figure 14A. [Figure 15A-1]Figures 15A-15F show gene set mutation analysis (GSVA) ​​and gene set enrichment analysis (GSEA) data for T cells expressing an anti-meso CAR and IL9Ra preincubated with either IL9 or IL2, and for T cells expressing an anti-meso CAR and an ortho-IL2Rβ-IL9Ra chimeric cytokine receptor (o9R) preincubated with ortho-IL2 or IL-2. The data in Figure 15A compares pathways significantly enriched in CAR T cells stimulated with IL9 versus IL2. Figure 15B provides a table of enriched pathways along with GSEA statistics. Figure 15C provides an enrichment plot and analysis of the interferon gamma response in T cells expressing an anti-meso CAR and IL9Ra preincubated with IL9 compared to IL2. Figure 15D provides an enrichment plot and analysis of the interferon alpha response in T cells expressing an anti-meso CAR and IL9Ra preincubated with IL9 compared to IL2. Figure 15E provides enrichment plots and analysis of 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 15F provides enrichment plots and analysis of 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 15A-2] See the description of Figure 15A-1. [Figure 15B] See the description of Figure 15A-1. [Figure 15C-1] See the description of Figure 15A-1. [Figure 15C-2] See the description of Figure 15A-1. [Figure 15D] See the description of Figure 15A-1. [Figure 15E-1]See the description of Figure 15A-1. [Figure 15E-2] See the description of Figure 15A-1. [Figure 15F] See the description of Figure 15A-1. [Figure 16A-1] Figures 16A-16D relate to the establishment of an in vivo syngeneic mouse model of PDA. Figure 16A shows a schematic of the protocol, a chart of tumor volume, and mesothelin expression data for PDA7940b cells. Figure 16B shows the experimental design for dose-finding of an adenoviral vector expressing mIL9 (Ad-mIL9) in a syngeneic PDA mouse model. Figure 16C provides transduction efficiency data for Ad-mIL9. Figure 16D provides dose-finding tumor growth data for the indicated conditions in a syngeneic PDA mouse model. [Figure 16A-2] See the description of Figure 16A-1. [Figure 16B-1] See the description of Figure 16A-1. [Figure 16B-2] See the description of Figure 16A-1. [Figure 16C-1] See the description of Figure 16A-1. [Figure 16C-2] See the description of Figure 16A-1. [Figure 16D-1] See the description of Figure 16A-1. [Figure 16D-2] See the description of Figure 16A-1. DETAILED DESCRIPTION OF THE INVENTION

[0177] Detailed Description The present disclosure provides several approaches for enabling IL-9 signaling in immune cells expressing chimeric antigen receptors (CARs), thereby enhancing the efficacy of CAR immune cell therapy. In one aspect, an IL9Ra receptor or chimeric cytokine receptor containing the IL9Ra intracellular signaling domain (ICD) and its use are provided in conjunction with adenoviral delivery of cytokine ligands at tumor sites. This approach improves chimeric antigen receptor (CAR) cell immunotherapy for treating cancer by (1) enabling IL-9 signaling in immune cells to improve effector function in situ, (2) selectively expressing cytokine ligands in tumor cells, thereby achieving higher intratumoral cytokine concentrations compared to systemic administration, and (3) promoting the spread of tumor antigens via viral oncolysis.

[0178] An alternative approach to enable IL-9 signaling in CAR-expressing immune cells provides CAR-expressing immune cells in which expression of Cullin 5 has been reduced and / or eliminated in the cells via genetic engineering techniques or by the introduction of inhibitory RNA.

[0179] The present disclosure also provides chimeric cytokine receptors and uses thereof to improve chimeric antigen receptor (CAR) cell immunotherapy for treating cancer by (1) utilizing molecules naturally occurring in tumors (i.e., ligands and tumor antigens) and checkpoint inhibitors in T cells to convert immunosuppressive signals to immunostimulatory signals in immune cells (e.g., T cells), (2) altering the phenotype of immune cells expressing the chimeric cytokine receptor and CAR upon binding of the ligand and / or checkpoint inhibitor at the tumor site, and (3) enabling IL-9 signaling in immune cells expressing the chimeric cytokine receptor and CAR to improve effector function in situ and / or downregulate immune cell exhaustion.

[0180] By repurposing IL-9R signaling in CAR T cells, these cells acquire new functions through the concomitant activation of STAT1, STAT3, and STAT5. Such T cells take on stem cell memory (Tscm) characteristics with improved trafficking and effector function, thereby resulting in improved antitumor activity against difficult-to-treat solid tumors.

[0181] Because orthogonal chimeric cytokine receptors containing the IL-9R ICD resulted in potent activation (e.g., phosphorylation) of STAT1, STAT3, and STAT5 in T cells expressing the orthogonal chimeric cytokine receptor, it is contemplated herein that receptors containing the IL-9 intracellular domain (ICD) of the present disclosure are distinct from receptors containing the IL-4R ICD, IL-7R ICD, or IL-21R ICD. See Kalbasi, et al. Nature, 607: 360-365 (2022). Indeed, CAR T cells expressing the oIL2Rβ-IL9Rα chimeric cytokine receptor took on characteristics of stem cell memory T cells and effector T cells and, for example, showed superior antitumor efficacy in two refractory syngeneic mouse solid tumor models of melanoma and pancreatic cancer when compared with cells expressing orthogonal receptors containing the IL-2R ICD. Furthermore, the antitumor effect of receptors containing the IL-9R ICD was effective in the absence of lymphodepletion conditioning. Additionally, CAR T cells expressing orthogonal chimeric cytokine receptors containing the IL-9R ICD proliferated less than, for example, cells expressing the IL-2R ICD.

[0182] Thus, the present disclosure provides novel CAR-expressing cells (e.g., CAR T cells) that express IL9Ra or a chimeric cytokine receptor comprising IL9Ra ICD, and a novel process for engineering CAR T cells with a stem-like phenotype that does not require the administration of orthogonal cytokines, or in some embodiments, does not require the administration of any exogenous cytokines. It is contemplated herein that the cells of the present invention exhibit superior anti-tumor activity. A stem-like phenotype in T cells is demonstrated herein by expressing wild-type IL9Ra together with a 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).

[0183] The novelty of the CAR-expressing cells co-expressing a chimeric cytokine receptor and IL9Ra or a chimeric cytokine receptor disclosed herein is highlighted by the fact that IL-9-naive T cells are insensitive to IL-9 and that 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 an important 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 effects of IL-9R signaling on T cells have 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., potent activation), and the role of IL-9 in stem cell memory T (T) cells have been well documented. SCM ) The acquisition of cellular characteristics was surprising and unexpected.

[0184] Thus, in one aspect, the present invention provides a chimeric cytokine receptor comprising: an extracellular domain comprising the ligand binding domain (LBD) of a receptor selected from interleukin-13 receptor alpha type 2 (IL13Ra2), interleukin-2 receptor beta (IL2Rb), interleukin-18 receptor alpha (IL18Ra), and interleukin-18 receptor beta (IL18Rb); a transmembrane domain; and an intracellular domain comprising the intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra).

[0185] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) a first nucleotide sequence encoding a chimeric cytokine receptor comprising: (i) an extracellular domain comprising the ligand binding domain (LBD) of a receptor selected from interleukin-13 receptor alpha type 2 (IL13Ra2), interleukin-2 receptor beta (IL2Rb), interleukin-18 receptor alpha (IL18Ra), and interleukin-18 receptor beta (IL18Rb), (ii) a first transmembrane domain, and (iii) an intracellular domain comprising the intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra); and b) a second nucleotide sequence encoding a chimeric antigen receptor (CAR) comprising an extracellular tumor antigen binding domain, a second transmembrane domain, and a second intracellular domain; The present invention provides an isolated nucleic acid comprising:

[0186] In another aspect, the present invention provides a modified cell, wherein the cell is an immune cell or a precursor cell thereof, and the cell comprises: a) interleukin-9 receptor alpha (IL9Ra), or A chimeric cytokine receptor comprising: (i) an extracellular domain comprising the ligand binding domain (LBD) of a receptor selected from interleukin-13 receptor alpha type 2 (IL13Ra2), interleukin-2 receptor beta (IL2Rb), interleukin-18 receptor alpha (IL18Ra), and interleukin-18 receptor beta (IL18Rb), (ii) a first transmembrane domain, and (iii) an intracellular domain comprising the intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra); and b) a chimeric antigen receptor (CAR) comprising an extracellular tumor antigen-binding domain, a second transmembrane domain, and a second intracellular domain; The modified cell is engineered to express the

[0187] In another aspect, the present invention provides a system for enabling IL9 signaling in a cell, comprising: (a)(i) interleukin-9 receptor alpha (IL9Ra), or a chimeric cytokine receptor comprising an extracellular domain comprising the ligand binding domain (LBD) of a receptor selected from interleukin-13 receptor alpha type 2 (IL13Ra2), interleukin-2 receptor beta (IL2Rb), interleukin-18 receptor alpha (IL18Ra), and interleukin-18 receptor beta (IL18Rb), and an intracellular domain comprising a first transmembrane domain and the intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra); and (ii) a chimeric antigen receptor (CAR) comprising an extracellular tumor antigen-binding domain, a second transmembrane domain, and a second intracellular domain; modified immune cells that have been engineered to express the (b) a vector comprising a nucleotide sequence encoding a cytokine selected from IL9, IL13, IL2, and IL18; The system includes:

[0188] In another aspect, the present invention provides a method of treating cancer in a subject in need thereof, comprising the steps of: (a) a population of modified cells, wherein the cells are immune cells or precursor cells thereof, and the cells comprise: (i) interleukin-9 receptor alpha (IL9Ra), or a chimeric cytokine receptor comprising an extracellular domain comprising the ligand binding domain (LBD) of a receptor selected from interleukin-13 receptor alpha type 2 (IL13Ra2), interleukin-2 receptor beta (IL2Rb), interleukin-18 receptor alpha (IL18Ra), and interleukin-18 receptor beta (IL18Rb), and an intracellular domain comprising a first transmembrane domain and the intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra); and (ii) a chimeric antigen receptor (CAR) comprising an extracellular tumor antigen-binding domain, a second transmembrane domain, and a second intracellular domain; a population of the modified cells that have been engineered to express (b) a vector comprising a nucleotide sequence encoding a cytokine selected from IL9, IL13, IL2, and IL18; to said subject.

[0189] In another aspect, the present invention provides a modified cell, wherein the cell is an immune cell or a precursor cell thereof, and the cell has been engineered to express a chimeric antigen receptor (CAR) comprising a tumor antigen-binding domain, a transmembrane domain, and an intracellular domain, and further wherein expression of Cullin 5 in the cell has been reduced and / or eliminated via genetic engineering techniques or by introduction of an inhibitory RNA.

[0190] In another aspect, the present invention provides a method of treating cancer in a subject in need thereof, comprising the step of administering to the subject a population of modified cells, wherein the cells are immune cells or precursor cells thereof and have been engineered to express a chimeric antigen receptor (CAR) comprising a tumor antigen-binding domain, a transmembrane domain, and an intracellular domain, and further wherein expression of Cullin 5 in the cells has been reduced and / or eliminated via genetic engineering techniques or by introduction of inhibitory RNA.

[0191] In one aspect, the present invention provides a chimeric cytokine receptor comprising an extracellular domain comprising the ligand-binding domain of an inhibitory immunoreceptor or an anti-checkpoint inhibitor antigen-binding domain, a transmembrane domain, and an intracellular domain comprising the intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra). In various embodiments, the inhibitory immunoreceptor is selected from programmed cell death protein 1 (PD1), transforming growth factor beta receptor I (TGFbRI), transforming growth factor beta receptor II (TGFbRII), T cell immunoreceptor with Ig and ITIM domains (TIGIT), and T cell immunoglobulin and mucin domain-containing 3 (TIM3). In various embodiments, the checkpoint inhibitor is selected from cytotoxic T lymphocyte-associated protein 4 (CTLA4), programmed cell death protein 1 (PD1), and programmed death-ligand-1 (PD-L1).

[0192] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) a first nucleotide sequence encoding a chimeric cytokine receptor comprising an extracellular domain comprising a ligand-binding domain of an inhibitory immunoreceptor or an anti-checkpoint inhibitor antigen-binding domain, a first transmembrane domain, and an intracellular domain comprising the intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra); and b) a second nucleotide sequence encoding a chimeric antigen receptor (CAR) comprising an extracellular tumor antigen-binding domain, a second transmembrane domain, and a second intracellular domain. In various embodiments, the inhibitory immunoreceptor is selected from programmed cell death protein 1 (PD1), transforming growth factor beta receptor I (TGFbRI), transforming growth factor beta receptor II (TGFbRII), T cell immunoreceptor with Ig and ITIM domains (TIGIT), and T cell immunoglobulin and mucin domain-containing 3 (TIM3). In various embodiments, the checkpoint inhibitor is selected from cytotoxic T lymphocyte-associated protein 4 (CTLA4), programmed cell death protein 1 (PD1), and programmed death-ligand-1 (PD-L1).

[0193] In another aspect, the present invention provides modified cells, which are immune cells or precursor cells thereof, engineered to express: (a) a chimeric cytokine receptor comprising an extracellular domain comprising the ligand-binding domain of an inhibitory immunoreceptor or an anti-checkpoint inhibitor antigen-binding domain, a first transmembrane domain, and an intracellular domain comprising the intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra); and (b) a chimeric antigen receptor (CAR) comprising an extracellular tumor antigen-binding domain, a second transmembrane domain, and a second intracellular domain. In various embodiments, the inhibitory immunoreceptor is selected from programmed cell death protein 1 (PD1), transforming growth factor beta receptor I (TGFbRI), transforming growth factor beta receptor II (TGFbRII), T cell immunoreceptor with Ig and ITIM domains (TIGIT), and T cell immunoglobulin and mucin domain-containing 3 (TIM3). In various embodiments, the checkpoint inhibitor is selected from cytotoxic T-lymphocyte-associated protein 4 (CTLA4), programmed cell death protein 1 (PD1), and programmed death-ligand-1 (PD-L1).

[0194] In another aspect, the present invention provides 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 have been engineered to express: (a) a chimeric cytokine receptor comprising an extracellular domain comprising the ligand-binding domain of an inhibitory immunoreceptor or an anti-checkpoint inhibitor antigen-binding domain, a first transmembrane domain, and an intracellular domain comprising the intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra); and (b) a chimeric antigen receptor (CAR) comprising an extracellular tumor antigen-binding domain, a second transmembrane domain, and a second intracellular domain. In various embodiments, the inhibitory immunoreceptor is selected from programmed cell death protein 1 (PD1), transforming growth factor beta receptor I (TGFbRI), transforming growth factor beta receptor II (TGFbRII), T cell immunoreceptor with Ig and ITIM domains (TIGIT), and T cell immunoglobulin and mucin domain-containing 3 (TIM3). In various embodiments, the checkpoint inhibitor is selected from cytotoxic T-lymphocyte-associated protein 4 (CTLA4), programmed cell death protein 1 (PD1), and programmed death-ligand-1 (PD-L1).

[0195] In other aspects, related compositions (eg, pharmaceutical compositions) and kits are provided herein.

[0196] 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.

[0197] Furthermore, unless otherwise indicated, the experiments described herein use conventional molecular biological, cell biological and immunological techniques within the skill of those skilled in the art.Such techniques are well known to those skilled in the art and are fully described in literature.See, for example, Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2008), including all supplements; 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).

[0198] 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 herein by reference in their entireties.

[0199] A. definition Unless otherwise defined, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In the case of potential ambiguity, definitions provided herein take precedence over 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 stated. The use of the term "including" and other forms such as "includes" and "included" is not limiting.

[0200] In general, the nomenclatures used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional methods known in the art and as described in the 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 nomenclatures used in connection with analytical chemistry, synthetic organic chemistry, and medicinal chemistry and drug discovery chemistry described herein, as well as the experimental procedures 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 patient treatment.

[0201] In order that the present disclosure may be more readily understood, selected terms are defined below.

[0202] 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.

[0203] As used herein, "about" when referring to a measurable value such as amount, duration, and the like, is intended to encompass a variation of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value, such variations being suitable for practicing the disclosed methods.

[0204] 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.

[0205] As used herein, "alleviating" a disease means reducing the severity of one or more symptoms of the disease.

[0206] As used herein, the term "antigen" is defined as a molecule that provokes an immune response. This immune response may include antibody production, or activation of specific immunocompetent cells, or both. Those skilled in the art will understand that virtually any macromolecule can function as an antigen, including any protein or peptide.

[0207] Furthermore, antigens can be derived from recombinant or genomic DNA. Thus, those skilled in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response encodes an "antigen," as that term is used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of multiple genes, and that these nucleotide sequences can be arranged in various combinations to elicit a desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be synthetically produced or derived from a biological sample. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0208] As used herein, the term "autologous" is intended to refer to any material that originates from an individual and is later reintroduced into the same individual.

[0209] A "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors.

[0210] 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-regulation or down-regulation of key molecules.

[0211] "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, an animal's "disorder" 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 did not exist.If left untreated, the disorder does not necessarily cause further deterioration of the animal's health condition.

[0212] As used herein, the term "downregulation" refers to the reduction or elimination of gene expression of one or more genes.

[0213] "Effective amount" or "therapeutically effective amount," as used interchangeably herein, refer to an amount of a compound, formulation, material, or composition described herein that is effective to achieve a particular biological result or provide 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. Immune responses can be readily assessed by a number of art-recognized methods. Those skilled in the art will understand that the amount of a 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.

[0214] "Encoding" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the biological properties resulting therefrom. Thus, a gene encodes a protein when transcription and translation of the mRNA corresponding to that gene produces the 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 encode the protein or other product of that gene or cDNA.

[0215] As used herein, "endogenous" refers to any substance that is produced from or within an organism, cell, tissue, or system.

[0216] As used herein, the term "epitope" 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, epitopes are approximately 10 amino acids and / or sugars in size. Preferably, epitopes are approximately 4-18 amino acids, more preferably approximately 5-16 amino acids, even more preferably approximately 6-14 amino acids, more preferably approximately 7-12 amino acids, and most preferably approximately 8-10 amino acids. Those skilled in the art will understand that, in general, the overall three-dimensional structure of the molecule, rather than the specific linear sequence, 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.

[0217] As used herein, the term "exogenous" refers to any substance that is introduced into or produced outside of an organism, cell, tissue, or system.

[0218] As used herein, the term "expand" 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. As used herein, the term "ex vivo" refers to cells removed from a living organism (e.g., a human) and grown outside of the organism (e.g., in a culture dish, test tube, or bioreactor).

[0219] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0220] "Expression vector" refers to a vector containing a recombinant polynucleotide comprising 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 supplied 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) incorporating recombinant polynucleotides.

[0221] As used herein, "identity" refers to the identity of the subunit sequence between two polymer molecules, particularly between two amino acid molecules, for example, between two polypeptide molecules. If two amino acid sequences have the same residue at the same position, for example, if each position of two polypeptide molecules is occupied by arginine, they are identical at that position. The identity or degree to which two amino acid sequences have the same residue at the same position in 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; for example, 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 identical or identical, the two amino acid sequences are 90% identical.

[0222] 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.

[0223] The term "immunosuppressive" is used herein to refer to reducing the overall immune response.

[0224] "Isolated" means changed or removed from the natural state. For example, a nucleic acid or peptide naturally occurring in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or it can exist in a non-native environment, such as a host cell.

[0225] " Lentivirus " as used herein refers to a genus of Retroviridae family.Lentivirus is unique among retroviruses in that it can infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of host cells, so they are one of the most efficient methods of gene delivery vectors.HIV, SIV and FIV are all examples of lentivirus.Vector derived from lentivirus provides a means to achieve significant levels of gene transfer in vivo.

[0226] The term "modified" as used herein refers to an altered state or structure of a molecule or cell of the present invention. Molecules can be modified in many ways, including chemical, structural, and functional ways. Cells can be modified through the introduction of nucleic acids.

[0227] 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.

[0228] 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.

[0229] 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 (i.e., A, U, C, G) in which "U" replaces "T".

[0230] 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 that encodes the protein may, in some versions, contain introns.

[0231] "Parenteral" administration of the immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection, or infusion techniques.

[0232] The term "polynucleotide" as used herein is defined as a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Therefore, as used herein, "nucleic acid" and "polynucleotide" are interchangeable. Those skilled in the art have the general knowledge that 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 by synthetic means.

[0233] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, with no limit on the maximum number of amino acids that a protein or peptide sequence can contain. A polypeptide includes any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains, commonly referred to in the art as peptides, oligopeptides, and oligomers, and the many types of longer chains commonly referred to in the art as proteins. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins, among others. A polypeptide includes natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0234] The term "specifically binds" as used herein with respect to antibodies refers to an antibody that recognizes a specific 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 antibody's classification as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross-reactivity does not in itself change the antibody's classification as specific. In some cases, the terms "specific binding" or "specifically binds" can 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 specific structure (e.g., an antigenic determinant or epitope) in the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than proteins generally. If an antibody is specific for epitope "A," in a reaction involving labeled "A" and the antibody, the presence of a molecule containing epitope A (or free, unlabeled A) reduces the amount of labeled A that binds to the antibody.

[0235] The term "stimulation" refers to a primary response induced by the binding of a stimulatory molecule (e.g., a TCR / CD3 complex) to 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 specific molecules, such as downregulation of TGF-beta and / or rearrangement of cytoskeletal structure.

[0236] "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.

[0237] 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.), is capable of specifically binding to a cognate binding partner (referred to herein 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, among others, peptide-loaded MHC class I molecules, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies.

[0238] The term "subject" is intended to include a living organism (e.g., a mammal) in which an immune response can be elicited. As used herein, a "subject" or "patient" can be a human or non-human mammal. Non-human mammals include, for example, livestock 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.

[0239] "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.

[0240] 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 antigen presentation. TCRs are responsible for recognizing antigens bound to major histocompatibility complex molecules. TCRs are composed of a heterodimer of an alpha (α) chain and a beta (β) chain, 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 containing 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.

[0241] The term "therapeutic" as used herein means treatment and / or prophylaxis. A therapeutic effect is achieved by suppressing, ameliorating, or eradicating a disease state.

[0242] As used herein, the terms "transfected" or "transformed" or "transduced" 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.

[0243] "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.

[0244] A "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. This term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds and liposomes. Examples of viral vectors include, but are not limited to, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, etc.

[0245] Ranges: Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0246] B. Chimeric cytokine receptors The present invention provides a chimeric cytokine receptor comprising an extracellular domain comprising the ligand binding domain (LBD) of a receptor selected from interleukin-13 receptor alpha type 2 (IL13Ra2), interleukin-2 receptor beta (IL2Rb), interleukin-18 receptor alpha (IL18Ra), and interleukin-18 receptor beta (IL18Rb); a transmembrane domain; and an intracellular domain comprising the intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra). In various embodiments, the transmembrane domain is derived from IL9Ra.

[0247] In some embodiments, the chimeric cytokine receptor comprises a human IL13Ra2 LBD, a human IL9Ra transmembrane domain, and a human IL9Ra intracellular signaling domain.

[0248] In some embodiments, the chimeric cytokine receptor comprises a human IL2Rb LBD, a human IL9Ra transmembrane domain, and a human IL9Ra intracellular signaling domain.

[0249] In some embodiments, the chimeric cytokine receptor comprises a human IL18Ra LBD, a human IL9Ra transmembrane domain, and a human IL9Ra intracellular signaling domain.

[0250] In some embodiments, the chimeric cytokine receptor comprises a human IL18Rb LBD, a human IL9Ra transmembrane domain, and a human IL9Ra intracellular signaling domain.

[0251] In some embodiments, the chimeric cytokine receptor comprises a murine IL9Ra LBD, a murine IL9Ra transmembrane domain, and a murine IL9Ra intracellular signaling domain.

[0252] In some embodiments, the chimeric cytokine receptor comprises a murine IL13Ra2 LBD, a murine IL9Ra transmembrane domain, and a murine IL9Ra intracellular signaling domain.

[0253] In some embodiments, the chimeric cytokine receptor comprises a mouse IL2Rb LBD, a mouse IL9Ra transmembrane domain, and a mouse IL9Ra intracellular signaling domain.

[0254] In some embodiments, the chimeric cytokine receptor comprises a murine IL18Ra LBD, a murine IL9Ra transmembrane domain, and a murine IL9Ra intracellular signaling domain.

[0255] In some embodiments, the chimeric cytokine receptor comprises a mouse IL18Rb LBD, a mouse IL9Ra transmembrane domain, and a mouse IL9Ra intracellular signaling domain.

[0256] The chimeric cytokine receptor of the present invention may also include a leader sequence, a hinge domain, and / or one or more spacer or linker sequences described herein that serve to link one domain of the chimeric cytokine receptor to the next. The chimeric cytokine receptor may also include a tag (e.g., a chemical or biological tag) or be fused to another protein (e.g., a fluorescent protein such as GFP). Such a tag may be, for example, at the N-terminus or C-terminus, or may be incorporated between the two domains of the chimeric cytokine receptor. Techniques for post-transcriptional site-selective tagging of polypeptides are also well known in the art. Those skilled in the art will be able to select such sequences and tags that are appropriate for inclusion in the chimeric cytokine receptor of the present invention.

[0257] The amino acid and nucleotide sequences for certain embodiments of the chimeric cytokine receptors and their domains are set forth below. TIFF2024534471000001.tif71149TIFF2024534471000002.tif227149TIFF2024534471000003.tif215149TIFF2024534471000004.tif220149TIFF2024534471000005.tif227149TIFF2024534471000006.tif227149TIFF2024534471000007.tif228149TIFF2024534471000008.tif227149TIFF2024534471000009.tif220149TIFF2024534471000010.tif227149TIFF2024534471000011.tif226149TIFF2024534471000012.tif227149TIFF2024534471000013.tif228149TIFF2024534471000014.tif228149TIFF2024534471000015.tif228149TIFF2024534471000016.tif220149TIFF2024534471000017.tif222149TIFF2024534471000018.tif227149TIFF2024534471000019.tif227149TIFF2024534471000020.tif227149TIFF2024534471000021.tif227149TIFF2024534471000022.tif227149TIFF2024534471000023.tif227149TIFF2024534471000024.tif228149TIFF2024534471000025.tif227149TIFF2024534471000026.tif220149TIFF2024534471000027.tif226149TIFF2024534471000028.tif221149TIFF2024534471000029.tif228149TIFF2024534471000030.tif118149

[0258] In some embodiments, the immune cells are engineered to express IL-9Ra and a CAR. In some embodiments, the IL9Ra is human IL9Ra. In some embodiments, the intracellular signaling domain (ICD) of human IL9Ra 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 SEQ ID NO: 1. As is understood in the art, the genetic code is degenerate and any nucleotide sequence that encodes 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: 1 can be used to encode the ICD of human IL9Ra. In some embodiments, the ICD of human IL9Ra 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 SEQ ID NO: 2 or SEQ ID NO: 73. In some embodiments, the ICD of human IL9Ra comprises SEQ ID NO: 1.In some embodiments, the ICD of human IL9Ra is encoded by a nucleic acid comprising SEQ ID NO: 2 or SEQ ID NO: 73.

[0259] In some embodiments, the immune cells are engineered to express IL-9Ra and a CAR. In some embodiments, the IL9Ra is human IL9Ra. In some embodiments, the transmembrane domain (TM) of human IL9Ra 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 SEQ ID NO: 3. As is understood in the art, the genetic code is degenerate and any nucleotide sequence that encodes 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: 3 can be used to encode the TM of human IL9Ra. In some embodiments, the TM of human IL9Ra 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 SEQ ID NO: 4 or SEQ ID NO: 74. In some embodiments, the TM of human IL9Ra comprises SEQ ID NO: 3.In some embodiments, the TM of human IL9Ra is encoded by a nucleic acid comprising SEQ ID NO: 4 or SEQ ID NO: 74.

[0260] In some embodiments, the immune cells are engineered to express IL-9Ra and a CAR. In some embodiments, the IL9Ra is human IL9Ra. In some embodiments, the ligand binding domain (LBD) of human IL9Ra 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 SEQ ID NO: 5. As is understood in the art, the genetic code is degenerate and any nucleotide sequence that encodes 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: 5 can be used to encode the LBD of human IL9Ra. In some embodiments, the LBD of human IL9Ra 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 SEQ ID NO: 6. In some embodiments, the LBD of human IL9Ra comprises SEQ ID NO: 5. In some embodiments, the LBD of human IL9Ra is encoded by a nucleic acid comprising SEQ ID NO: 6.

[0261] In some embodiments, the IL13Ra2 is human IL13Ra2. In some embodiments, the ligand binding domain (LBD) of human IL13Ra2 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 SEQ ID NO:7. As is understood in the art, the genetic code is degenerate and any nucleotide sequence encoding 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: 7 can be used to encode the LBD of human IL13Ra2. In some embodiments, the LBD of human IL13Ra2 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 SEQ ID NO: 8. In some embodiments, the LBD of human IL13Ra2 comprises SEQ ID NO: 7. In some embodiments, the LBD of human IL13Ra2 is encoded by a nucleic acid comprising SEQ ID NO: 8.

[0262] In some embodiments, the IL2Rb is human IL2Rb. In some embodiments, the ligand binding domain (LBD) of human IL2Rb 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 SEQ ID NO: 9. As is understood in the art, the genetic code is degenerate and any nucleotide sequence encoding 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: 9 can be used to encode the LBD of human IL2Rb. In some embodiments, the LBD of human IL2Rb 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 SEQ ID NO: 10. In some embodiments, the LBD of human IL2Rb comprises SEQ ID NO: 9. In some embodiments, the LBD of human IL2Rb is encoded by a nucleic acid comprising SEQ ID NO: 10.

[0263] In some embodiments, the IL18Ra is human IL18Ra. In some embodiments, the ligand binding domain (LBD) of human IL18Ra 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 SEQ ID NO: 11. As is understood in the art, the genetic code is degenerate and any nucleotide sequence encoding 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: 11 can be used to encode the LBD of human IL18Ra. In some embodiments, the LBD of human IL18Ra 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 SEQ ID NO: 12. In some embodiments, the LBD of human IL18Ra comprises SEQ ID NO: 11. In some embodiments, the LBD of human IL18Ra is encoded by a nucleic acid comprising SEQ ID NO: 12.

[0264] In some embodiments, the IL18Rb is human IL18Rb. In some embodiments, the ligand binding domain (LBD) of human IL18Rb 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 SEQ ID NO: 13. As is understood in the art, the genetic code is degenerate and any nucleotide sequence encoding 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: 13 can be used to encode the LBD of human IL18Rb. In some embodiments, the LBD of human IL18Rb 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 SEQ ID NO: 14. In some embodiments, the LBD of human IL18Rb comprises SEQ ID NO: 13. In some embodiments, the LBD of human IL18Rb is encoded by a nucleic acid comprising SEQ ID NO: 14.

[0265] In some embodiments, the immune cells are engineered to express a CAR and a human IL9Ra, wherein the human IL9Ra comprises a human IL9Ra LBD fused to a human IL9Ra TM fused to a human IL9Ra ICD. In some embodiments, the human IL9Ra 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 SEQ ID NO: 15. As is understood in the art, the genetic code is degenerate and any nucleotide sequence that encodes 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: 15 can be used to encode human IL9Ra. In some embodiments, the human IL9Ra 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 SEQ ID NO: 16. In some embodiments, the human IL9Ra comprises SEQ ID NO: 15. In some embodiments, the human IL9Ra is encoded by a nucleic acid comprising SEQ ID NO: 16.

[0266] In some embodiments, the chimeric cytokine receptor comprises a human IL13Ra2 LBD fused to a human IL9Ra TM fused to a human IL9Ra ICD. In some embodiments, the chimeric cytokine receptor 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 SEQ ID NO: 17. As is understood in the art, the genetic code is degenerate and any nucleotide sequence that encodes 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: 17 can be used to encode a chimeric cytokine receptor. In some embodiments, the chimeric cytokine receptor 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 SEQ ID NO: 18. In some embodiments, the chimeric cytokine receptor comprises SEQ ID NO: 17. In some embodiments, the chimeric cytokine receptor is encoded by a nucleic acid comprising SEQ ID NO: 18.

[0267] In some embodiments, the chimeric cytokine receptor comprises a human IL2Rb LBD fused to a human IL9Ra TM fused to a human IL9Ra ICD. In some embodiments, the chimeric cytokine receptor 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 SEQ ID NO: 19. As is understood in the art, the genetic code is degenerate and any nucleotide sequence that encodes 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: 19 can be used to encode a chimeric cytokine receptor. In some embodiments, the chimeric cytokine receptor 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 SEQ ID NO: 20. In some embodiments, the chimeric cytokine receptor comprises SEQ ID NO: 19. In some embodiments, the chimeric cytokine receptor is encoded by a nucleic acid comprising SEQ ID NO: 20.

[0268] In some embodiments, the chimeric cytokine receptor comprises a human IL18Ra LBD fused to a human IL9Ra TM fused to a human IL9Ra ICD. In some embodiments, the chimeric cytokine receptor 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 SEQ ID NO: 21. As is understood in the art, the genetic code is degenerate and any nucleotide sequence that encodes 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: 21 can be used to encode a chimeric cytokine receptor. In some embodiments, the chimeric cytokine receptor 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 SEQ ID NO: 22. In some embodiments, the chimeric cytokine receptor comprises SEQ ID NO: 21. In some embodiments, the chimeric cytokine receptor is encoded by a nucleic acid comprising SEQ ID NO: 22.

[0269] In some embodiments, the chimeric cytokine receptor comprises a human IL18Rb LBD fused to a human IL9Ra TM fused to a human IL9Ra ICD. In some embodiments, the chimeric cytokine receptor 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 SEQ ID NO: 23. As is understood in the art, the genetic code is degenerate and any nucleotide sequence that encodes 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: 23 can be used to encode a chimeric cytokine receptor. In some embodiments, the chimeric cytokine receptor 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 SEQ ID NO: 24. In some embodiments, the chimeric cytokine receptor comprises SEQ ID NO: 23. In some embodiments, the chimeric cytokine receptor is encoded by a nucleic acid comprising SEQ ID NO: 24.

[0270] In some embodiments, the IL9 is human IL9. In some embodiments, the human IL9 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 SEQ ID NO: 25. As is understood in the art, the genetic code is degenerate and any nucleotide sequence encoding 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: 25 can be used to encode human IL9. In some embodiments, human IL9 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 SEQ ID NO: 26. In some embodiments, human IL9 comprises SEQ ID NO: 25. In some embodiments, human IL9 is encoded by a nucleic acid comprising SEQ ID NO: 26.

[0271] In some embodiments, the IL13 is human IL13. In some embodiments, the human IL13 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 SEQ ID NO: 27. As is understood in the art, the genetic code is degenerate and any nucleotide sequence encoding 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: 27 can be used to encode human IL13. In some embodiments, human IL13 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 SEQ ID NO: 28. In some embodiments, human IL13 comprises SEQ ID NO: 27. In some embodiments, human IL13 is encoded by a nucleic acid comprising SEQ ID NO: 28.

[0272] In some embodiments, the IL13 is human IL13-TQM. Human IL13-TQM is an IL-13 variant containing four point mutations (E13K, R66D, S69D, and K105R) that improve its binding affinity to the IL-13Rα2 receptor (Kd approximately 5 nM) while decreasing affinity to the IL-13 receptor α1 subunit. In some embodiments, human IL13-TQM 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 SEQ ID NO: 29. As is understood in the art, the genetic code is degenerate and any nucleotide sequence encoding 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: 29 can be used to encode human IL13-TQM. In some embodiments, human IL13-TQM 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 SEQ ID NO: 30.In some embodiments, the human IL13-TQM comprises SEQ ID NO: 29. In some embodiments, the human IL13-TQM is encoded by a nucleic acid comprising SEQ ID NO: 30.

[0273] In some embodiments, the IL2 is human IL2. In some embodiments, the human IL2 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 SEQ ID NO: 31. As is understood in the art, the genetic code is degenerate and any nucleotide sequence encoding 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: 31 can be used to encode human IL2. In some embodiments, human IL2 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 SEQ ID NO: 32. In some embodiments, human IL2 comprises SEQ ID NO: 31. In some embodiments, human IL2 is encoded by a nucleic acid comprising SEQ ID NO: 32.

[0274] In some embodiments, the IL2 is human IL2. In some embodiments, the IL2 is human IL2 F42A. In some embodiments, the human IL2 F42A 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 SEQ ID NO: 33. As is understood in the art, the genetic code is degenerate and any nucleotide sequence encoding 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: 33 can be used to encode human IL2 F42A. In some embodiments, human IL2 F42A 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 SEQ ID NO: 34. In some embodiments, human IL2 F42A comprises SEQ ID NO: 33. In some embodiments, human IL2 F42A is encoded by a nucleic acid comprising SEQ ID NO: 34.

[0275] In some embodiments, the IL18 is human IL18. In some embodiments, the human IL18 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 SEQ ID NO: 35. As is understood in the art, the genetic code is degenerate and any nucleotide sequence encoding 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: 35 can be used to encode human IL18. In some embodiments, human IL18 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 SEQ ID NO: 36. In some embodiments, human IL18 comprises SEQ ID NO: 35. In some embodiments, human IL18 is encoded by a nucleic acid comprising SEQ ID NO: 36.

[0276] In some embodiments, the immune cells are engineered to express IL-9Ra and a CAR. In some embodiments, the IL9Ra is murine IL9Ra. In some embodiments, the intracellular signaling domain (ICD) of murine IL9Ra 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 SEQ ID NO: 37. As is understood in the art, the genetic code is degenerate and any nucleotide sequence that encodes 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: 37 can be used to encode the ICD of murine IL9Ra. In some embodiments, the ICD of murine IL9Ra 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 SEQ ID NO: 38, SEQ ID NO: 75, or SEQ ID NO: 76. In some embodiments, the ICD of murine IL9Ra comprises SEQ ID NO: 37.In some embodiments, the ICD of mouse IL9Ra is encoded by a nucleic acid comprising SEQ ID NO: 38, SEQ ID NO: 75, or SEQ ID NO: 76.

[0277] In some embodiments, the immune cells are engineered to express IL-9Ra and a CAR. In some embodiments, the IL9Ra is murine IL9Ra. In some embodiments, the transmembrane domain (TM) of murine IL9Ra 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 SEQ ID NO: 39. As is understood in the art, the genetic code is degenerate and any nucleotide sequence that encodes 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: 39 can be used to encode the TM of murine IL9Ra. In some embodiments, the TM of mouse IL9Ra 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 SEQ ID NO: 40, SEQ ID NO: 77, or SEQ ID NO: 78. In some embodiments, the TM of mouse IL9Ra comprises SEQ ID NO: 39.In some embodiments, the TM of mouse IL9Ra is encoded by a nucleic acid comprising SEQ ID NO: 40, SEQ ID NO: 77, or SEQ ID NO: 78.

[0278] In some embodiments, the immune cells are engineered to express IL-9Ra and a CAR. In some embodiments, the IL9Ra is murine IL9Ra. In some embodiments, the ligand binding domain (LBD) of murine IL9Ra 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 SEQ ID NO: 41. As is understood in the art, the genetic code is degenerate and any nucleotide sequence that encodes 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: 41 can be used to encode the LBD of murine IL9Ra. In some embodiments, the LBD of murine IL9Ra 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 SEQ ID NO: 42. In some embodiments, the LBD of murine IL9Ra comprises SEQ ID NO: 41.In some embodiments, the LBD of mouse IL9Ra is encoded by a nucleic acid comprising SEQ ID NO:42.

[0279] In some embodiments, the IL13Ra2 is murine IL13Ra2. In some embodiments, the ligand binding domain (LBD) of murine IL13Ra2 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 SEQ ID NO: 43. As is understood in the art, the genetic code is degenerate and any nucleotide sequence encoding 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: 43 can be used to encode the LBD of mouse IL13Ra2. In some embodiments, the LBD of mouse IL13Ra2 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 SEQ ID NO: 44. In some embodiments, the LBD of mouse IL13Ra2 comprises SEQ ID NO: 43. In some embodiments, the LBD of mouse IL13Ra2 is encoded by a nucleic acid comprising SEQ ID NO: 44.

[0280] In some embodiments, the IL2Rb is murine IL2Rb. In some embodiments, the ligand binding domain (LBD) of murine IL2Rb 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 SEQ ID NO: 45. As is understood in the art, the genetic code is degenerate, and any nucleotide sequence that encodes 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: 45 can be used to encode the LBD of mouse IL2Rb. In some embodiments, the LBD of mouse IL2Rb 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 SEQ ID NO: 46. In some embodiments, the LBD of mouse IL2Rb comprises SEQ ID NO: 45. In some embodiments, the LBD of mouse IL2Rb is encoded by a nucleic acid comprising SEQ ID NO: 46.

[0281] In some embodiments, the IL18Ra is murine IL18Ra. In some embodiments, the ligand binding domain (LBD) of murine IL18Ra 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 SEQ ID NO: 47. As is understood in the art, the genetic code is degenerate and any nucleotide sequence that encodes 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: 47 can be used to encode the LBD of murine IL18Ra. In some embodiments, the LBD of mouse IL18Ra 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 SEQ ID NO: 48. In some embodiments, the LBD of mouse IL18Ra comprises SEQ ID NO: 47. In some embodiments, the LBD of mouse IL18Ra is encoded by a nucleic acid comprising SEQ ID NO: 48.

[0282] In some embodiments, the IL18Rb is murine IL18Rb. In some embodiments, the ligand binding domain (LBD) of murine IL18Rb 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 SEQ ID NO: 49. As is understood in the art, the genetic code is degenerate and any nucleotide sequence encoding 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: 49 can be used to encode the LBD of mouse IL18Rb. In some embodiments, the LBD of mouse IL18Rb 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 SEQ ID NO: 50. In some embodiments, the LBD of mouse IL18Rb comprises SEQ ID NO: 49. In some embodiments, the LBD of mouse IL18Rb is encoded by a nucleic acid comprising SEQ ID NO: 50.

[0283] In some embodiments, the immune cells are engineered to express a CAR and a murine IL9Ra, wherein the murine IL9Ra comprises a murine IL9Ra LBD fused to a murine IL9Ra TM fused to a murine IL9Ra ICD. In some embodiments, the murine IL9Ra 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 SEQ ID NO: 51. As is understood in the art, the genetic code is degenerate and any nucleotide sequence that encodes 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: 51 can be used to encode murine IL9Ra. In some embodiments, the murine IL9Ra 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 SEQ ID NO: 52. In some embodiments, the murine IL9Ra comprises SEQ ID NO: 51.In some embodiments, the mouse IL9Ra is encoded by a nucleic acid comprising SEQ ID NO: 52.

[0284] In some embodiments, the chimeric cytokine receptor comprises a murine IL13Ra2 LBD fused to a murine IL9Ra TM fused to a murine IL9Ra ICD. In some embodiments, the chimeric cytokine receptor 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 SEQ ID NO: 53. As is understood in the art, the genetic code is degenerate and any nucleotide sequence that encodes 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: 53 can be used to encode a chimeric cytokine receptor. In some embodiments, the chimeric cytokine receptor 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 SEQ ID NO: 54. In some embodiments, the chimeric cytokine receptor comprises SEQ ID NO: 53. In some embodiments, the chimeric cytokine receptor is encoded by a nucleic acid comprising SEQ ID NO: 54.

[0285] In some embodiments, the chimeric cytokine receptor comprises a murine IL2Rb LBD fused to a murine IL9Ra TM fused to a murine IL9Ra ICD. In some embodiments, the chimeric cytokine receptor 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 SEQ ID NO: 55. As is understood in the art, the genetic code is degenerate and any nucleotide sequence that encodes 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: 55 can be used to encode a chimeric cytokine receptor. In some embodiments, the chimeric cytokine receptor 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 SEQ ID NO: 56. In some embodiments, the chimeric cytokine receptor comprises SEQ ID NO: 55. In some embodiments, the chimeric cytokine receptor is encoded by a nucleic acid comprising SEQ ID NO: 56.

[0286] In some embodiments, the chimeric cytokine receptor comprises a murine IL18Ra LBD fused to a murine IL9Ra TM fused to a murine IL9Ra ICD. In some embodiments, the chimeric cytokine receptor 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 SEQ ID NO: 57. As is understood in the art, the genetic code is degenerate and any nucleotide sequence that encodes 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: 57 can be used to encode a chimeric cytokine receptor. In some embodiments, the chimeric cytokine receptor 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 SEQ ID NO: 58. In some embodiments, the chimeric cytokine receptor comprises SEQ ID NO: 57. In some embodiments, the chimeric cytokine receptor is encoded by a nucleic acid comprising SEQ ID NO: 58.

[0287] In some embodiments, the chimeric cytokine receptor comprises a murine IL18Rb LBD fused to a murine IL9Ra TM fused to a murine IL9Ra ICD. In some embodiments, the chimeric cytokine receptor 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 SEQ ID NO: 59. As is understood in the art, the genetic code is degenerate and any nucleotide sequence that encodes 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: 59 can be used to encode a chimeric cytokine receptor. In some embodiments, the chimeric cytokine receptor 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 SEQ ID NO: 60. In some embodiments, the chimeric cytokine receptor comprises SEQ ID NO: 59. In some embodiments, the chimeric cytokine receptor is encoded by a nucleic acid comprising SEQ ID NO: 60.

[0288] In some embodiments, the IL9 is murine IL9. In some embodiments, the murine IL9 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 SEQ ID NO: 61. As is understood in the art, the genetic code is degenerate and any nucleotide sequence encoding 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: 61 can be used to encode murine IL9. In some embodiments, the murine IL9 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 SEQ ID NO: 62. In some embodiments, the murine IL9 comprises SEQ ID NO: 61. In some embodiments, the murine IL9 is encoded by a nucleic acid comprising SEQ ID NO: 62.

[0289] In some embodiments, the IL13 is murine IL13. In some embodiments, the murine IL13 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 SEQ ID NO: 63. As is understood in the art, the genetic code is degenerate and any nucleotide sequence encoding 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: 63 can be used to encode murine IL13. In some embodiments, the murine IL13 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 SEQ ID NO: 64. In some embodiments, the murine IL13 comprises SEQ ID NO: 63. In some embodiments, the murine IL13 is encoded by a nucleic acid comprising SEQ ID NO: 64.

[0290] In some embodiments, the IL2 is murine IL2. In some embodiments, the murine IL2 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 SEQ ID NO: 67. As is understood in the art, the genetic code is degenerate and any nucleotide sequence encoding 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: 67 can be used to encode murine IL2. In some embodiments, the mouse IL2 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 SEQ ID NO: 68. In some embodiments, the mouse IL2 comprises SEQ ID NO: 67. In some embodiments, the mouse IL2 is encoded by a nucleic acid comprising SEQ ID NO: 68.

[0291] In some embodiments, the IL18 is murine IL18. In some embodiments, the murine IL18 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 SEQ ID NO: 71. As is understood in the art, the genetic code is degenerate and any nucleotide sequence encoding 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: 71 can be used to encode murine IL18. In some embodiments, murine IL18 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 SEQ ID NO: 72. In some embodiments, murine IL18 comprises SEQ ID NO: 71. In some embodiments, murine IL18 is encoded by a nucleic acid comprising SEQ ID NO: 72.

[0292] In some embodiments, the IL9Ra or chimeric cytokine receptor described herein is co-expressed on an immune cell (e.g., a T cell) with any CAR that targets a tumor antigen, such as any of the CARs described herein.

[0293] The IL9Ra and chimeric cytokine receptor of the present invention enable IL9 signal transduction in immune cells expressing CAR. In some embodiments, the chimeric cytokine receptor of the present invention is a switch receptor that switches the signal derived from the binding of a ligand to the ligand binding domain (LBD) to an immunostimulatory signal transmitted by the intracellular signaling domain (ICD) of IL9Ra. The ligand that binds to the LBD is a cytokine that is delivered into tumors (e.g., by intratumoral injection) via an adenoviral vector. In some embodiments, the adenoviral vector is a serotype 5 adenoviral vector. In some embodiments, the adenoviral vector is an oncolytic adenoviral vector.

[0294] In some embodiments, the immune cells express a CAR and a human IL9Ra comprising a human IL9Ra LBD, a human IL9Ra transmembrane domain, and a human IL9Ra intracellular signaling domain, and the cytokine is IL9.

[0295] In some embodiments, the chimeric cytokine receptor comprises a human IL13Ra2 LBD, a human IL9Ra transmembrane domain, and a human IL9Ra intracellular signaling domain, and the cytokine is IL13.

[0296] In some embodiments, the chimeric cytokine receptor comprises a human IL2Rb LBD, a human IL9Ra transmembrane domain, and a human IL9Ra intracellular signaling domain, and the cytokine is IL2.

[0297] In some embodiments, the chimeric cytokine receptor comprises a human IL18Ra LBD, a human IL9Ra transmembrane domain, and a human IL9Ra intracellular signaling domain, and the cytokine is IL18.

[0298] In some embodiments, the chimeric cytokine receptor comprises a human IL18Rb LBD, a human IL9Ra transmembrane domain, and a human IL9Ra intracellular signaling domain, and the cytokine is IL18.

[0299] In some embodiments, the immune cells express a CAR and a mouse IL9Ra comprising a mouse IL9Ra LBD, a mouse IL9Ra transmembrane domain, and a mouse IL9Ra intracellular signaling domain, and the cytokine is IL9.

[0300] In some embodiments, the chimeric cytokine receptor comprises a murine IL13Ra2 LBD, a murine IL9Ra transmembrane domain, and a murine IL9Ra intracellular signaling domain, and the cytokine is IL13.

[0301] In some embodiments, the chimeric cytokine receptor comprises a mouse IL2Rb LBD, a mouse IL9Ra transmembrane domain, and a mouse IL9Ra intracellular signaling domain, and the cytokine is IL2.

[0302] In some embodiments, the chimeric cytokine receptor comprises a murine IL18Ra LBD, a murine IL9Ra transmembrane domain, and a murine IL9Ra intracellular signaling domain, and the cytokine is IL18.

[0303] In some embodiments, the chimeric cytokine receptor comprises a mouse IL18Rb LBD, a mouse IL9Ra transmembrane domain, and a mouse IL9Ra intracellular signaling domain, and the cytokine is IL18.

[0304] In some embodiments, the cytokines of the present disclosure are encoded by nucleic acid sequences contained within an oncolytic adenoviral vector, such as a conditionally replicating oncolytic adenoviral vector. An example of a conditionally replicating oncolytic adenoviral vector is a serotype 5 adenoviral vector (Ad5) with modifications to the early genes E1A and E3 to enable cancer cell-specific replication and transgene expression, respectively. E1A is modified by deleting 24 base pairs of DNA from the CR2 region (also known as the D24 variant), resulting in a virus that can selectively replicate in cancer cells with p16-Rb pathway mutations. The cytokine transgene can be placed in the E3 region. Furthermore, the viral capsid is modified to include a chimeric 5 / 3 fiber, which allows for improved tumor cell transduction efficiency.

[0305] The present invention provides chimeric cytokine receptors comprising an extracellular domain comprising the ligand-binding domain (LBD) of an inhibitory immunoreceptor or an anti-checkpoint inhibitor antigen-binding domain, a transmembrane domain (TM), and an intracellular domain (ICD) comprising the intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra). In various embodiments, the inhibitory immunoreceptor is selected from programmed cell death protein 1 (PD1), transforming growth factor beta receptor I (TGFbRI), transforming growth factor beta receptor II (TGFbRII), T cell immunoreceptor with Ig and ITIM domains (TIGIT), and T cell immunoglobulin and mucin domain-containing 3 (TIM3). In various embodiments, the checkpoint inhibitor is selected from cytotoxic T lymphocyte-associated protein 4 (CTLA4), programmed cell death protein 1 (PD1), and programmed death-ligand-1 (PD-L1).

[0306] In some embodiments, the chimeric cytokine receptor comprises an extracellular domain comprising the ligand binding domain of PD1, a transmembrane domain, and an intracellular signaling domain of IL9Ra. In some embodiments, the chimeric cytokine receptor comprises an extracellular domain comprising the ligand binding domain of TGFbRI, a transmembrane domain, and an intracellular signaling domain of IL9Ra. In some embodiments, the chimeric cytokine receptor comprises an extracellular domain comprising the ligand binding domain of TGFbRII, a transmembrane domain, and an intracellular signaling domain of IL9Ra. In some embodiments, the chimeric cytokine receptor comprises an extracellular domain comprising the ligand binding domain of TIGIT, a transmembrane domain, and an intracellular signaling domain of IL9Ra. In some embodiments, the chimeric cytokine receptor comprises an extracellular domain comprising the ligand binding domain of TIM3, a transmembrane domain, and an intracellular signaling domain of IL9Ra. In some embodiments, the chimeric cytokine receptor comprises an extracellular domain comprising an anti-CTLA4 antigen binding domain, a transmembrane domain, and an intracellular signaling domain of IL9Ra. In some embodiments, the chimeric cytokine receptor comprises an extracellular domain comprising an anti-PD1 antigen-binding domain, a transmembrane domain, and the intracellular signaling domain of IL9Ra. In some embodiments, the chimeric cytokine receptor comprises an extracellular domain comprising an anti-PD-L1 antigen-binding domain, a transmembrane domain, and the intracellular signaling domain of IL9Ra.

[0307] The anti-checkpoint inhibitor antigen-binding domain of the chimeric cytokine receptor can comprise any domain that binds to a checkpoint inhibitor, including, but 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 fragment thereof or an scFv thereof. In some embodiments, the antigen-binding domain is an scFv.

[0308] In some embodiments, the anti-checkpoint inhibitor antigen-binding domain of the chimeric cytokine receptor comprises a light chain and a heavy chain, wherein the light chain comprises three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3), and the heavy chain comprises three heavy chain complementarity determining regions (HCDR1, CDR2, and HCDR3). In some embodiments, the heavy chain lacks a CH3 region. In some embodiments, the light chain is encoded by a first nucleotide sequence, and the heavy chain is encoded by a second nucleotide sequence. In some embodiments, the first nucleotide sequence and the second nucleotide sequence are linked by a nucleotide sequence encoding a 2A self-cleaving peptide, such as a P2A sequence.

[0309] 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 an immunoglobulin (e.g., murine 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., a tumor antigen-binding domain) comprises an scFv with a VH-linker-VL configuration from N-terminus to C-terminus. In some embodiments, the antigen-binding domain comprises an scFv with a VL-linker-VH or VH-linker-VL configuration from N-terminus to C-terminus. Those skilled in the art will be able to select an appropriate configuration for use in the present invention.

[0310] Linkers are typically glycine-rich for flexibility and serine- or threonine-rich for solubility. The linker can connect the heavy chain variable region and light chain variable region 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, including, but not limited to, glycine-serine (GS) linkers, are known in the art. Those skilled in the art will be able to select an appropriate linker sequence 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), where the VH and VL are separated by a linker sequence.

[0311] 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 2(10): 31-40). Agonist 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).

[0312] As used herein, "Fab" refers to the fragment of an antibody structure that binds to an antigen but is monovalent and does not have the 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 to antigen).

[0313] As used herein, "F(ab')2" refers to an antibody fragment produced by pepsin digestion of a whole IgG antibody, which fragment has two antigen-binding (ab') (bivalent) regions, each (ab') region comprising two separate amino acid chains, a portion of a heavy chain and a light (L) chain linked by an S-type disulfide bond for antigen binding, with the remaining heavy chain portions linked together. The "F(ab')2" fragment can be divided into two individual Fab' fragments.

[0314] 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).

[0315] 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.

[0316] 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.

[0317] In some embodiments, the anti-checkpoint inhibitor antigen-binding domain binds CTLA4 and is derived from ipilimumab. In some embodiments, the anti-checkpoint inhibitor antigen-binding domain binds PD1 and is derived from nivolumab, pembrolizumab, or cemiplimab. In some embodiments, the anti-checkpoint inhibitor antigen-binding domain binds PD-L1 and is derived from atezolizumab, avelumab, or durvalumab.

[0318] The transmembrane domain (TM) of the chimeric cytokine receptor can be derived from an inhibitory immunoreceptor or IL9Ra, or can comprise any other suitable transmembrane domain. In various embodiments, the transmembrane domain is derived from IL9Ra. In some embodiments, the chimeric cytokine receptor comprises an extracellular domain comprising the ligand binding domain of an inhibitory immunoreceptor or an anti-checkpoint inhibitor antigen binding domain, a transmembrane domain of IL9Ra, and an intracellular signaling domain of IL9Ra. In some embodiments, the chimeric cytokine receptor comprises an extracellular domain comprising the ligand binding domain of PD1, a transmembrane domain of IL9Ra, and an intracellular signaling domain of IL9Ra. In some embodiments, the chimeric cytokine receptor comprises an extracellular domain comprising the ligand binding domain of TGFbRI, a transmembrane domain of IL9Ra, and an intracellular signaling domain of IL9Ra. In some embodiments, the chimeric cytokine receptor comprises an extracellular domain comprising the ligand binding domain of TGFbRII, a transmembrane domain of IL9Ra, and an intracellular signaling domain of IL9Ra. In some embodiments, the chimeric cytokine receptor comprises an extracellular domain comprising the ligand binding domain of TIGIT, a transmembrane domain of IL9Ra, and an intracellular signaling domain of IL9Ra. In some embodiments, the chimeric cytokine receptor comprises an extracellular domain comprising the ligand binding domain of TIM3, a transmembrane domain of IL9Ra, and an intracellular signaling domain of IL9Ra. In some embodiments, the chimeric cytokine receptor comprises an extracellular domain comprising an anti-CTLA4 antigen binding domain, a transmembrane domain of IL9Ra, and an intracellular signaling domain of IL9Ra. In some embodiments, the chimeric cytokine receptor comprises an extracellular domain comprising an anti-PD1 antigen binding domain, a transmembrane domain of IL9Ra, and an intracellular signaling domain of IL9Ra. In some embodiments, the chimeric cytokine receptor comprises an extracellular domain comprising an anti-PD-L1 antigen binding domain, a transmembrane domain of IL9Ra, and an intracellular signaling domain of IL9Ra.

[0319] In some embodiments, the chimeric cytokine receptor comprises an extracellular domain comprising the ligand-binding domain of PD1, a transmembrane domain, and an intracellular signaling domain of IL9Ra, wherein the ligand-binding domain binds programmed death-ligand 1 (PD-L1). PD-1 is a ligand expressed by tumor cells, including, but not limited to, tumor cells from non-small cell lung cancer, small cell lung cancer, Merkel cell carcinoma, melanoma, Hodgkin's lymphoma, urothelial carcinoma, gastric cancer, cervical cancer, cutaneous squamous cell carcinoma, renal cell carcinoma, and triple-negative breast cancer.

[0320] In some embodiments, the chimeric cytokine receptor comprises an extracellular domain comprising the ligand-binding domain of TGFbRI or TGFbRII, a transmembrane domain, and an intracellular signaling domain of IL9Ra, wherein the ligand-binding domain binds transforming growth factor-β (TGF-β), a ligand expressed by tumor cells, including but not limited to, tumor cells from breast, colon, esophageal, gastric, liver, lung, kidney, pancreatic, prostate, brain, and melanoma cancers.

[0321] In some embodiments, the chimeric cytokine receptor comprises an extracellular domain comprising the ligand-binding domain of TIGIT, a transmembrane domain, and an intracellular signaling domain of IL9Ra, wherein the ligand-binding domain binds CD155, a ligand expressed by tumor cells, including, but not limited to, tumor cells from colon cancer, lung adenocarcinoma, melanoma, pancreatic cancer, glioblastoma, and hepatocellular carcinoma.

[0322] In some embodiments, the chimeric cytokine receptor comprises an extracellular domain comprising the ligand-binding domain of TIM3, a transmembrane domain of IL9Ra, and an intracellular signaling domain of IL9Ra, wherein the ligand-binding domain binds galectin-9, a ligand expressed by tumor cells, including, but not limited to, tumor cells from breast cancer, gallbladder cancer, colon cancer, cervical squamous cell carcinoma, and hepatocellular carcinoma.

[0323] In some embodiments, the chimeric cytokine receptor comprises an extracellular domain comprising an anti-CTLA4 antigen-binding domain, a transmembrane domain of IL9Ra, and an intracellular signaling domain of IL9Ra, wherein the anti-CTLA4 antigen-binding domain binds CTLA4, which is a checkpoint inhibitor expressed on the surface of T cells.

[0324] In some embodiments, the chimeric cytokine receptor comprises an extracellular domain comprising an anti-PD1 antigen-binding domain, a transmembrane domain of IL9Ra, and an intracellular signaling domain of IL9Ra, wherein the anti-PD1 antigen-binding domain binds PD1. PD1 is a checkpoint inhibitor expressed on the surface of T cells.

[0325] In some embodiments, the chimeric cytokine receptor comprises an extracellular domain comprising an anti-PD-L1 antigen-binding domain, a transmembrane domain of IL9Ra, and an intracellular signaling domain of IL9Ra, wherein the anti-PD-L1 antigen-binding domain binds PD-L1, which is a checkpoint inhibitor expressed on the surface of T cells.

[0326] The chimeric cytokine receptor of the present invention may also include a leader sequence, a hinge domain, and / or one or more spacer or linker sequences described herein that serve to link one domain of the chimeric cytokine receptor to the next. The chimeric cytokine receptor may also include a tag (e.g., a chemical or biological tag) or be fused to another protein (e.g., a fluorescent protein such as GFP). Such a tag may be, for example, at the N-terminus or C-terminus, or may be incorporated between the two domains of the chimeric cytokine receptor. Techniques for post-transcriptional site-selective tagging of polypeptides are also well known in the art. Those skilled in the art will be able to select such sequences and tags that are appropriate for inclusion in the chimeric cytokine receptor of the present invention.

[0327] The amino acid and nucleotide sequences for certain embodiments of the chimeric cytokine receptors and their domains are set forth below. TIFF2024534471000031.tif59149TIFF2024534471000032.tif227149TIFF2024534471000033.ti f227149TIFF2024534471000034.tif227149TIFF2024534471000035.tif228149TIFF20245344710 00036.tif228149TIFF2024534471000037.tif228149TIFF2024534471000038.tif228149TIFF202 4534471000039.tif228149TIFF2024534471000040.tif227149TIFF2024534471000041.tif215148 TIFF2024534471000042.tif228149TIFF2024534471000043.tif220149TIFF2024534471000044.t if214149TIFF2024534471000045.tif230149TIFF2024534471000046.tif221149TIFF2024534471 000047.tif228149TIFF2024534471000048.tif221149TIFF2024534471000049.tif229149TIFF20 24534471000050.tif222149TIFF2024534471000051.tif226149TIFF2024534471000052.tif77149

[0328] In some embodiments, the IL9Ra is human IL9Ra. In some embodiments, the intracellular signaling domain (ICD) of human IL9Ra 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 SEQ ID NO: 1. As is understood in the art, the genetic code is degenerate and any nucleotide sequence that encodes 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: 1 can be used to encode the ICD of human IL9Ra. In some embodiments, the ICD of human IL9Ra 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 SEQ ID NO: 2. In some embodiments, the ICD of human IL9Ra comprises SEQ ID NO: 1. In some embodiments, the ICD of human IL9Ra is encoded by a nucleic acid comprising SEQ ID NO: 2.

[0329] In some embodiments, the IL9Ra is human IL9Ra. In some embodiments, the transmembrane domain (TM) of human IL9Ra 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 SEQ ID NO:3. As is understood in the art, the genetic code is degenerate and any nucleotide sequence that encodes 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: 3 can be used to encode the TM of human IL9Ra. In some embodiments, the TM of human IL9Ra 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 SEQ ID NO: 4. In some embodiments, the TM of human IL9Ra comprises SEQ ID NO: 3. In some embodiments, the TM of human IL9Ra is encoded by a nucleic acid comprising SEQ ID NO: 4.

[0330] In some embodiments, the PD1 is human PD1. In some embodiments, the ligand binding domain (LBD) of human PD1 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 SEQ ID NO:205. As is understood in the art, the genetic code is degenerate, and any nucleotide sequence that encodes 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: 205 can be used to encode the LBD of human PD1. In some embodiments, the LBD of human PD1 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 SEQ ID NO: 206. In some embodiments, the LBD of human PD1 comprises SEQ ID NO: 205. In some embodiments, the LBD of human PD1 is encoded by a nucleic acid comprising SEQ ID NO: 206.

[0331] In some embodiments, the TGFbRI is human TGFbRI. In some embodiments, the ligand binding domain (LBD) of human TGFbRI 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 SEQ ID NO:207. As is understood in the art, the genetic code is degenerate and any nucleotide sequence encoding 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: 207 can be used to encode the LBD of human TGFbRI. In some embodiments, the LBD of human TGFbRI 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 SEQ ID NO: 208. In some embodiments, the LBD of human TGFbRI comprises SEQ ID NO: 207. In some embodiments, the LBD of human TGFbRI is encoded by a nucleic acid comprising SEQ ID NO: 208.

[0332] In some embodiments, the TGFbRII is human TGFbRII. In some embodiments, the ligand binding domain (LBD) of human TGFbRII 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 SEQ ID NO:209. As is understood in the art, the genetic code is degenerate and any nucleotide sequence encoding 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: 209 can be used to encode the LBD of human TGFbRII. In some embodiments, the LBD of human TGFbRII 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 SEQ ID NO: 210. In some embodiments, the LBD of human TGFbRII comprises SEQ ID NO: 209. In some embodiments, the LBD of human TGFbRII is encoded by a nucleic acid comprising SEQ ID NO: 210.

[0333] In some embodiments, the TIGIT is human TIGIT. In some embodiments, the ligand-binding domain (LBD) of human TIGIT 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 SEQ ID NO: 211. As is understood in the art, the genetic code is degenerate, and any nucleotide sequence encoding 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: 211 can be used to encode the LBD of human TIGIT. In some embodiments, the LBD of human TIGIT 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 SEQ ID NO: 212. In some embodiments, the LBD of human TIGIT comprises SEQ ID NO: 211. In some embodiments, the LBD of human TIGIT is encoded by a nucleic acid comprising SEQ ID NO: 212.

[0334] In some embodiments, the TIM3 is human TIM3. In some embodiments, the ligand binding domain (LBD) of human TIM3 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 SEQ ID NO: 213. As is understood in the art, the genetic code is degenerate, and any nucleotide sequence encoding 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: 213 can be used to encode the LBD of human TIM3. In some embodiments, the LBD of human TIM3 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 SEQ ID NO: 214. In some embodiments, the LBD of human TIM3 comprises SEQ ID NO: 213. In some embodiments, the LBD of human TIM3 is encoded by a nucleic acid comprising SEQ ID NO: 214.

[0335] In some embodiments, the chimeric cytokine receptor comprises a human PD1 LBD fused to a human IL9Ra TM fused to a human IL9Ra ICD. In some embodiments, the chimeric cytokine receptor 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 SEQ ID NO: 215. As is understood in the art, the genetic code is degenerate and any nucleotide sequence that encodes 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: 215 can be used to encode a chimeric cytokine receptor. In some embodiments, the chimeric cytokine receptor 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 SEQ ID NO: 216. In some embodiments, the chimeric cytokine receptor comprises SEQ ID NO: 215. In some embodiments, the chimeric cytokine receptor is encoded by a nucleic acid comprising SEQ ID NO: 216.

[0336] In some embodiments, the chimeric cytokine receptor comprises a human TGFRbI LBD fused to a human IL9Ra TM fused to a human IL9Ra ICD. In some embodiments, the chimeric cytokine receptor 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 SEQ ID NO: 217. As is understood in the art, the genetic code is degenerate and any nucleotide sequence that encodes 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: 217 can be used to encode a chimeric cytokine receptor. In some embodiments, the chimeric cytokine receptor 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 SEQ ID NO: 218. In some embodiments, the chimeric cytokine receptor comprises SEQ ID NO: 217.In some embodiments, the chimeric cytokine receptor is encoded by a nucleic acid comprising SEQ ID NO:218.

[0337] In some embodiments, the chimeric cytokine receptor comprises a human TGFRbII LBD fused to a human IL9Ra TM fused to a human IL9Ra ICD. In some embodiments, the chimeric cytokine receptor 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 SEQ ID NO: 219. As is understood in the art, the genetic code is degenerate and any nucleotide sequence that encodes 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: 219 can be used to encode a chimeric cytokine receptor. In some embodiments, the chimeric cytokine receptor 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 SEQ ID NO: 220. In some embodiments, the chimeric cytokine receptor comprises SEQ ID NO: 219.In some embodiments, the chimeric cytokine receptor is encoded by a nucleic acid comprising SEQ ID NO:220.

[0338] In some embodiments, the chimeric cytokine receptor comprises a human TIGIT LBD fused to a human IL9Ra TM fused to a human IL9Ra ICD. In some embodiments, the chimeric cytokine receptor 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 SEQ ID NO: 221. As is understood in the art, the genetic code is degenerate and any nucleotide sequence that encodes 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: 221 can be used to encode a chimeric cytokine receptor. In some embodiments, the chimeric cytokine receptor 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 SEQ ID NO: 222. In some embodiments, the chimeric cytokine receptor comprises SEQ ID NO: 221. In some embodiments, the chimeric cytokine receptor is encoded by a nucleic acid comprising SEQ ID NO: 222.

[0339] In some embodiments, the chimeric cytokine receptor comprises a human TIM3 LBD fused to a human IL9Ra TM fused to a human IL9Ra ICD. In some embodiments, the chimeric cytokine receptor 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 SEQ ID NO: 223. As is understood in the art, the genetic code is degenerate and any nucleotide sequence that encodes 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: 223 can be used to encode a chimeric cytokine receptor. In some embodiments, the chimeric cytokine receptor 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 SEQ ID NO: 224. In some embodiments, the chimeric cytokine receptor comprises SEQ ID NO: 223. In some embodiments, the chimeric cytokine receptor is encoded by a nucleic acid comprising SEQ ID NO: 224.

[0340] In some embodiments, the IL9Ra is murine IL9Ra. In some embodiments, the intracellular signaling domain (ICD) of murine IL9Ra 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 SEQ ID NO:225. As is understood in the art, the genetic code is degenerate and any nucleotide sequence that encodes 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: 225 can be used to encode the ICD of murine IL9Ra. In some embodiments, the ICD of murine IL9Ra 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 SEQ ID NO: 226 or SEQ ID NO: 302. In some embodiments, the ICD of murine IL9Ra comprises SEQ ID NO: 225.In some embodiments, the ICD of mouse IL9Ra is encoded by a nucleic acid comprising SEQ ID NO: 226 or SEQ ID NO: 302.

[0341] In some embodiments, the IL9Ra is murine IL9Ra. In some embodiments, the transmembrane domain (TM) of murine IL9Ra 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 SEQ ID NO:227. As is understood in the art, the genetic code is degenerate and any nucleotide sequence that encodes 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: 227 can be used to encode the TM of murine IL9Ra. In some embodiments, the TM of mouse IL9Ra 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 SEQ ID NO: 228. In some embodiments, the TM of mouse IL9Ra comprises SEQ ID NO: 227. In some embodiments, the TM of mouse IL9Ra is encoded by a nucleic acid comprising SEQ ID NO: 228.

[0342] In some embodiments, the PD1 is mouse PD1. In some embodiments, the ligand binding domain (LBD) of mouse PD1 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 SEQ ID NO:229. As is understood in the art, the genetic code is degenerate, and any nucleotide sequence that encodes 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: 229 can be used to encode the LBD of mouse PD1. In some embodiments, the LBD of mouse PD1 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 SEQ ID NO: 230. In some embodiments, the LBD of mouse PD1 comprises SEQ ID NO: 229. In some embodiments, the LBD of mouse PD1 is encoded by a nucleic acid comprising SEQ ID NO: 230.

[0343] In some embodiments, the TGFbRI is mouse TGFbRI. In some embodiments, the ligand binding domain (LBD) of mouse TGFbRI 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 SEQ ID NO:231. As is understood in the art, the genetic code is degenerate and any nucleotide sequence encoding 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: 231 can be used to encode the LBD of mouse TGFbRI. In some embodiments, the LBD of mouse TGFbRI 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 SEQ ID NO: 232. In some embodiments, the LBD of mouse TGFbRI comprises SEQ ID NO: 231. In some embodiments, the LBD of mouse TGFbRI is encoded by a nucleic acid comprising SEQ ID NO: 232.

[0344] In some embodiments, the TGFbRII is mouse TGFbRII. In some embodiments, the ligand binding domain (LBD) of mouse TGFbRII 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 SEQ ID NO:233. As is understood in the art, the genetic code is degenerate and any nucleotide sequence encoding 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: 233 can be used to encode the LBD of mouse TGFbRII. In some embodiments, the LBD of mouse TGFbRII 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 SEQ ID NO: 234. In some embodiments, the LBD of mouse TGFbRII comprises SEQ ID NO: 233. In some embodiments, the LBD of mouse TGFbRII is encoded by a nucleic acid comprising SEQ ID NO: 234.

[0345] In some embodiments, the TIGIT is mouse TIGIT. In some embodiments, the ligand binding domain (LBD) of mouse TIGIT 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 SEQ ID NO: 235. As is understood in the art, the genetic code is degenerate, and any nucleotide sequence encoding 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: 235 can be used to encode the LBD of mouse TIGIT. In some embodiments, the LBD of mouse TIGIT 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 SEQ ID NO: 236. In some embodiments, the LBD of mouse TIGIT comprises SEQ ID NO: 235. In some embodiments, the LBD of mouse TIGIT is encoded by a nucleic acid comprising SEQ ID NO: 236.

[0346] In some embodiments, the TIM3 is mouse TIM3. In some embodiments, the ligand binding domain (LBD) of mouse TIM3 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 SEQ ID NO: 237. As is understood in the art, the genetic code is degenerate, and any nucleotide sequence encoding 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: 237 can be used to encode the LBD of mouse TIM3. In some embodiments, the LBD of mouse TIM3 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 SEQ ID NO: 238. In some embodiments, the LBD of mouse TIM3 comprises SEQ ID NO: 237. In some embodiments, the LBD of mouse TIM3 is encoded by a nucleic acid comprising SEQ ID NO: 238.

[0347] In some embodiments, the chimeric cytokine receptor comprises a mouse PD1 LBD fused to a mouse IL9Ra TM fused to a mouse IL9Ra ICD. In some embodiments, the chimeric cytokine receptor 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 SEQ ID NO: 239. As is understood in the art, the genetic code is degenerate and any nucleotide sequence that encodes 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: 239 can be used to encode a chimeric cytokine receptor. In some embodiments, the chimeric cytokine receptor 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 SEQ ID NO: 240. In some embodiments, the chimeric cytokine receptor comprises SEQ ID NO: 239.In some embodiments, the chimeric cytokine receptor is encoded by a nucleic acid comprising SEQ ID NO:240.

[0348] In some embodiments, the chimeric cytokine receptor comprises a mouse TGFRbI LBD fused to a mouse IL9Ra TM fused to a mouse IL9Ra ICD. In some embodiments, the chimeric cytokine receptor 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 SEQ ID NO: 241. As is understood in the art, the genetic code is degenerate and any nucleotide sequence that encodes 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: 241 can be used to encode a chimeric cytokine receptor. In some embodiments, the chimeric cytokine receptor 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 SEQ ID NO: 242. In some embodiments, the chimeric cytokine receptor comprises SEQ ID NO: 241.In some embodiments, the chimeric cytokine receptor is encoded by a nucleic acid comprising SEQ ID NO:242.

[0349] In some embodiments, the chimeric cytokine receptor comprises a mouse TGFRbII LBD fused to a mouse IL9Ra TM fused to a mouse IL9Ra ICD. In some embodiments, the chimeric cytokine receptor 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 SEQ ID NO: 243. As is understood in the art, the genetic code is degenerate and any nucleotide sequence that encodes 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: 243 can be used to encode a chimeric cytokine receptor. In some embodiments, the chimeric cytokine receptor 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 SEQ ID NO: 244. In some embodiments, the chimeric cytokine receptor comprises SEQ ID NO: 243.In some embodiments, the chimeric cytokine receptor is encoded by a nucleic acid comprising SEQ ID NO:244.

[0350] In some embodiments, the chimeric cytokine receptor comprises a mouse TIGIT LBD fused to a mouse IL9Ra TM fused to a mouse IL9Ra ICD. In some embodiments, the chimeric cytokine receptor 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 SEQ ID NO: 245. As is understood in the art, the genetic code is degenerate and any nucleotide sequence that encodes 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: 245 can be used to encode a chimeric cytokine receptor. In some embodiments, the chimeric cytokine receptor 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 SEQ ID NO: 246. In some embodiments, the chimeric cytokine receptor comprises SEQ ID NO: 245.In some embodiments, the chimeric cytokine receptor is encoded by a nucleic acid comprising SEQ ID NO:246.

[0351] In some embodiments, the chimeric cytokine receptor comprises a mouse TIM3 LBD fused to a mouse IL9Ra TM fused to a mouse IL9Ra ICD. In some embodiments, the chimeric cytokine receptor 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 SEQ ID NO: 247. As is understood in the art, the genetic code is degenerate and any nucleotide sequence that encodes 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:247 can be used to encode a chimeric cytokine receptor. In some embodiments, the chimeric cytokine receptor 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 SEQ ID NO: 248. In some embodiments, the chimeric cytokine receptor comprises SEQ ID NO: 247.In some embodiments, the chimeric cytokine receptor is encoded by a nucleic acid comprising SEQ ID NO:248.

[0352] In some embodiments, the anti-checkpoint inhibitor antigen-binding domain is an anti-CTLA4 antigen-binding domain. In some embodiments, the anti-CTLA4 antigen-binding domain is derived from ipilimumab and comprises an LCDR1 comprising SEQ ID NO: 294, an LCDR2 comprising SEQ ID NO: 295, an LCDR3 comprising SEQ ID NO: 296, an HCDR1 comprising SEQ ID NO: 297, an HCDR2 comprising SEQ ID NO: 298, and an HCDR3 comprising SEQ ID NO: 299.

[0353] In some embodiments, the anti-checkpoint inhibitor antigen-binding domain is an anti-CTLA4 antigen-binding domain. In some embodiments, the anti-CTLA4 antigen-binding domain is derived from ipilimumab and comprises a light chain having 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: 289. As is understood in the art, the genetic code is degenerate, and any nucleotide sequence encoding 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: 289 can be used to encode an anti-CTLA4 light chain. In some embodiments, the anti-CTLA4 light chain comprises SEQ ID NO: 289.

[0354] In some embodiments, the anti-checkpoint inhibitor antigen-binding domain is an anti-CTLA4 antigen-binding domain. In some embodiments, the anti-CTLA4 antigen-binding domain is derived from ipilimumab and comprises a heavy chain having 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: 290. As is understood in the art, the genetic code is degenerate, and any nucleotide sequence encoding 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: 290 can be used to encode an anti-CTLA4 heavy chain. In some embodiments, the anti-CTLA4 heavy chain comprises SEQ ID NO: 290.

[0355] In some embodiments, the anti-checkpoint inhibitor antigen-binding domain is an anti-CTLA4 antigen-binding domain. In some embodiments, the anti-CTLA4 antigen-binding domain is derived from ipilimumab and 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 SEQ ID NO: 293. As is understood in the art, the genetic code is degenerate, and any nucleotide sequence encoding 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: 293 can be used to encode the anti-CTLA4 antigen-binding domain. In some embodiments, the anti-CTLA4 antigen-binding domain comprises SEQ ID NO: 293.

[0356] In some embodiments, the chimeric cytokine receptor comprises an extracellular domain comprising an anti-CTLA4 antigen binding domain, a hIL9Ra transmembrane domain, and a hIL9Ra intracellular signaling domain. In some embodiments, the chimeric cytokine receptor 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 SEQ ID NO: 300. As is understood in the art, the genetic code is degenerate, and any nucleotide sequence encoding 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: 300 can be used to encode a chimeric cytokine receptor. In some embodiments, the chimeric cytokine receptor comprises SEQ ID NO: 300. In some embodiments, the chimeric cytokine receptor is encoded by a nucleic acid comprising SEQ ID NO: 301.

[0357] In some embodiments, the chimeric cytokine receptor of the present invention is a switch receptor that switches the signal derived from the binding of a ligand to the ligand binding domain (LBD) of an inhibitory immunoreceptor to an immunostimulatory signal transduction by the intracellular signaling domain (ICD) of IL9Ra. The ligand that binds to the LBD is naturally expressed by tumor cells. In some embodiments, the chimeric cytokine receptor of the present invention activates IL9Ra signaling in immune cells when a checkpoint inhibitor binds to the anti-checkpoint inhibitor antigen-binding domain. The checkpoint inhibitor is expressed by T cells.

[0358] In one aspect, the present invention provides modified cells (e.g., immune cells or their precursor cells) engineered to express a chimeric cytokine receptor and a chimeric antigen receptor (CAR). The chimeric cytokine receptor binds to a ligand or an immune checkpoint inhibitor, and the CAR binds to a tumor antigen, where the ligand and tumor antigen are naturally expressed by tumor cells, and the checkpoint inhibitor is expressed by T cells. Thus, the chimeric cytokine receptor disclosed herein and its use improves chimeric antigen receptor (CAR) cell immunotherapy for treating cancer by (1) utilizing molecules naturally present in tumors (i.e., ligands and tumor antigens) and / or checkpoint inhibitors in T cells to convert immunosuppressive signals into immunostimulatory signals in immune cells (e.g., T cells), (2) changing the phenotype of immune cells expressing the chimeric cytokine receptor and CAR upon binding of the ligand and / or checkpoint inhibitor at the tumor site, and (3) enabling IL-9 signaling in immune cells expressing the chimeric cytokine receptor and CAR to improve effector function in situ and / or downregulate immune cell exhaustion.

[0359] In some embodiments, the chimeric cytokine receptor described herein is co-expressed on an immune cell (e.g., a T cell) with any CAR that targets a tumor antigen, such as any of the CARs described herein.

[0360] C. Chimeric antigen receptor (CAR) The present invention provides modified immune cells or their precursor cells (e.g., modified T cells) engineered to express a CAR and a chimeric cytokine receptor comprising IL9Ra or an IL9Ra ICD. The present invention also provides modified immune cells or their precursor cells (e.g., modified T cells) that express a CAR, wherein expression of Cullin 5 in the cells is reduced and / or eliminated via genetic engineering techniques or by introduction of inhibitory RNA. In each aspect, the CAR 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.

[0361] 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.

[0362] The CAR of the present invention may also comprise a leader sequence as described herein. The CAR of the present invention may also comprise a hinge domain as described herein. The CAR of the present invention may also comprise one or more spacer domains or linkers as described herein, which may serve to link one domain of the CAR to the next domain.

[0363] antigen-binding domain The antigen binding domain of CAR is the extracellular region of CAR that binds to a specific target antigen, including proteins, carbohydrates, and glycolipids.The CAR of the present invention comprises an antigen binding domain that can bind to 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, GPC2, TnMuc1, CD70, PMSA, and EGFRvIII.

[0364] 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 fragment thereof or an scFv thereof. In some embodiments, the tumor antigen-binding domain is an scFv.

[0365] 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 an immunoglobulin (e.g., murine 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., a tumor antigen-binding domain) comprises an scFv with a VH-linker-VL configuration from N-terminus to C-terminus. In some embodiments, the antigen-binding domain comprises an scFv with a VL-linker-VH or VH-linker-VL configuration from N-terminus to C-terminus. Those skilled in the art will be able to select an appropriate configuration for use in the present invention.

[0366] Linkers are typically glycine-rich for flexibility and serine- or threonine-rich for solubility. The linker can connect the heavy chain variable region and light chain variable region 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 International Publication No. 2014 / 087010, the contents of which are incorporated herein by reference in their entirety. Various linker sequences, including but not limited to glycine-serine (GS) linkers, are known in the art. Those skilled in the art will be able to select an appropriate linker sequence 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), where the VH and VL are separated by a linker sequence.

[0367] 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, as well as 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 2(10): 31-40). Agonist 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).

[0368] As used herein, "Fab" refers to the fragment of an antibody structure that binds to an antigen but is monovalent and does not have the 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 to antigen).

[0369] As used herein, "F(ab')2" refers to an antibody fragment produced by pepsin digestion of a whole IgG antibody, which fragment 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-type disulfide bond for antigen binding, with the remaining heavy chain portions linked together. The "F(ab')2" fragment can be divided into two individual Fab' fragments.

[0370] 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).

[0371] 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.

[0372] 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.

[0373] In some embodiments, the light chain is encoded by a first nucleotide sequence, and the heavy chain is encoded by a second nucleotide sequence. In some embodiments, the first nucleotide sequence and the second nucleotide sequence are linked by a nucleotide sequence encoding a 2A self-cleaving peptide, such as a P2A sequence. In some embodiments, the heavy chain lacks a CH3 region.

[0374] Transmembrane domain The CAR of the present invention can 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.

[0375] In some embodiments, the transmembrane domain is naturally associated with one or more of the domains in the CAR. In some embodiments, the transmembrane domain can be selected to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins, or can be modified by one or more amino acid substitutions, to minimize interaction with other members of the receptor complex.

[0376] Transmembrane domain can be derived from either natural or synthetic sources.When source is natural, domain can be derived from any membrane-binding protein or transmembrane protein, for example, type I transmembrane protein.When source is synthetic, transmembrane domain can be any artificial sequence, for example, artificial hydrophobic sequence, that facilitates the insertion of CAR into 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).

[0377] 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.

[0378] 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.

[0379] 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.

[0380] 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 a 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 a 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, the CD8a hinge, an artificial hinge made of a polypeptide that may be as small as three glycines (Gly), and the CH1 and CH3 domains of IgG (such as human IgG4).

[0381] 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 in recognizing and binding 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 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 the hinge region to adopt many different conformations.

[0382] 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).

[0383] The hinge region can have a length of about 4 amino acids to about 50 amino acids, e.g., 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.

[0384] A suitable hinge region can be readily selected and can be any of several suitable lengths, for example, from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 to 15 amino acids, from 3 to 12 amino acids, for example, 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).

[0385] For example, the hinge region may be a glycine polymer (G) nFlexible linkers include 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 can therefore 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 comprise 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 comprise an amino acid sequence derived from human CD8 or a variant thereof.

[0386] 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 a 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.

[0387] 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 molecules that act in concert to initiate signaling in T cells, as well as any derivatives or variants of these elements, and any synthetic sequences that have the same functional capabilities.

[0388] 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, FcsRIγ and β chains, MB1 (Iga) chain, B29 (Ig) chain, etc., 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.

[0389] 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 that has the same functional capability, and any combination thereof.

[0390] 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 to 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, ITGB1, 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, fragments or domains derived from one or more molecules or receptors, including, but 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, 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.

[0391] 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 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.

[0392] Intracellular signaling domains suitable for use in the CARs 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; changes in transcription of a target gene; changes in protein activity; changes in cellular behavior, such as cell death; cell proliferation; cell differentiation; cell survival; modulation of a cell signaling response, 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 ITAM motif (e.g., 1, 2, 3, 4, 5, 6, etc.) 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 linked to the membrane-bound CAR, but instead is diffused within the cytoplasm.

[0393] Intracellular signaling domains suitable for use in the CARs of the present 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 to 8 amino acids. In one embodiment, the intracellular signaling domain of the CAR comprises three ITAM motifs.

[0394] In some embodiments, the intracellular signaling domain comprises a...

Claims

1. (a) an extracellular domain comprising the ligand binding domain (LBD) of a receptor selected from the group consisting of interleukin-13 receptor alpha type 2 (IL13Ra2), interleukin-2 receptor beta (IL2Rb), interleukin-18 receptor alpha (IL18Ra), and interleukin-18 receptor beta (IL18Rb); (b) transmembrane domain and; (c) an intracellular domain containing the intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra); A chimeric cytokine receptor comprising:

2. The chimeric cytokine receptor of claim 1, wherein the transmembrane domain is an IL9Ra transmembrane domain.

3. (a) Human IL13Ra2 LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (b) human IL2Rb LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (c) human IL18Ra LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (d) human IL18Rb LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (e) mouse IL13Ra2 LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; (f) mouse IL2Rb LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; (g) mouse IL18Ra LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; or (h) Mouse IL18Rb LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain The chimeric cytokine receptor of claim 1 , comprising:

4. 2. The chimeric cytokine receptor 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: 15, 17, 19, 21, 23, 51, 53, 55, 57, and 59.

5. 2. The chimeric cytokine receptor of claim 1, 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: 16, 18, 20, 22, 24, 52, 54, 56, 58, and 60.

6. An isolated nucleic acid comprising a nucleotide sequence encoding the chimeric cytokine receptor described in claim 1.

7. A vector comprising the isolated nucleic acid of claim 6.

8. 8. The vector of claim 7, which is a retroviral vector or a lentiviral vector. Claim 9: (a) a first nucleotide sequence encoding a chimeric cytokine receptor comprising: (i) an extracellular domain comprising the ligand binding domain (LBD) of a receptor selected from the group consisting of interleukin-13 receptor alpha type 2 (IL13Ra2), interleukin-2 receptor beta (IL2Rb), interleukin-18 receptor alpha (IL18Ra), and interleukin-18 receptor beta (IL18Rb), (ii) a first transmembrane domain, and (iii) an intracellular domain comprising the intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra); and (b) a second nucleotide sequence encoding a chimeric antigen receptor (CAR) comprising an extracellular tumor antigen-binding domain, a second transmembrane domain, and a second intracellular domain; An isolated nucleic acid comprising:

10. 10. The isolated nucleic acid of claim 9, wherein the transmembrane domain is an IL9Ra transmembrane domain.

11. the chimeric cytokine receptor (a) Human IL13Ra2 LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (b) human IL2Rb LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (c) human IL18Ra LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (d) human IL18Rb LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (e) mouse IL13Ra2 LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; (f) mouse IL2Rb LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; (g) mouse IL18Ra LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; or (h) Mouse IL18Rb LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain 10. The isolated nucleic acid of claim 9, comprising:

12. 10. The isolated nucleic acid of claim 9, wherein the chimeric cytokine receptor 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 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 15, 17, 19, 21, 23, 51, 53, 55, 57, and 59.

13. 10. The isolated nucleic acid of claim 9, wherein the first nucleotide sequence has 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: 16, 18, 20, 22, 24, 52, 54, 56, 58, and 60.

14. The extracellular tumor antigen-binding domain is selected from the group consisting of 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, and EpC.

10. The isolated nucleic acid of claim 9, wherein the nucleic acid binds to a tumor antigen selected from the group consisting of 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, 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.

15. 10. The isolated nucleic acid of claim 9, wherein the tumor antigen-binding domain is a single-chain variable fragment (scFv).

16. 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: 79 or SEQ ID NO: 95; (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: 117; (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: 119 or SEQ ID NO: 121; (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: 123; (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: 125; (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: 129; 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 100% sequence identity to SEQ ID NO:

131.

10. The isolated nucleic acid of claim 9, selected from the group consisting of:

17. 10. The isolated nucleic acid of claim 9, wherein the intracellular domain of the CAR 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).

18. 10. The isolated nucleic acid of claim 9, wherein the intracellular domain of the CAR 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.

19. 10. The isolated nucleic acid of claim 9, wherein the intracellular domain of the CAR comprises a costimulatory domain of CD28, a costimulatory domain of 4-1BB, an intracellular signaling domain of CD3 zeta, or any combination thereof.

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

21. 21. The vector of claim 20, which is a retroviral vector or a lentiviral vector.

22. (a) a chimeric cytokine receptor according to any one of claims 1 to 5, (b) an isolated nucleic acid according to any one of claims 6 or 9 to 19, and / or (c) The vector according to any one of claims 7 to 8 or 20 to 21.

1. A modified cell comprising:

23. The modified cell of claim 22, which is a T cell, an autologous cell, a human cell, or any combination thereof.

24. A pharmaceutical composition comprising the modified cells of claim 22 for use in a method for treating cancer in a subject in need thereof.

25. A modified cell, wherein the cell is an immune cell or a precursor cell thereof, and the cell comprises: (a) interleukin-9 receptor alpha (IL9Ra), or A chimeric cytokine receptor comprising: (i) an extracellular domain comprising the ligand binding domain (LBD) of a receptor selected from the group consisting of interleukin-13 receptor alpha type 2 (IL13Ra2), interleukin-2 receptor beta (IL2Rb), interleukin-18 receptor alpha (IL18Ra), and interleukin-18 receptor beta (IL18Rb), (ii) a first transmembrane domain, and (iii) an intracellular domain comprising the intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra); and (b) a chimeric antigen receptor (CAR) comprising an extracellular tumor antigen-binding domain, a second transmembrane domain, and a second intracellular domain. The modified cell is engineered to express the

26. the chimeric cytokine receptor (a) Human IL13Ra2 LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (b) human IL2Rb LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (c) human IL18Ra LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (d) human IL18Rb LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (e) mouse IL13Ra2 LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; (f) mouse IL2Rb LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; (g) mouse IL18Ra LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; or (h) Mouse IL18Rb LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain 26. The modified cell of claim 25, comprising:

27. (a) the intracellular domain of the CAR comprises a costimulatory domain of CD28, a costimulatory domain of 4-1BB, an intracellular signaling domain of CD3 zeta, or any combination thereof; and / or (b) the IL9Ra or chimeric cytokine receptor is capable of activating STAT1, STAT3, STAT5, or any combination thereof in the cell; and / or (c) the transmembrane domain is an IL9Ra transmembrane domain; 26. The modified cell of claim 25.

28. 26. A pharmaceutical composition comprising the population of modified cells of claim 25 and at least one pharmaceutically acceptable carrier.

29. 1. A system for enabling IL9 signaling in a cell, comprising: (a) (i) interleukin-9 receptor alpha (IL9Ra), or a chimeric cytokine receptor comprising an extracellular domain comprising the ligand binding domain (LBD) of a receptor selected from the group consisting of interleukin-13 receptor alpha type 2 (IL13Ra2), interleukin-2 receptor beta (IL2Rb), interleukin-18 receptor alpha (IL18Ra), and interleukin-18 receptor beta (IL18Rb), and an intracellular domain comprising a first transmembrane domain and the intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra); and (ii) a chimeric antigen receptor (CAR) comprising an extracellular tumor antigen-binding domain, a second transmembrane domain, and a second intracellular domain; modified immune cells that have been engineered to express the (b) a vector comprising a nucleotide sequence encoding a cytokine selected from the group consisting of IL9, IL13, IL2, and IL18; The system comprising:

30. A composition for use in a method for treating cancer in a subject in need thereof, comprising the system of claim 29. (a) the subject is a human; and / or (b) the cancer is 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; and / or (c) the cancer is B-cell lymphoma or leukemia; 31. The composition of claim 30.

32. A modified immune cell or its precursor cell that has been engineered to express a chimeric antigen receptor (CAR) comprising a tumor antigen-binding domain, a transmembrane domain, and an intracellular domain, and further wherein expression of Cullin 5 in said modified immune cell or its precursor cell has been reduced and / or eliminated via genetic engineering techniques or by the introduction of inhibitory RNA.

33. A chimeric cytokine receptor comprising an extracellular domain comprising the ligand-binding domain (LBD) of an inhibitory immunoreceptor or an anti-checkpoint inhibitor antigen-binding domain, a transmembrane domain, and an intracellular domain comprising the intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra).

34. (a) the inhibitory immunoreceptor is selected from the group consisting of programmed cell death protein 1 (PD1), transforming growth factor beta receptor I (TGFbRI), transforming growth factor beta receptor II (TGFbRII), T cell immunoreceptor with Ig and ITIM domains (TIGIT), and T cell immunoglobulin and mucin domain containing 3 (TIM3), and the checkpoint inhibitor is selected from the group consisting of cytotoxic T lymphocyte-associated protein 4 (CTLA4), programmed cell death protein 1 (PD1), and programmed death ligand-1 (PD-L1); and / or (b) the transmembrane domain is an IL9Ra transmembrane domain; 34. The chimeric cytokine receptor of claim 33.

35. (a) Human PD1 LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (b) human TGFbRI LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (c) human TGFbRII LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (d) human TIGIT LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (e) human TIM3 LBD, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (f) mouse PD1 LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; (g) mouse TGFbRI LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; (h) mouse TGFbRII LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; (i) mouse TIGIT LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; (j) mouse TIM3 LBD, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; (k) anti-human CTLA4 antigen-binding domain, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (l) anti-human PD1 antigen-binding domain, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (m) anti-human PD-L1 antigen-binding domain, human IL9Ra transmembrane domain, and human IL9Ra intracellular signaling domain; (n) anti-mouse CTLA4 antigen-binding domain, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain; (o) an anti-mouse PD1 antigen-binding domain, a mouse IL9Ra transmembrane domain, and a mouse IL9Ra intracellular signaling domain; or (p) Anti-mouse PD-L1 antigen-binding domain, mouse IL9Ra transmembrane domain, and mouse IL9Ra intracellular signaling domain 34. The chimeric cytokine receptor of claim 33, comprising:

36. 34. The chimeric cytokine receptor of claim 33, 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: 215, 217, 219, 221, 223, 239, 241, 243, 245, 247, and 300.

37. 34. The chimeric cytokine receptor of claim 33, 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: 216, 218, 220, 222, 224, 240, 242, 244, 246, 248, and 301.

38. An isolated nucleic acid comprising a nucleotide sequence encoding the chimeric cytokine receptor of any one of claims 33 to 37.

39. (a) a first nucleotide sequence encoding a chimeric cytokine receptor comprising an extracellular domain comprising a ligand-binding domain of an inhibitory immunoreceptor or an anti-checkpoint inhibitor antigen-binding domain, a first transmembrane domain, and an intracellular domain comprising the intracellular signaling domain of interleukin-9 receptor alpha (IL9Ra); and (b) a second nucleotide sequence encoding a chimeric antigen receptor (CAR) comprising an extracellular tumor antigen-binding domain, a second transmembrane domain, and a second intracellular domain; An isolated nucleic acid comprising: (a) a chimeric cytokine receptor according to any one of claims 33 to 37, or (b) the isolated nucleic acid of claim 38; A modified immune cell or a precursor thereof, comprising:

41. 41. A pharmaceutical composition comprising a population of modified immune cells or progenitor cells thereof according to claim 40 and at least one pharmaceutically acceptable carrier.

42. A pharmaceutical composition comprising the modified immune cells or precursor cells thereof described in claim 40, for use in a method for treating cancer in a subject in need thereof.

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