Chimeric antigen receptor with 4-1BB costimulatory domain

Abstract

To provide immune cells containing a chimeric antigen receptor (CAR).SOLUTION: Provided are CAR-T compositions that are directed to a CAR including (i) an extracellular domain comprising an antigen-binding domain; (ii) a transmembrane domain; and (iii) an intracellular domain comprising a costimulatory endodomain, wherein the costimulatory endodomain comprises an intracellular signaling domain from 4-1BB / CD137 and five additional amino acids. The disclosure also provides vectors, compositions, and methods of treatment using the antigen binding molecules and engineered immune cells comprising the CAR. The CAR compositions provided herein can be used for the treatment of certain cancers.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. patent application No. 62 / 867,503, filed June 27, 2019, International patent application No. PCT / KR2019 / 010244, filed August 12, 2019, U.S. patent application No. 16 / 715,462, filed December 16, 2019, U.S. patent application No. 62 / 991,493, filed March 18, 2020, U.S. patent application No. 63 / 004,827, filed April 3, 2020, and U.S. patent application No. 63 / 043,237, filed June 24, 2020, the disclosures of each of which are incorporated herein by reference in their entirety. [Background technology]

[0002] background Cancer remains one of the leading causes of death worldwide. Recent statistics indicate that 13% of the world's population dies from cancer. According to estimates by the International Agency for Research on Cancer (IARC), there were 14.1 million new cases of cancer and 8.2 million cancer-related deaths worldwide in 2012. Due to population growth and aging, as well as exposure to risk factors such as smoking, unhealthy diets, and physical inactivity, the number of new cancer cases and cancer-related deaths worldwide is expected to increase to 21.7 million and 13 million, respectively, by 2030. Furthermore, the pain and medical costs associated with cancer treatment reduce the quality of life of cancer patients and their families.

[0003] Chimeric antigen receptor-engineered T cells (CAR-T) have great therapeutic potential in the treatment of diseases such as cancer. CAR-T therapeutics confer strong target affinity and signaling function to T cells. However, the remarkable efficacy of CAR-T therapy is often accompanied by severe side effects, such as cytokine release syndrome (CRS). Therefore, there remains an unmet need for the development of CAR-T therapeutics and strategies with reduced side effects. Summary of the Invention

[0004] overview Provided herein is an immune cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises: (a) an extracellular domain comprising an antigen-binding domain; (b) a transmembrane domain; and (c) an intracellular domain comprising a costimulatory endodomain, wherein the costimulatory endodomain comprises an intracellular signaling domain derived from 4-1BB / CD137 and five additional amino acids.

[0005] In some embodiments, the chimeric antigen receptor is a single polypeptide, hi some embodiments, the chimeric antigen receptor is composed of two polypeptides.

[0006] In some embodiments, the costimulatory endodomain comprises an intracellular signaling domain derived from 4-1BB / CD137 and five additional amino acids, wherein the five additional amino acids are encoded by SEQ ID NO: 1. In some embodiments, the costimulatory endodomain comprises SEQ ID NO: 2.

[0007] In some embodiments, the antigen-binding domain is humanized. In some embodiments, the antigen-binding domain is human. In some embodiments, the antigen-binding domain is an scFv. In some embodiments, the antigen-binding domain specifically binds to an antigen associated with a disease. In some embodiments, the antigen-binding domain specifically binds to a tumor antigen. In some embodiments, the antigen-binding domain specifically binds to an antigen selected from the group consisting of glypican 3 (GPC3), malignant tumor variant receptor (MVR), and CD19.

[0008] In some embodiments, the transmembrane domain is selected from the group consisting of 4-1BB / CD137, activating NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8 alpha, CD8 beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, ligand specifically binding to CD83, LIGHT, LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1), M The transmembrane domain is selected from the group consisting of HC class 1 molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1), SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor proteins, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, and VLA-6. In some embodiments, the transmembrane domain is derived from CD8α. In some embodiments, the intracellular domain further comprises an intracellular domain derived from CD3ζ.

[0009] In some embodiments, the chimeric antigen receptor further comprises a signal peptide or leader sequence. In some embodiments, the chimeric antigen receptor further comprises a hinge region. In some embodiments, the hinge region is a CD8α hinge. In some embodiments, the chimeric antigen receptor further comprises an additional antigen-binding domain. In some embodiments, the additional antigen-binding domain is an scFv.

[0010] In some embodiments, the immune cells are human immune cells. In some embodiments, the human immune cells are autologous human immune cells. In some embodiments, the human immune cells are allogeneic human immune cells. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are NK cells.

[0011] Provided herein is a nucleic acid encoding a chimeric antigen receptor (CAR), the chimeric antigen receptor comprising: (a) an extracellular domain comprising an antigen-binding domain; (b) a transmembrane domain; and (c) an intracellular domain comprising a costimulatory endodomain, the costimulatory endodomain comprising an intracellular signaling domain derived from 4-1BB / CD137 and five additional amino acids.

[0012] In some embodiments, the chimeric antigen receptor is a single polypeptide, hi some embodiments, the chimeric antigen receptor is composed of two polypeptides.

[0013] In some embodiments, the costimulatory endodomain comprises an intracellular signaling domain derived from 4-1BB / CD137 and five additional amino acids, wherein the five additional amino acids are encoded by the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the costimulatory endodomain comprises SEQ ID NO:2.

[0014] In some embodiments, the antigen-binding domain is humanized. In some embodiments, the antigen-binding domain is human. In some embodiments, the antigen-binding domain is an scFv. In some embodiments, the antigen-binding domain specifically binds to an antigen associated with a disease. In some embodiments, the antigen-binding domain specifically binds to a tumor antigen. In some embodiments, the antigen-binding domain specifically binds to an antigen selected from the group consisting of glycan 3 (GPC3), malignant tumor variant receptor (MVR), and CD19.

[0015] In some embodiments, the transmembrane domain is selected from the group consisting of 4-1BB / CD137, activating NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8 alpha, CD8 beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, ligand specifically binding to CD83, LIGHT, LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1), M The transmembrane domain is selected from a protein selected from the group consisting of HC class 1 molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1), SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor proteins, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, and VLA-6. In some embodiments, the transmembrane domain is a transmembrane domain derived from CD8 alpha.

[0016] In some embodiments, the intracellular domain further comprises an intracellular domain derived from CD3ζ. In some embodiments, the chimeric antigen receptor further comprises a signal peptide or leader sequence. In some embodiments, the chimeric antigen receptor further comprises a hinge region. In some embodiments, the hinge region is a CD8α hinge.

[0017] Provided herein is a vector comprising any one of the nucleic acids described herein. In some embodiments, the vector further comprises a promoter operably linked to the nucleic acid. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a lentiviral vector.

[0018] Provided herein are methods of producing engineered immune cells, comprising introducing any one of the nucleic acids described herein or any one of the vectors described herein into immune cells, thereby producing the engineered immune cells. In some embodiments, the method further comprises culturing the engineered immune cells after the introducing step. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are NK cells.

[0019] In some embodiments, the method further comprises obtaining immune cells from the subject prior to the introducing step. In some embodiments, the method further comprises administering the engineered immune cells to the subject. In some embodiments, the subject has been diagnosed or identified as having cancer.

[0020] Provided herein is an engineered immune cell produced by any one of the methods described herein.

[0021] Provided herein is a pharmaceutical composition comprising any one of the engineered immune cells described herein and a pharmaceutically acceptable carrier.

[0022] Provided herein are methods of treating cancer in a subject, comprising administering to the subject any one of the engineered immune cells described herein or any one of the pharmaceutical compositions described herein. In some embodiments, the cancer is an anti-glypican 3-associated cancer, an anti-CD19-associated cancer, or an anti-MVR-associated cancer. In some embodiments, the cancer is carcinoma, lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma), blastoma, sarcoma, leukemia, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastric cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, other lymphoproliferative disorders, and various types of head and neck cancer. In some embodiments, the subject has previously received one or more additional anti-cancer therapies, wherein the anti-cancer therapies are selected from the group consisting of ionizing radiation, chemotherapeutic agents, therapeutic antibodies, and checkpoint inhibitors. In some embodiments, the subject has been identified or diagnosed as having cancer. [The present invention 1001] An immune cell comprising a chimeric antigen receptor (CAR), the CAR comprising: (a) an extracellular domain comprising an antigen-binding domain; (b) a transmembrane domain; (c) an intracellular domain comprising a costimulatory endodomain, wherein the costimulatory endodomain comprises an intracellular signaling domain derived from 4-1BB / CD137 and five additional amino acids; and The immune cell comprising: [The present invention 1002] 1001. The immune cell of the present invention, wherein said chimeric antigen receptor is a single polypeptide. [The present invention 1003] 1001. The immune cell of the present invention, wherein said chimeric antigen receptor is composed of two polypeptides. [The present invention 1004] Any of the immune cells of the present invention 1001 to 1003, wherein the costimulatory endodomain comprises an intracellular signaling domain derived from 4-1BB / CD137 and five additional amino acids, wherein the five additional amino acids are encoded by SEQ ID NO: 1. [The present invention 1005] 1004. The immune cell of the present invention, wherein said costimulatory endodomain comprises SEQ ID NO:2. [The present invention 1006] The immune cell of any of claims 1001 to 1005, wherein the antigen-binding domain is humanized. [The present invention 1007] The immune cell of any of claims 1001 to 1006, wherein the antigen-binding domain is human. [The present invention 1008] The immune cell of any of claims 1001 to 1007, wherein the antigen-binding domain is an scFv. [The present invention 1009] The immune cell of any of claims 1001 to 1008, wherein the antigen-binding domain specifically binds to an antigen associated with a disease. [The present invention 1010] The immune cell of any of claims 1001 to 1009, wherein the antigen-binding domain specifically binds to a tumor antigen. [The present invention 1011] The immune cell of any of 1001 to 1010 of the present invention, wherein the antigen-binding domain specifically binds to an antigen selected from the group consisting of glypican 3 (GPC3), malignant tumor variant receptor (MVR), and CD19. [The present invention 1012] The transmembrane domain is selected from the group consisting of 4-1BB / CD137, activating NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8 alpha, CD8 beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, and CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, ligand specifically binding to CD83, LIGHT, LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1), MHC class 1 molecule, NK any one of 1001 to 1011 immune cells of the present invention, wherein the transmembrane domain is selected from a protein selected from the group consisting of G2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1), SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, and VLA-6. [The present invention 1013] The immune cell of any one of claims 1001 to 1012, wherein the transmembrane domain is a transmembrane domain derived from CD8α. [The present invention 1014] The immune cell of any of claims 1001 to 1013, wherein the intracellular domain further comprises an intracellular domain derived from CD3ζ. [The present invention 1015] The immune cell of any of claims 1001 to 1014, wherein the chimeric antigen receptor further comprises a signal peptide or a leader sequence. [The present invention 1016] The immune cell of any of claims 1001 to 1015, wherein the chimeric antigen receptor further comprises a hinge region. [The present invention 1017] 1016. The immune cell of the present invention, wherein the hinge region is a CD8α hinge. [The present invention 1018] The immune cell of any of claims 1001 to 1017, wherein the chimeric antigen receptor further comprises an additional antigen-binding domain. [The present invention 1019] The immune cell of the present invention, wherein the additional antigen-binding domain is an scFv. [The present invention 1020] The immune cell of any one of claims 1001 to 1019 of the present invention, wherein the immune cell is a human immune cell. [The present invention 1021] The immune cell of the present invention 1020, wherein the human immune cell is an autologous human immune cell. [The present invention 1022] The immune cell of the present invention 1020, wherein said human immune cell is an allogeneic human immune cell. [The present invention 1023] The immune cell of any one of 1001 to 1022 of the present invention, wherein the immune cell is a T cell. [The present invention 1024] The immune cell of any one of claims 1001 to 1023, wherein the immune cell is a NK cell. [The present invention 1025] A nucleic acid encoding a chimeric antigen receptor (CAR), the chimeric antigen receptor comprising: (a) an extracellular domain comprising an antigen-binding domain; (b) a transmembrane domain; (c) an intracellular domain comprising a costimulatory endodomain, wherein the costimulatory endodomain comprises an intracellular signaling domain derived from 4-1BB / CD137 and five additional amino acids; and The nucleic acid comprising: [The present invention 1026] 1025. The nucleic acid of claim 1025, wherein the chimeric antigen receptor is a single polypeptide. [The present invention 1027] 1025. The nucleic acid of the present invention, wherein the chimeric antigen receptor is composed of two polypeptides. [The present invention 1028] Any of the nucleic acids 1025 to 1027 of the present invention, wherein the costimulatory endodomain comprises an intracellular signaling domain derived from 4-1BB / CD137 and five additional amino acids, wherein the five additional amino acids are encoded by the nucleotide sequence of SEQ ID NO: 1. [The present invention 1029] 1028. The nucleic acid of claim 1028, wherein the costimulatory endodomain comprises SEQ ID NO:2. [The present invention 1030] The nucleic acid of any one of claims 1025 to 1029, wherein the antigen-binding domain is humanized. [The present invention 1031] The nucleic acid of any one of 1025 to 1030 of the present invention, wherein the antigen-binding domain is human. [The present invention 1032] The nucleic acid of any one of claims 1025 to 1031, wherein the antigen-binding domain is an scFv. [The present invention 1033] The nucleic acid of any one of claims 1025 to 1032, wherein the antigen-binding domain specifically binds to an antigen associated with a disease. [The present invention 1034] The nucleic acid of any of claims 1025 to 1033, wherein the antigen-binding domain specifically binds to a tumor antigen. [This invention 1035] The nucleic acid of any one of claims 1025 to 1034, wherein the antigen-binding domain specifically binds to an antigen selected from the group consisting of glycan 3 (GPC3), malignant tumor variant receptor (MVR), and CD19. [The present invention 1036] The transmembrane domain is selected from the group consisting of 4-1BB / CD137, activating NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8 alpha, CD8 beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, and CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, ligand specifically binding to CD83, LIGHT, LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1), MHC class 1 molecule, N Any of the nucleic acids 1025 to 1035 of the present invention, wherein the nucleic acid is a transmembrane domain selected from a protein selected from the group consisting of KG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1), SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, and VLA-6. [This invention 1037] The nucleic acid of any one of claims 1025 to 1036, wherein the transmembrane domain is a transmembrane domain derived from CD8α. [The present invention 1038] The nucleic acid of any one of 1025 to 1037, wherein the intracellular domain further comprises an intracellular domain derived from CD3ζ. [This invention 1039] The nucleic acid of any one of 1025 to 1038, wherein the chimeric antigen receptor further comprises a signal peptide or leader sequence. [The present invention 1040] The nucleic acid of any one of 1025 to 1039, wherein the chimeric antigen receptor further comprises a hinge region. [The present invention 1041] 1040. The nucleic acid of claim 10, wherein said hinge region is a CD8α hinge. [The present invention 1042] A vector comprising any one of the nucleic acids of the present inventions 1025 to 1041. [This invention 1043] 1042. The vector of claim 1042, further comprising a promoter operably linked to said nucleic acid. [This invention 1044] 1043. The vector of the present invention, wherein the promoter is a constitutive promoter. [This invention 1045] 1043. The vector of the present invention, wherein the promoter is an inducible promoter. [The present invention 1046] The vector of any one of 1042 to 1045 of the present invention, which is a viral vector. [This invention 1047] The vector of the present invention 1046, wherein the viral vector is a lentiviral vector. [This invention 1048] A method for producing engineered immune cells, comprising introducing a nucleic acid according to any one of claims 1025 to 1041 or a vector according to any one of claims 1042 to 1047 into immune cells, thereby producing the engineered immune cells. [This invention 1049] 1048. The method of claim 1048, further comprising culturing said engineered immune cells after the introducing step. [The present invention 1050] The method of any one of claims 1048 to 1049, wherein the immune cells are T cells. [This invention 1051] The method of any one of claims 1048 to 1050, wherein the immune cells are NK cells. [This invention 1052] The method of any one of claims 1048 to 1051, further comprising obtaining said immune cells from a subject prior to the introducing step. [This invention 1053] The method of claim 1052, further comprising administering said engineered immune cells to said subject. [This invention 1054] The method of any one of claims 1052 to 1053, wherein said subject has been diagnosed or identified as having cancer. [This invention 1055] An engineered immune cell produced by any of the methods of the present inventions 1048 to 1054. [The present invention 1056] A pharmaceutical composition comprising an engineered immune cell of the present invention 1055 and a pharmaceutically acceptable carrier. [This invention 1057] A method for treating cancer in a subject, comprising administering to said subject an engineered immune cell of invention 1055 or a pharmaceutical composition of invention 1056. [This invention 1058] 1057. The method of claim 1057, wherein said cancer is an anti-glypican 3 associated cancer, an anti-CD19 associated cancer, or an anti-MVR associated cancer. [This invention 1059] The method of the present invention 1057, wherein the cancer is carcinoma, lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma), blastoma, sarcoma, leukemia, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastric cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, other lymphoproliferative disorders, and various types of head and neck cancer. [The present invention 1060] Any of the methods of claims 1057 to 1059, wherein the subject has previously been administered one or more additional anti-cancer treatments, wherein the anti-cancer treatments are selected from the group consisting of ionizing radiation, chemotherapeutic agents, therapeutic antibodies, and checkpoint inhibitors. [The present invention 1061] The method of any of claims 1057 to 1060, wherein said subject has been identified or diagnosed as having said cancer. [Brief explanation of the drawings]

[0023] [Figure 1] 1 shows a schematic diagram of an exemplary MVR CAR construct. [Figure 2] 1 shows exemplary enzyme mapping results after MVRL2H2-4-1BB cloning. [Figure 3] The results of restriction enzyme digestion of huGC33(VH-VL)-euBBz are shown, with predicted sizes indicated on a DNA ladder and gel electrophoresis images of the results. [Figure 4] The results of restriction enzyme digestion of huGC33(VH-VL)-BBz are shown, with predicted sizes indicated on a DNA ladder and gel electrophoresis images of the results. [Figure 5] Figure 5A is a graph showing the total fold expansion of CAR-T cells over 11 days in vitro. Figure 5B is a graph comparing the fold expansion of CAR-T cells in vitro. Figure 5C is a graph showing the cell viability of CAR-T cells in vitro. [Figure 6] 1 shows an analysis of CAR expression in T cells transduced with huGC33(VH-VL)-euBBz and huGC33(VH-VL)-BBz. [Figure 7] Figure 7A is a graph showing an LDH-based cytotoxicity assay using target cells derived from Huh-7 cell line, and Figure 7B is a graph showing an LDH-based cytotoxicity assay using target cells derived from PLC / PRF / 5 cell line. [Figure 8]1 is a set of graphs comparing the in vivo efficacy of huGC33(VH-VL)-euBBz and huGC33(VH-VL)-BBz CAR-T cells. [Figure 9] Figure 9A is a graph showing the number of CAR-T cells from a mouse model 5 weeks after infusion of huGC33(VH-VL)-euBBz and huGC33(VH-VL)-BBz CAR-T cells. Figure 9B is a set of graphs comparing the number of CAR-T cells from a mouse model 5 weeks after infusion of huGC33(VH-VL)-euBBz and huGC33(VH-VL)-BBz CAR-T cells. [Figure 10] 1 shows an analysis of CAR-T cells in the blood, bone marrow, spleen, and liver in mice 5 weeks after infusion of huGC33(VH-VL)-euBBz and huGC33(VH-VL)-BBz CAR-T cells using FACS staining. [Figure 11] Figure 11A shows CAR expression in CD19-BBz and CD19-euBBz transduced T cells, and Figure 11B is a graph of a luciferase-based cytotoxicity assay showing killing activity in CD19-BBz and CD19-euBBz transduced T cells. [Figure 12] The results of IVIS imaging of the effects of CD19-BBz CAR-T cells and CD19-euBBz CAR-T cells using an animal model are shown. [Figure 13] Graph showing photon values of cancer cells in an animal model after injection of CD19-BBz CAR-T cells and CD19-euBBz CAR-T cells. [Figure 14]Figure 14A is a set of graphs showing the percentage of total CD19 CAR-T cells present in the blood after mice were bled orbitally every 3-4 days using FACS. Figure 14B is a set of graphs showing the percentage of CD4 / CD8 CAR-T cells present in the blood after mice were bled orbitally every 3-4 days using FACS. Figure 14C is a graph showing the number of total CD19 CAR-T cells present in the blood after mice were bled orbitally every 3-4 days using FACS. [Figure 15] Figure 15A shows a schematic diagram of an exemplary GPC3 CAR construct. Figure 15B shows a schematic diagram of an exemplary GPC3 CAR construct. DETAILED DESCRIPTION OF THE INVENTION

[0024] Detailed Description The present disclosure describes chimeric antigen receptors (CARs) comprising the 4-1BB costimulatory endodomain, and methods for making and using same.

[0025] definition About: The term "about," when used herein in reference to a value, refers to a value that is similar in context to the referenced value. Generally, a person of ordinary skill in the art familiar with the context will understand the appropriate degree of variation encompassed by "about" in that context. For example, in some embodiments, the term "about" can encompass a range of values within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referenced value.

[0026] Administration: As used herein, the term "administration" typically refers to administering a composition to a subject or system to achieve delivery of the composition itself or an agent contained therein. Those skilled in the art will recognize various routes that may be utilized for administration to a subject, e.g., a human, in appropriate circumstances. For example, in some embodiments, administration can be ocular, oral, parenteral, topical, etc. In some specific embodiments, administration can be bronchial (e.g., bronchial instillation), buccal, transdermal (e.g., which may be or include one or more of topical to the dermis, intradermal, interdermal, transdermal, etc.), enteral, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, intraspecific organ (e.g., intrahepatic), mucosal, intranasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., intratracheal instillation), vaginal, vitreous, etc. In some embodiments, administration can involve a single dose, multiple doses, or a fixed number of doses. In some embodiments, administration can involve dosing that is intermittent (e.g., multiple doses separated by a period of time) and / or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration can involve continuous dosing (e.g., perfusion) for at least a selected period of time.

[0027] Affinity: As known in the art, "affinity" is a measure of the strength with which a particular ligand binds to its partner. Affinity can be measured in a variety of ways. In some embodiments, affinity is measured by a quantitative assay. In some embodiments, the binding partner concentration can be fixed in excess of the ligand concentration to mimic physiological conditions. Alternatively, or additionally, in some embodiments, the binding partner concentration and / or the ligand concentration can be varied. In some such embodiments, affinity can be compared to a standard under equivalent conditions (e.g., concentrations).

[0028] Antibody drug: As used herein, the term "antibody drug" can refer to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex that contains sufficient immunoglobulin structural elements to confer specific binding. Exemplary antibody drugs include, but are not limited to, monoclonal antibodies, polyclonal antibodies, and fragments thereof. In some embodiments, an antibody drug can include one or more sequence elements that are humanized, primatized, chimerized, etc., as known in the art. In many embodiments, the term "antibody drug" is used to refer to one or more constructs or formats known or developed in the art for utilizing the structural and functional characteristics of antibodies in alternative presentations. For example, in some embodiments, antibody agents utilized in accordance with the present invention include, but are not limited to, intact IgA, IgG, IgE, or IgM antibodies; bi- or multispecific antibodies (e.g., Zybodies®, etc.); antibody fragments, such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fv; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); S mall M odd I mmuno PThe antibody drug may take a format selected from harmaceuticals ("SMIP™"); single-chain or tandem diabodies (TandAb®); VHH; Anticalins®; Nanobodies® minibodies; BiTE®; ankyrin repeat proteins or DARPIN®; Avimers®; DART; TCR-like antibodies; Adnectins®; Affilins®, Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®. In some embodiments, the antibody drug may lack covalent modifications (e.g., glycan attachments) that it would have if produced naturally. In some embodiments, the antibody drug may include covalent modifications (e.g., glycan attachments), a payload (e.g., detectable moiety, therapeutic moiety, catalytic moiety, etc.), or other pendant groups (e.g., poly-ethylene glycol, etc.). In some embodiments, an antibody agent is or comprises a polypeptide having an amino acid sequence that includes one or more structural elements recognized by those skilled in the art as complementarity-determining regions (CDRs). In some embodiments, an antibody agent is or comprises a polypeptide that includes at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) whose amino acid sequence is substantially identical to that found in a reference antibody. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that they are sequence identical or contain one to five amino acid substitutions compared to the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that they exhibit at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the reference CDR.In some embodiments, the included CDRs are substantially identical to the reference CDRs in that they exhibit at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity to the reference CDRs. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that at least one amino acid in the included CDRs has been deleted, added, or substituted relative to the reference CDRs, but the included CDRs otherwise have the same amino acid sequence as the reference CDRs. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that one to five amino acids in the included CDRs have been deleted, added, or substituted relative to the reference CDRs, but the included CDRs otherwise have the same amino acid sequence as the reference CDRs. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that at least one amino acid in the included CDRs has been substituted relative to the reference CDRs, but the included CDRs otherwise have the same amino acid sequence as the reference CDRs. In some embodiments, the included CDR is substantially identical to the reference CDR in that one to five amino acids within the included CDR have been deleted, added, or substituted relative to the reference CDR, but the included CDR otherwise has the same amino acid sequence as the reference CDR. In some embodiments, the antibody drug is or comprises a polypeptide having an amino acid sequence that includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, the antibody drug is a polypeptide protein having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain. In some embodiments, the antibody drug is or comprises at least a portion of a chimeric antigen receptor (CAR).

[0029] Antigen: As used herein, the term "antigen" can refer to an agent that binds to an antibody drug. In some embodiments, an antigen binds to an antibody drug and may or may not induce a specific physiological response in an organism. Generally, an antigen can be or include any chemical entity (e.g., a small molecule, a nucleic acid, a polypeptide, a carbohydrate, a lipid, a polymer (including biological polymers (e.g., nucleic acid and / or amino acid polymers) and polymers other than biological polymers (e.g., polymers other than nucleic acids or amino acid polymers)). In some embodiments, an antigen can be or include a polypeptide. In some embodiments, an antigen can be or include a glycan. Those skilled in the art will understand that generally, antigens can be obtained in isolated or pure form, or alternatively, in crude form (e.g., with other materials (e.g., extracts such as cell extracts or relatively crude preparations of antigen-containing sources)). In some certain embodiments, the antigen is present in a cellular context (e.g., the antigen is expressed on the surface of or intracellularly with a cell). In some embodiments, the antigen is a recombinant antigen.

[0030] Antigen-binding domain: As used herein, the term "antigen-binding domain" refers to an antibody drug or portion thereof that specifically binds to a target site or entity. Typically, the interaction between the antigen-binding domain and its target is non-covalent. In some embodiments, the target site or entity can be any chemical class, including, for example, carbohydrates, lipids, nucleic acids, metals, polypeptides, or small molecules. In some embodiments, the antigen-binding domain can be or comprise a polypeptide (or a complex thereof). In some embodiments, the antigen-binding domain is part of a fusion polypeptide. In some embodiments, the antigen-binding domain is part of a chimeric antigen receptor (CAR).

[0031] Associated with: As used herein, two events or entities are "associated" with one another when the presence, level, and / or form of one event or entity correlates with the other. For example, a particular entity (e.g., a polypeptide, gene signature, metabolite, microorganism, etc.) is considered associated with a particular disease, disorder, or condition when its presence, level, and / or form correlates with the incidence and / or susceptibility of that disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically "associated" with one another when they directly or indirectly interact with one another to bring them into and / or maintain their physical proximity to one another. In some embodiments, two or more entities that are physically associated with one another are covalently bound to one another. In some embodiments, two or more entities that are physically associated with one another are not covalently bound to one another, but are non-covalently bound by, for example, hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0032] Binding: As used herein, the term "binding" will be understood to typically refer to a non-covalent association between or among two or more entities. "Direct" binding involves physical contact between the entities or moieties. Indirect binding involves physical interaction through physical contact with one or more intermediate entities. Binding between two or more entities can typically be assessed in any of a variety of contexts, including when the interacting entities or moieties are studied alone or in the context of a more complex system (e.g., covalently or otherwise associated with a carrier entity and / or in a biological system or cell).

[0033] Cancer: The terms "cancer," "malignancy," "neoplasm," "tumor," and "carcinoma" are used herein to refer to cells that exhibit relatively abnormal, uncontrolled, and / or autonomous growth, resulting in an abnormal growth phenotype characterized by a significant loss of cell proliferation control. In some embodiments, a tumor is or can include cells that are pre-cancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic. The present disclosure specifically identifies certain cancers for which the teachings may be particularly appropriate. In some embodiments, a suitable cancer may be characterized as a solid tumor. In some embodiments, a suitable cancer may be characterized as a hematological tumor. Generally, various types of cancers known in the art include, for example, hematopoietic cancers including leukemia, lymphomas (Hodgkin's and non-Hodgkin's), myeloma and myeloproliferative disorders, sarcoma, melanoma, adenoma, cancer of solid tissue, squamous cell carcinoma of the oral cavity, throat, pharynx, and lung, liver cancer, genitourinary cancers (e.g., prostate, cervical, bladder, uterine, and endometrial cancer), as well as renal cell carcinoma, bone cancer, pancreatic cancer, skin cancer, cutaneous and intraocular melanoma, endocrine system cancers, thyroid cancer, parathyroid cancer, head and neck cancer, breast cancer, gastrointestinal cancer, and nervous system cancers, benign lesions (e.g., papillomas), and the like.

[0034] CDR: As used herein, "CDR" can refer to a complementarity-determining region within the variable region of an antibody drug. There are three CDRs in each heavy and light chain variable region, designated CDR1, CDR2, and CDR3. A "set of CDRs" or "CDR set" refers to a group of three or six CDRs present in either a single variable region capable of binding to an antigen or the CDRs of cognate heavy and light chain variable regions capable of binding to an antigen. Certain systems for defining CDR boundaries (e.g., Rabat, Chothia, etc.) have been established in the art. Those skilled in the art will recognize the differences between these systems and will be able to understand CDR boundaries to the extent necessary to understand and practice the claimed invention.

[0035] Chemotherapeutic Agent: As used herein, the term "chemotherapeutic agent" has its art-recognized meaning and refers to one or more pro-apoptotic, cytostabilizing, and / or cytotoxic agents, including, for example, agents specifically available or recommended for use in treating one or more diseases, disorders, or conditions associated with unwanted cell proliferation. In many embodiments, the chemotherapeutic agent is useful in the treatment of cancer. In some embodiments, the chemotherapeutic agent is or can include one or more alkylating agents, one or more anthracyclines, one or more cytoskeletal disruptors (e.g., microtubule-targeting agents (e.g., taxanes, maytansine, and analogs thereof)), one or more epothilones, one or more histone deacetylase inhibitors (HDACs), one or more topoisomerase inhibitors (e.g., inhibitors of topoisomerase I and / or topoisomerase II), one or more kinase inhibitors, one or more nucleotide analogs or nucleotide precursor analogs, one or more peptide antibiotics, one or more platinum-based agents, one or more retinoids, one or more vinca alkaloids, and / or one or more analogs of the following (i.e., those sharing related antiproliferative activity): In some specific embodiments, the chemotherapeutic agent is actinomycin, all-trans retinoic acid, auristatin, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, curcumin, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, gemcitabine, The therapeutic agent may be or include one or more of hydroxyurea, idarubicin, imatinib, irinotecan, maytansine and / or its analogs (e.g., DM1), mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, maytansinoids, oxaliplatin, paclitaxel, pemetrexed, teniposide, thioguanine, topotecan, barbican, vinblastine, vincristine, vindesine, vinorelbine, and combinations thereof.In some embodiments, a chemotherapeutic agent can be utilized in the context of an antibody-drug conjugate. In some embodiments, the chemotherapeutic agent can be hLL1-doxorubicin, hRS7-SN-38, hMN-14-SN-38, hLL2-SN-38, hA20-SN-38, hPAM4-SN-38, hLL1-SN-38, hRS7-Pro-2-P-Dox, hMN-14-Pro-2-P-Dox, hLL2-Pro-2-P-Dox, hA20-Pro-2-P-Dox, hPAM4-Pro-2-P-Dox, hLL1-Pro-2-P-Dox, P 4 / D10-doxorubicin, gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, glembatumumab vedotin, SAR3419, SAR566658, BIIB015, BT062, SGN-75, SGN-CD19A, AMG-172, AMG-595, BAY-94-9343, ASG-5ME, ASG-22ME, ASG-16M8F, MDX-1203, MLN-0264, anti-PSMA A chemotherapeutic agent found in an antibody-drug conjugate selected from the group consisting of ADC, RG-7450, RG-7458, RG-7593, RG-7596, RG-7598, RG-7599, RG-7600, RG-7636, ABT-414, IMGN-853, IMGN-529, borsetuzumab mafodotin, and lorvotuzumab mertansine.

[0036] Engineered: In general, the term "engineered" can refer to aspects of being manipulated by the hand of man. For example, a polypeptide is considered "engineered" if its sequence has been engineered by the hand of man. For example, in some embodiments of the present invention, an engineered polypeptide includes a sequence containing one or more amino acid mutations, deletions, and / or insertions introduced by the hand of man into a reference polypeptide sequence. In some embodiments, an engineered polypeptide includes a polypeptide that has been fused (i.e., covalently linked) by the hand of man to one or more additional polypeptides to form a fusion polypeptide that does not naturally occur in vivo. Similarly, a cell or organism is considered "engineered" if it has been engineered to alter its genetic information (e.g., by introducing new genetic material not previously present, through transformation, mating, somatic hybridization, transfection, transduction, or other mechanisms, or by altering or removing previously present genetic material through substitution or deletion mutations, or mating protocols). This is conventional, and one of skill in the art will understand that derivatives or progeny of engineered polypeptides or cells are typically still referred to as "engineered," even though the actual manipulation was performed on the prior entity.

[0037] Host cell: As used herein, the term "host cell" can refer to a cell of an organism that has been selected, modified, transformed, grown, used, or treated in any way for the production of a material by the cell, e.g., the expression of a gene, DNA or RNA sequence, protein, or enzyme by the cell. Host cells include immune cells, including, but not limited to, lymphocytes (e.g., T cells, B cells, and NK cells), neutrophils, and monocytes / macrophages.

[0038] In vitro: As used herein, the term "in vitro" refers to events that take place in an artificial environment, e.g., a test tube or reaction vessel, cell culture, etc., rather than within a multicellular organism.

[0039] In vivo: As used herein, the term "in vivo" refers to events that occur in multicellular organisms (e.g., humans and non-human animals). In the context of cell-based systems, the term can be used to refer to events that occur within living cells (e.g., as opposed to in vitro systems).

[0040] Isolated: As used herein, the term "isolated" can refer to (1) substances and / or entities that are separated from at least some of the components with which they are associated when originally produced (whether in nature and / or in an experimental setting) and / or (2) substances and / or entities that are designed, produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities can be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they are originally associated. In some embodiments, an isolated agent is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. In some embodiments, as one of skill in the art will understand, a substance can still be considered "isolated" or even "pure" even after being combined with other components, such as, for example, one or more carriers or excipients (e.g., buffers, solvents, water, etc.), and in such embodiments, the percent isolation or purity of the substance is calculated without including such carriers or excipients. By way of example only, in some embodiments, a biological polymer (e.g., a naturally occurring polypeptide or polynucleotide) is considered to be "isolated" if: a) by virtue of its origin or source, it is not associated with some or all of the components that accompany it in its native state in nature; b) it is substantially free from other polypeptides or nucleic acids of the same species from the species that produces it in nature; or c) it is expressed by or otherwise associated with components from a cell or other expression system other than the species that produces it in nature. Thus, for example, in some embodiments, a chemically synthesized polypeptide or a polypeptide synthesized in a cellular system other than the cellular system that produces it in nature is considered an "isolated" polypeptide.Alternatively, or additionally, a polypeptide that has been subjected to one or more purification techniques may be considered to be an "isolated" polypeptide to the extent that it has been separated from a) other components with which it is associated in nature, and / or b) other components with which it was associated when originally produced.

[0041] Operably linked: As used herein, the term "operably linked" can refer to a juxtaposition where the components described are in a relationship permitting them to function in their intended manner. A control element "operably linked" to a functional element is associated in such a way that expression and / or activation of the functional element is achieved under conditions compatible with the control element. In some embodiments, an "operably linked" control element is contiguous (e.g., covalently linked) with a coding element of interest. In some embodiments, the control element acts in trans or otherwise with the functional element of interest.

[0042] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to a composition in which an active agent is formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the composition is suitable for administration to a human or animal subject. In some embodiments, the active agent is present in a unit dose suitable for administration in a treatment regimen that exhibits a statistically significant likelihood of achieving a predetermined therapeutic effect when administered to an appropriate population.

[0043] Polypeptide: As used herein, the term "polypeptide" generally has its art-recognized meaning and refers to a polymer of at least three amino acids. Those of skill in the art will understand that the term "polypeptide" is sufficiently general and intended to encompass not only polypeptides having the complete sequences described herein, but also polypeptides corresponding to functional fragments of such complete polypeptides (i.e., fragments that retain at least one activity). Furthermore, those of skill in the art will understand that protein sequences generally tolerate some degree of substitution without destroying activity. Thus, polypeptides that retain activity and share at least 30-40% overall sequence identity, often greater than about 50%, 60%, 70%, or 80% sequence identity, with other polypeptides of the same class, and further include at least one region with much higher identity, often greater than 90%, or even 95%, 96%, 97%, 98%, or 99%, usually within a single highly conserved region (usually encompassing at least 3-4, often 20 or more amino acids), are encompassed by the related term "polypeptide" as used herein. Polypeptides can contain L-amino acids, D-amino acids, or both, and can include any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, and the like. In some embodiments, proteins can include natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to polypeptides that are less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids in length. In some embodiments, the protein is an antibody drug, an antibody fragment, a biologically active portion thereof, and / or a characteristic portion thereof.

[0044] Prevent or Prevention: As used herein, the terms "prophylaxis" or "prevention," when used in reference to the occurrence of a disease, disorder, and / or condition, can refer to a reduction in the risk of developing the disease, disorder, and / or condition and / or a delay in the onset and / or severity of one or more characteristics or symptoms of the disease, disorder, or condition. In some embodiments, prevention is assessed on a population basis, and an agent is considered to "prevent" a particular disease, disorder, or condition if there is a statistically significant reduction in the occurrence, frequency, and / or intensity of one or more symptoms of the disease, disorder, or condition in a population susceptible to the disease, disorder, or condition.

[0045] Recombinant: As used herein, the term "recombinant" can refer to a polypeptide that is designed, engineered, prepared, created, manufactured, and / or isolated by recombinant means, e.g., a polypeptide expressed using a recombinant expression vector transfected into a host cell; a polypeptide isolated from a recombinant combinatorial human polypeptide library; a polypeptide isolated or otherwise engineered from an animal (e.g., mouse, rabbit, sheep, fish, etc.) that is transgenic for a gene(s) or genetic components that encode and / or directly express the polypeptide, or one or more components, portions, elements, or domains thereof; and / or a polypeptide prepared, expressed, created, or isolated by any other means that involves splicing or ligating selected nucleic acid sequence elements together, chemically synthesizing selected sequence elements, and / or otherwise generating nucleic acids that encode and / or direct the expression of the polypeptide, or one or more components, portions, elements, or domains thereof. In some embodiments, one or more of such selected sequence elements are found in nature. In some embodiments, one or more of such selected sequence elements are designed in silico. In some embodiments, one or more such selected sequence elements result from mutagenesis (e.g., in vivo or in vitro) of known sequence elements derived from natural or synthetic sources (e.g., the germline of a source organism of interest (e.g., human, mouse, etc.)).

[0046] Specific binding: As used herein, the term "specific binding" can refer to the ability to distinguish between a potential binding partner within an environment in which binding can occur. A binding agent that interacts with one specific target in the presence of other potential targets is said to "specifically bind" to the interacting target. In some embodiments, specific binding is assessed by detecting or measuring the degree of association between the binding agent and its partner. In some embodiments, specific binding is assessed by detecting or measuring the degree of dissociation of the binding agent-partner complex. In some embodiments, specific binding is assessed by detecting or measuring the ability of a binding agent to compete with an alternative interaction between its partner and another entity. In some embodiments, specific binding is assessed by performing such detection or measurement over a range of concentrations.

[0047] Subject: As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human, including in some embodiments prenatal human forms). In some embodiments, the subject is suffering from the relevant disease, disorder, or condition. In some embodiments, the subject is susceptible to the disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of the disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of the disease, disorder, or condition. In some embodiments, the subject is one who possesses one or more characteristics characteristic of susceptibility to or risk for a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual for whom diagnosis and / or treatment is and / or has been administered.

[0048] Therapeutic Agent: As used herein, the term "therapeutic agent" generally refers to any agent that induces a desired pharmacological effect when administered to an organism. In some embodiments, an agent is considered a therapeutic agent if it exhibits a statistically significant effect across a suitable population. In some embodiments, the suitable population may be a population of model organisms. In some embodiments, the suitable population may be defined by various criteria (e.g., a particular age group, gender, genetic background, pre-existing clinical conditions, etc.). In some embodiments, a therapeutic agent is a substance that can be used to alleviate, ameliorate, mitigate, suppress, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition. In some embodiments, a "therapeutic agent" is a drug that has been approved, or requires approval, by a government agency before being marketed for administration to humans. In some embodiments, a "therapeutic agent" is a drug that requires a medical prescription for administration to humans.

[0049] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" refers to an amount that, when administered to a population suffering from or susceptible to a disease, disorder, and / or condition, is sufficient to treat the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is an amount that reduces the incidence and / or severity of, stabilizes one or more characteristics of, and / or delays the onset of, one or more symptoms of the disease, disorder, and / or condition. Those skilled in the art will appreciate that the term "therapeutically effective amount" does not actually require that a successful treatment be achieved in a particular individual. Rather, a therapeutically effective amount is considered to be an amount that, when administered to patients in need of such treatment, results in a specific, desired pharmacological response in a significant number of subjects. For example, in some embodiments, the term "therapeutically effective amount," in the context of the treatment of the present invention, refers to an amount that, when administered to an individual in need thereof, blocks, stabilizes, attenuates, or reverses cancer-supporting processes occurring in said individual, or enhances or increases cancer-suppressing processes in said individual. In the context of cancer treatment, a "therapeutically effective amount" is an amount that, when administered to an individual diagnosed with cancer, prevents, stabilizes, inhibits, or reduces further development of cancer in that individual. Particularly preferred "therapeutically effective amounts" of the compositions described herein reverse (therapeutic treatment) the development of a malignant tumor, such as pancreatic cancer, or help achieve or prolong remission of the malignant tumor. The therapeutically effective amount administered to an individual to treat cancer may be the same as or different from the therapeutically effective amount administered to promote remission or inhibit metastasis. As with most cancer treatments, the therapeutic methods described herein are not intended to be construed as, limited to, or otherwise confined to "curing" cancer; rather, the therapeutic methods are directed toward "treating" cancer using the described compositions, i.e., effecting a desired or beneficial change in the health of an individual with cancer.Such benefits are recognized by skilled medical providers in the field of oncology and include, but are not limited to, stabilization of a patient's condition, reduction in tumor size (tumor regression), improved functioning (e.g., improved function of cancerous tissue or organ), reduction or inhibition of further metastasis, reduction in opportunistic infections, increased survival, reduction in pain, improved motor function, improved cognitive function, improved energy (reduced fatigue), improved sense of well-being, restoration of normal appetite, restoration of healthy weight gain, and combinations thereof. Additionally, regression of a particular tumor in an individual (e.g., as a result of a treatment described herein) can also be assessed by taking samples of cancer cells from the site of the tumor, such as a pancreatic adenocarcinoma (e.g., over the course of treatment), and testing the cancer cells for levels of metabolic and signaling markers to monitor the state of the cancer cells and confirm at a molecular level that the cancer cells have regressed to a less aggressive phenotype. For example, tumor regression induced by the methods of the present invention is indicated by a decrease in any of the angiogenic markers discussed above, an increase in the anti-angiogenic markers described herein, or normalization (i.e., a change to that observed in normal individuals without cancer) of metabolic, intercellular, or intracellular signaling pathways that exhibit aberrant activity in individuals diagnosed with cancer. One of skill in the art will appreciate that in some embodiments, a therapeutically effective amount can be formulated and / or administered in a single dose. In some embodiments, a therapeutically effective amount can be formulated and / or administered in multiple doses, e.g., as part of a dosing regimen.

[0050] Transfection: As used herein, the term "transfection" can refer to the introduction of foreign nucleic acid into a cell using recombinant DNA techniques. As used herein, the term "transformation" can refer to the introduction of foreign gene, DNA, or RNA sequences into a host cell, whereby the host cell expresses the introduced gene or sequence to produce the encoded protein or enzyme.

[0051] Transduction. As used herein, "transduction" can refer to the introduction of foreign nucleic acid into cells using a viral vector.

[0052] Variant: As used herein in the context of molecules (e.g., nucleic acids, proteins, small molecules), the term "variant" can refer to a molecule that exhibits significant structural identity with a reference molecule but differs structurally from the reference molecule, e.g., the presence, absence, or level of one or more chemical moieties that differ from the reference entity. In some embodiments, a variant also differs functionally from its reference molecule. Generally, whether a particular molecule is appropriately considered a "variant" of a reference molecule is based on the degree of structural identity with the reference molecule. As one of skill in the art will understand, biological or chemical reference molecules have certain characteristic structural elements. A variant, by definition, is a distinct molecule that shares one or more characteristic structural elements but differs in at least one respect from the reference molecule. For example, to cite just a few examples, a polypeptide may have characteristic sequence elements composed of multiple amino acids whose positions relative to each other in linear or three-dimensional space are specified and / or contribute to a particular structural motif and / or biological function, and a nucleic acid may have characteristic sequence elements composed of multiple nucleotide residues whose positions relative to each other in linear or three-dimensional space are specified. In some embodiments, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid sequence or nucleotide sequence. In some embodiments, a variant polypeptide or nucleic acid exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% overall sequence identity to the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with the reference polypeptide or nucleic acid. In some embodiments, the reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, a variant polypeptide or nucleic acid shares one or more biological activities of the reference polypeptide or nucleic acid.

[0053] Vector: As used herein, the term "vector" can refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors can include both non-viral and viral carriers for introducing nucleic acids into cells in vitro, ex vivo, or in vivo.

[0054] A vector can be a replicon to which another DNA fragment is attached so as to amplify that fragment. The term "replicon" refers to any genetic element (e.g., a plasmid, phage, cosmid, chromosome, or virus) that can act as an autonomous unit of DNA replication in vivo. Many vectors known in the art can be used to manipulate nucleic acids, incorporate response elements and promoters into genes, etc. Preferred vectors include, but are not limited to, plasmids (e.g., PBR322 or pUC plasmid derivatives), modified viruses (e.g., adenovirus, retrovirus, adeno-associated virus, or herpes virus), or Bluescript vectors. For example, a DNA fragment corresponding to the response element and promoter can be inserted into an appropriate vector by combining it with a selected vector having complementary cohesive ends. In some embodiments, the ends of the DNA molecules are enzymatically modified, or arbitrary sites can be generated by ligating nucleotide sequences to the DNA termini via linkers. In some embodiments, vectors can be engineered to contain a selectable marker gene for screening cells that have integrated the marker into the cellular genome. Such markers allow for the identification and / or screening of host cells that express the protein encoded by the marker.

[0055] One type of vector, a "plasmid," refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector, a viral vector, allows additional DNA segments to be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell and replicate in conjunction with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors." Non-limiting examples of expression vectors and packaging constructs that can be used to deliver the chimeric antigen receptors described herein include retroviral vectors (e.g., SFG, pMX, pSAMEN, pMP71, pLXSN, pMSCV, pMSGV), lentiviral vectors (e.g., epHIV7, pLenO, pSIN, pSIEW, pELPS, pELNS, pHR), and packaging constructs (psPAX2, pRDF, pEQ-PAM3(-E), pVSVg, pCL, pMEVSVg, pMD2G, pMDLg / p.RRE, pRSV.REV, pTSV.rev, pCHGP, pCMV-g, pCMV-Rev2, pCMVdR8.91, pGALV).

[0056] In some embodiments, the vector provides the necessary regulatory sequences (e.g., transcription and translation elements) to regulate expression of the fusion protein in a suitable host cell. Regulatory sequences may include promoter regions, enhancer regions, transcription termination sites, ribosome binding sites, initiation codons, splice signals, introns, polyadenylation signals, Shine / Dalgarno translation sequences, and Kozak consensus sequences. Regulatory sequences are selected taking into account the host cell in which the fusion protein will be produced. In some embodiments, suitable bacterial promoters include, but are not limited to, bacteriophage λpL or pR, T6, T7, T7 / lacO, lac, recA, gal, trp, ara, hut, and trp-lac. In some embodiments, suitable eukaryotic promoters include, but are not limited to, PRBI, GAPDH, metallothionein, thymidine kinase, viral LTR, cytomegalovirus, SV40, or tissue-specific or tumor-specific promoters (e.g., alpha-fetoprotein, amylase, cathepsin E, M1 muscarinic receptor, gamma-glutamyltransferase).

[0057] In some embodiments, additional vectors include lipoplexes (cationic liposome-DNA complexes), polyplexes (cationic polymer-DNA complexes), and protein-DNA complexes. In addition to the nucleic acid, the vector may also contain one or more regulatory regions and / or selectable markers useful for selecting, measuring, and monitoring the outcome of nucleic acid delivery (e.g., delivery to a particular tissue or duration of expression).

[0058] Vectors can be introduced into cells by methods known in the art, such as injection, transfection, electroporation, microinjection, transduction, cell fusion, lipofection, calcium phosphate precipitation (Graham, Flet et al., Virology, 52:456 (1973); Chen and Okayama, Mol. Cell. Biol. 7:2745-2752 (1987)), liposome-mediated textured salt method (Wong, TK et al., Gene, 10:87 (1980); Nicolau and Sene, Biochim. Biophys. Acta, 721:185-190 (1982); Nicolau et al., Methods Enzymol., 149:157-176 (1987)), DEAE-dextran treatment (Gopal, Mol. Cell. Biol., 5:1188-1190 (1985)), gene bombardment (Yang et al., Proc. Natl. Acad. Sci., 87:9568-9572 (1990)), or the use of gene seeds or DNA vector transporters (Wu et al., J. Biol. Chem. 267:963 (1992); Wu et al., J. Biol. Chem. 263:14621 (1988); Hartmut et al., Canadian Patent Application No. 2,012,311).

[0059] In some embodiments, viral vectors have been used for a wide range of gene transfer applications in cells and live animal subjects. Viral vectors that can be used include, but are not limited to, adenovirus, retrovirus, vaccinia virus, poxvirus, adeno-associated virus, herpes simplex virus, lentivirus, baculovirus, Sendai virus, measles virus, Simian virus 40, and Epstein-Barr virus vectors. Non-viral vectors include plasmids, lipoplexes (cationic liposome-DNA complexes), polyplexes (cationic polymer-DNA complexes), and protein-DNA complexes. In addition to the nucleic acid, the vector may also contain one or more regulatory regions and / or selectable markers useful for screening, measuring, and monitoring the results of nucleic acid delivery (e.g., tissue delivery or persistence of expression).

[0060] In some embodiments, polynucleotides can be introduced in vivo by lipofection. Liposomes are increasingly being used to encapsulate and transfect nucleic acids in vitro. In some embodiments, liposomes for in vivo gene transfection can be prepared using synthetic cationic lipids designed to limit the difficulties and risks encountered with liposome-mediated transfection (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413 (1987); Mackey et al., Proc. Natl. Acad. Sci. USA 85:8027 (1988); Ulmer et al., Science 259:1745 (1993)). In some embodiments, the use of cationic lipids can facilitate encapsulation of negatively charged nucleic acids and can also facilitate fusion with negatively charged cell membranes (Feigner et al., Science 337:387 (1989)). Lipid compounds and compositions particularly useful for nucleic acid delivery are described in WO 95 / 18863, WO 96 / 17823, and U.S. Pat. No. 5,459,127, which are incorporated herein by reference in their entireties. In some embodiments, direct transfection of specific cell types is particularly desirable in tissues with apparent cellular heterogeneity (e.g., pancreas, liver, kidney, and brain). In some embodiments, lipids can be chemically conjugated to other molecules for targeting (Mackey et al., 1988). In some embodiments, targeted peptides, such as hormones or neurotransmitters, proteins, such as antibodies, or non-peptide molecules can be chemically conjugated to liposomes.

[0061] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures can generally be performed according to conventional methods known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 2 nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) (incorporated herein by reference for all purposes).

[0062] Engineered immune cells As used herein, "immune cells" refer to cells of the immune system, which are classified as lymphocytes (T cells, B cells, and NK cells), neutrophils, and monocytes / macrophages. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are NK cells. In some embodiments, the immune cells are macrophages. In some embodiments, the immune cells are engineered immune cells, meaning that they have been genetically modified to express a non-native protein (e.g., a chimeric antigen receptor) or to contain an exogenous nucleic acid.

[0063] Immune cells (e.g., T cells) can be modified in one way or in more than one way. Immune cells (e.g., T cells) can express at least one non-natural molecule that is a receptor for an antigen present on the surface of one or more types of cells. In some embodiments, the immune cells are engineered to contain or express at least one synthetic molecule not found in nature, thereby comprising immune cells (e.g., T cells) not found in nature. In certain embodiments, the immune cells (e.g., T cells) are engineered to express at least one chimeric antigen receptor (CAR), including a CAR that targets a specific tumor antigen (e.g., glypican 3 (GPC3), malignant tumor variant receptor (MVR), HLA-DR (human leukocyte antigen D-related), or CD19). In certain embodiments, the immune cells are T cells, e.g., CD4 + T cells, CD8 +The T cells may be a population of T cells, including T cells, Treg cells, Thl T cells, Th2 T cells, Thl7 T cells, non-specific T cells, or any combination thereof. Immune cells (e.g., T cells) engineered with chimeric antigen receptors (CARs) have great potential for cancer treatment. In CARs, the receptor recognizes an antigen, and when the antigen binds, the immune cells can be activated and programmed to kill cells expressing the antigen. Therefore, immune cells expressing CARs against an antigen expressed on tumor cells can target and kill tumor cells. For example, recent clinical trials of CD19-targeted CAR-transduced T cells (CD19-CAR T cells) for hematological malignancies have demonstrated the potent efficacy of CAR T technology (Kochenderfer, JNet al. (2010) Blood 116:4099-4102; Porter, DL, et al. (2011) N. Engl. J. Med. 365:725-733; Grupp, SA et al. (2013) N. Engl. J. Med. 368:1509-1518; Kochenderfer, JNet al. (2015) J. Clin. Oncol. 33:540-549; Brown, CE et al. (2016) A. Engl. J. Med. 375:2561-2569). The clinical success of CAR T is at least partly due to the fusion structure of the CAR, which is created by artificially combining a high-affinity antigen-binding domain with multiple signaling domains (Maus, MV et al. (2014) Blood 123:2625-2635; van der Stegen, SJ et al. (2015) Nat. Rev. Drug Discov. 14:499-509).

[0064] CARs comprise an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the extracellular antigen-binding domain comprises a single-chain variable fragment (scFv) capable of recognizing a tumor-associated antigen, the transmembrane domain uses a transmembrane domain derived from molecules such as CD8 and CD28, and the intracellular signaling domain uses an intracellular signaling domain from an immunoreceptor tyrosine-based activation motif (e.g., CD3ζ) and a costimulatory signal molecule (e.g., CD28 and CD137 (4-1BB)).

[0065] As used herein, "single-chain variable fragment (scFv)" refers to a fragment of an antibody defined as a recombinant protein comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) connected by a linker that joins the two domains to form an antigen-binding site.

[0066] In some embodiments, the transmembrane domain is selected from the group consisting of 4-1BB / CD137, activating NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8 alpha, CD8 beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, ligand specifically binding to CD83, LIGHT, LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA) -1), MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1), SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, and VLA-6.

[0067] In some embodiments, the intracellular signaling domain is selected from the group consisting of 4-1BB / CD137, activating NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8 alpha, CD8 beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, ligand specifically binding to CD83, LIGHT, LTBR, Ly9 (CD229), Lymphocyte function-associated antigen-1 (LFA-1), MHC class 1 The molecule comprises an intracellular signaling domain from a protein selected from NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1), SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, and VLA-6, or any combination thereof.

[0068] In some embodiments, the chimeric antigen receptor further comprises an additional antigen-binding domain. In some embodiments, the chimeric antigen receptor is a bispecific CAR (i.e., it targets two antigen-binding domains). In some embodiments, the chimeric antigen receptor is multivalent (i.e., it targets multiple antigen-binding domains). In some embodiments, the additional antigen-binding domain is an scFv.

[0069] Immune cells (e.g., T cells) can be obtained from any source known in the art. For example, immune (e.g., T) cells can be differentiated in vitro from a hematopoietic stem cell population, or immune (e.g., T) cells can be obtained from a subject. T cells can be obtained from peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, or tumor. In addition, immune (e.g., T) cells, etc., can be derived from one or more immune cell lines available in the art. In some embodiments, T cells can be obtained from blood drawn from a subject using any technique known to those skilled in the art (e.g., FICOLL™ separation and / or apheresis). Additional methods for isolating T cells for T cell therapy are disclosed in U.S. Patent Publication No. 2013 / 0287748. Other non-limiting examples can be found in International Patent Application No. PCT / US2015 / 014520 (published as WO2015 / 120096) and International Patent Application No. PCT / US2016 / 057983 (published as WO2017 / 070395), each of which is incorporated herein by reference in its entirety.

[0070] In some embodiments, the immune cells are autologous T cells. In some embodiments, the immune cells are obtained from a subject other than the patient. In some embodiments, the T cells used in the therapeutic methods are syngenic (identical twins, but different donor and recipient). In some embodiments, the T cells used in the therapeutic methods are allogeneic (same species, different donor) to the recipient subject. In some embodiments, the T cells are autologous stem cells (in the case of autologous stem cell therapy, i.e., ASCT). In some embodiments, the immune cells are non-autologous T cells. In some embodiments, the immune cells are obtained from a healthy donor. In some embodiments, the immune cells are obtained from a patient afflicted with cancer or a tumor.

[0071] T cells can be engineered to express, for example, chimeric antigen receptors (CARs). In some embodiments, CAR-T cells can be engineered to express an extracellular single-chain variable fragment (scFv). In some embodiments, CARs are engineered to express a costimulatory domain as a separate polypeptide chain. Exemplary CAR-T cell therapies and constructs are described in U.S. Patent Publication Nos. 2013 / 0287748, 2014 / 0227237, 2014 / 0099309, and 2014 / 0050708, which are incorporated by reference in their entireties.

[0072] CAR construct The present disclosure provides, at least in part, chimeric antigen receptor (CAR) polypeptides. As used herein, "chimeric antigen receptor (CAR)" refers to a receptor that does not exist in nature and can provide immune effector cells with specificity for a particular antigen. In some embodiments, CAR refers to a receptor used to deliver the specificity of a monoclonal antibody drug to T cells. Generally, a CAR comprises an extracellular binding domain (ectodomain), a transmembrane domain, and an intracellular signaling domain (endodomain).

[0073] In some embodiments, to achieve robust immune (e.g., CAR-T) cell proliferation, function, persistence, and anti-tumor activity, costimulatory signals can be provided by incorporating intracellular signaling domains from immune (e.g., T cell) cell costimulatory molecules into the CAR construct. In some embodiments, the selection and placement of costimulatory domains within the CAR construct influences immune (e.g., CAR-T) cell function and cell fate and can have different impacts on immune (e.g., CAR-T) cell kinetics, cytotoxic function, and potentially safety profile. Non-limiting examples of costimulatory molecules include CD28, ICOS, CD27, 4-1BB / CD137, OX40, and CD40L.

[0074] As used herein, 4-1BB / CD137 is an activation-induced T cell costimulatory molecule expressed on a subset of resting CD8+ T cells and is upregulated on both CD4+ and CD8+ T cells after activation. In some embodiments, T cells expressing CARs incorporating the 4-1BB / CD137 domain can express granzyme B, IFN-γ, TNF-α, GM-CSF, and the anti-apoptotic protein Bcl-XL (Zhong et al., Mol. Ther. 2010;18:413-420), and CARs incorporating the 4-1BB / CD137 costimulatory domain can exhibit longer CAR-T cell persistence (Zhao et al., Cancer Cell 2015;28:415-428). In some embodiments, the intracellular domain of the chimeric receptors described herein comprises a 4-1BB signaling domain followed by a five amino acid sequence, which can be further combined with any other desired extracellular, transmembrane, and / or intracellular domains useful in the context of the chimeric receptor.

[0075] In some embodiments, the CAR comprises (a) an extracellular domain comprising an antigen-binding domain, (b) a transmembrane domain, and (c) an intracellular domain comprising a costimulatory endodomain, wherein the costimulatory endodomain comprises an intracellular signaling domain derived from 4-1BB / CD137 and five additional amino acids. As contemplated herein, a CAR construct can comprise an extracellular domain directed to any desired antigen-binding domain. In some embodiments, the costimulatory endodomain comprises an intracellular signaling domain derived from 4-1BB / CD137 and five additional amino acids, wherein the five additional amino acids are encoded by SEQ ID NO: 1. In some embodiments, the costimulatory endodomain comprises SEQ ID NO: 2. In some embodiments, the costimulatory endodomain comprises a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2 or 4.

[0076] SEQ ID NO: 1 - 5 additional amino acids (DNA sequence) CGTTTCTCTGTTGTT SEQ ID NO:2 - 4-1BB costimulatory domain and 5 additional amino acids (DNA sequence) TIFF2025118764000002.tif18161 SEQ ID NO: 3 - 5 additional amino acids (amino acid sequence) RFSVV SEQ ID NO: 4 - 4-4-1BB costimulatory domain and 5 additional amino acids (amino acid sequence) TIFF2025118764000003.tif4139

[0077] In some embodiments, the extracellular binding domain of the CAR comprises an antigen-binding domain. In some embodiments, the antigen-binding domain specifically binds to an antigen associated with a disease. In some embodiments, the antigen-binding domain specifically binds to a tumor antigen. In some embodiments, the antigen-binding domain specifically binds to any number of targets, including surface antigens, cytoplasmic antigens, or nuclear antigens. For example, the antigen-binding domain may be any of the following: BCMA, CD2, CD3, CD4, CD8, CD10, CD19, CD20, CD22, CD23, CD33, CD38, CD44, CD52, CD70, CD99, CD138, CD123, CD274, TIM-3, members of the epidermal growth factor receptor family (erb1, erb2, erb3, erb4, and variants thereof), members of the ephrin receptor family (EphA1-10, EphB1-6), prostate-specific antigens (e.g., prostate stem cell antigen PSCA, prostate-specific membrane antigen PSMA), embryonic antigens (e.g., carcinoembryonic antigen CEA, fetal acetylcholine receptor), members of the vascular endothelial growth factor family (VEGFR1-3), and the like. ), epithelial cell adhesion molecule EpCAM, alpha-fetoprotein AFP, members of the mucin protein family (e.g., MUC1, MUC16), follicle-stimulating hormone receptor (FSHR), human high molecular weight melanoma-associated antigen (HMW-MAA), folate-binding protein FBP, α-folate receptor, ligands for the NKG2D receptor, members of the epithelial glycoprotein family (e.g., EGP-2, EGP-4), diasialogangliosides (e.g., GD2, GD3), members of the carbonic anhydrase family (e.g., CAIX), and members of the carbohydrate antigen family (e.g., Ley), including variants of the listed proteins and protein families. In some embodiments, the antigen-binding domain can bind to an antibody or fragment thereof that binds to a cytoplasmic or nuclear antigen, such as La / SSB antigen, a member of the Rho family of GTPases, a member of the high mobility group protein, or a fragment thereof. Similarly, the antigen-binding domain can bind to the alpha and beta chains or gamma and delta chains of the T cell receptor (TCR), or fragments thereof.In some embodiments, the antigen-binding domain can bind to peptides presented by the human leukocyte antigen class (HLA) I and II protein complexes. Examples include, but are not limited to, the EGFR family, survivin, the sry-like high mobility group box (SOX) protein family, melanoma-associated antigens (e.g., autoimmune cancer / testis antigen NY-ESO-1, melanoma antigen family A member MAGEA, melanoma-preferentially expressed antigens PRAME, gp100, MART-1), and leukemia-associated antigens (e.g., AML1-ETO, DEK-CAN, PML-RAR alpha, Flt3-ITD, NPM1, AurA, Bcl-2, Examples of antigen-binding domains include Bl-1, BMI1, BRAP, CML28, CML66, cyclin A, cyclin B1, cyclin E, CYP1B1, ETO / MTG8, G250 / CAIX, HOXA9, hTERT, Mcl-1, MAGE, mesothelin, mHAg, myeloperoxidase, MPP11, MUC1, NuSAP1, OFA / iLRP, PASD1, PRAME, proteinase 3, RAGE-1, RGS5, RHAMM, SSX2IP, survivin, and Wilms' tumor gene 1 (WT1). The antigen-binding domain can bind to a cytokine receptor (e.g., IL-13 receptor, IL-22 receptor), an NKG2D receptor (e.g., ULBP1, ULBP2), an EGFR family member, or an autoreactive TCR. In some embodiments, the antigen-binding domain specifically binds to a tumor antigen. Examples include, but are not limited to, glypican 3 (GPC), MVR (malignant tumor variant receptor), HLA-DR (human leukocyte antigen D-related), AFP, CEA, CA-125, MUC-1, ETA, tyrosinase, MAGE, immature laminin receptor, TAG-72, HPV E6, HPV E7, BING-4, calcium-activated chloride channel 2, cyclin B1, 9D7, Ep-CAM, EphA3, Her2 / neu, telomerase, mesothelin, SAP-1, survivin, NY-ESO-1 / LAGE-1, PRAME, SSX-2, BRCA1 / 2, CDK4, CML66, or CD 19. In some embodiments, the antigen-binding domain is or comprises an antibody drug.In some embodiments, the antigen binding domain is or comprises an antibody agent that specifically binds to GPC3, MVR, HLA-DR, or CD19.

[0078] In some embodiments, the transmembrane domain is selected from the group consisting of 4-1BB / CD137, activating NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8 alpha, CD8 beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, ligand specifically binding to CD83, LIGHT, LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA) In some embodiments, the transmembrane domain is derived from a protein selected from: MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1), SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, and VLA-6. In some embodiments, the transmembrane domain is derived from CD8α. In some embodiments, the transmembrane domain comprises SEQ ID NO:5.In some embodiments, the transmembrane domain comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:5.

[0079] SEQ ID NO:5 - CD8 / hinge / transmembrane TIFF2025118764000004.tif25160

[0080] In some embodiments, the intracellular domain further comprises an intracellular domain derived from CD3ζ. In some embodiments, the intracellular domain comprises SEQ ID NO: 6. In some embodiments, the intracellular domain comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 6.

[0081] SEQ ID NO: 6 - CD3ζ TIFF2025118764000005.tif46161

[0082] In some embodiments, the CAR further comprises a T2A self-cleaving peptide. In some embodiments, the CAR further comprises a signal peptide or leader sequence. In some embodiments, the CAR further comprises a CD8α leader sequence. In some embodiments, the CAR further comprises a flag tag sequence. In some embodiments, the CAR further comprises a hinge region. In some embodiments, the hinge region is a CD8α hinge. In some embodiments, the CAR further comprises SEQ ID NO: 7. In some embodiments, the CAR further comprises SEQ ID NO: 8. In some embodiments, the extracellular domain comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 7 or 8.

[0083] SEQ ID NO:7 - CD8α leader sequence TIFF2025118764000006.tif10159 SEQ ID NO: 8 - Flag-tag sequence TIFF2025118764000007.tif3128

[0084] GPC3 CAR Glypican 3 (GPC3), a cell surface protein encoded by the human GPC3 gene, is an oncofetal antigen frequently re-expressed on neoplastic hepatocytes (Vidali, et al., 2008, J Hepatol 48:399-406). GPC3 is highly expressed in fetal liver but not in normal adult liver tissue. However, its expression is reactivated in hepatocellular carcinoma (HCC), which is closely associated with the development of HCC. GPC3 expression is relatively high in the early stages of HCC and increases with progression. Furthermore, GPC3 is also expressed in tumors such as melanoma, ovarian clear cell carcinoma, yolk sac tumor, and neuroblastoma. Given its specific high expression in hepatocellular carcinoma, melanoma, and other tumors, GPC3 has emerged as a useful immunohistochemical diagnostic test (Anatelli, et al., 2008, Am J Clin Path 130:219-223) and a potential biomarker (Aburatani, 2005, J Gastroenterol 40.SI 6:1-6).

[0085] GPC3 is a member of the proteoglycan family and functions as an extracellular matrix for cell adhesion during organogenesis and as a receptor for cell growth factors. The GPC3 protein core contains two subunits: an N-terminal subunit and a C-terminal subunit. A glycosylphosphatidylinositol (GPI) anchor is attached to serine at position 560 on the carboxyl (C) terminus of GPC3. The GPI anchor localizes GPC3 to the cell surface by covalently binding to plasma membrane lipids. Furthermore, serine at positions 495 and 509 of GPC3 are modified with heparan sulfate (HS) chains. These HS chains are known to regulate multiple growth signaling pathways, including Wnt signaling, FGF signaling, and BMP signaling. The growth signaling pathways involved vary depending on the type of cancer. For example, in hepatocellular carcinoma (HCC), cell growth is mediated by stimulation of the Wnt signaling pathway.

[0086] The present disclosure provides, at least in part, a GPC3 CAR polypeptide. In some embodiments, the extracellular binding domain of the GPC3 CAR comprises an antigen-binding domain. In some embodiments, the antigen-binding domain is or comprises an antibody drug. In some embodiments, the antigen-binding domain is or comprises an antibody drug that specifically binds to GPC3.

[0087] In some embodiments, the chimeric antigen receptor (CAR) polypeptide comprises i) an extracellular antigen-binding domain comprising a light chain variable domain comprising a light chain CDR1 comprising SEQ ID NO: 9, a light chain CDR2 comprising SEQ ID NO: 10, and a light chain CDR3 comprising SEQ ID NO: 11, and a heavy chain variable domain comprising a heavy chain CDR1 comprising SEQ ID NO: 12, a heavy chain CDR2 comprising SEQ ID NO: 13, and a heavy chain CDR3 comprising SEQ ID NO: 14; ii) a transmembrane domain; and iii) an intracellular signaling domain that activates the T cell when an antigen is bound to the antibody drug.

[0088] TIFF2025118764000008.tif52161

[0089] In some embodiments, the CAR polypeptide comprises i) an extracellular antigen-binding domain comprising a light chain variable domain comprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 15 and a heavy chain variable domain comprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 16, ii) a transmembrane domain, and iii) an intracellular signaling domain that activates a T cell when an antigen is bound to the antibody drug. In some embodiments, the CAR polypeptide comprises i) an extracellular antigen-binding domain comprising a light chain variable domain comprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 17 and a heavy chain variable domain comprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 18, ii) a transmembrane domain, and iii) an intracellular signaling domain that activates a T cell when an antigen is bound to the antibody drug.

[0090] In some embodiments, the CAR polypeptide comprises i) an extracellular antigen-binding domain comprising a light chain variable domain comprising SEQ ID NO: 15 and a heavy chain variable domain comprising SEQ ID NO: 16, ii) a transmembrane domain, and iii) an intracellular signaling domain that activates the T cell when an antigen is bound to the antibody drug.

[0091] SEQ ID NO: 15 - Human GC33 light chain variable region (amino acid sequence) TIFF2025118764000009.tif11160 SEQ ID NO: 16 - Human GC33 heavy chain variable region (amino acid sequence) TIFF2025118764000010.tif18159 SEQ ID NO: 17 - Human GC33 light chain variable region (DNA sequence) TIFF2025118764000011.tif39161 SEQ ID NO: 18 - Human GC33 heavy chain variable region (DNA sequence) TIFF2025118764000012.tif46160

[0092] MYR CAR Human leukocyte antigen-DR (HLA-DR) is a classical major histocompatibility complex II molecule (Shackelford, DA et al., 1982 Immunol. Rev. 66:133-187). HLA-DR and its ligands, peptides of 9 or more amino acids in length, constitute ligands for the T cell receptor (TCR). HLA-DR molecules are upregulated in response to signals. In the case of infection, peptides (e.g., Staphylococcal enterotoxin I peptides) bind to DR molecules and are presented to T cell receptors found on T helper cells. These cells then bind antigens on the surface of B cells, stimulating B cell proliferation.

[0093] The primary function of HLA-DR is to present peptide antigens, potentially of foreign origin, to the immune system, eliciting or suppressing T (helper) cell responses and ultimately eliciting antibody production against the same peptide antigens. HLA-DR is an αβ heterodimer, a cell surface receptor, with each subunit containing two extracellular domains, a transmembrane domain, and a cytoplasmic tail. Both the α and β chains are membrane-anchored. The N-terminal domain of the mature protein forms an alpha helix that constitutes the exposed portion of the binding groove, while the C-terminal cytoplasmic region interacts with the other chain below the binding groove to form a β sheet that spans the cell membrane. The majority of peptide contact sites are located within the first 80 residues of each chain.

[0094] HLA-DR is expressed exclusively on antigen-presenting cells (e.g., dendritic cells, macrophages, monocytes, and B cells). Because the abundance of the HLA-DR "antigen" on the cell surface often increases in response to stimulation, HLA-DR is also a marker of immune stimulation. Because HLA-DR expression levels are high in B-cell malignancies and its spectrum is limited in normal cells, antibodies against HLA-DR have been developed and are being tested in B-cell malignancies in preclinical and clinical trials (Nagy, ZA, et al. (2002) Nat. Med. 8:801-807; DeNardo, GL, et al. (2005) Clin. Cancer Res. 11:7075s-7079s; Ivanov, A., et al. (2009) J. Clin. Invest. 119:2143-2159; Lin, TS, et al. (2009) Leuk. Lymphoma 50:1958-1963). Although toxicity was not severe in a Phase I / II trial, efficacy was limited and further testing was discontinued (Lin, TS, et al. (2009) Leuk. Lymphoma 50:1958-1963).

[0095] As used herein, a malignant tumor variant receptor (MVR) antibody agent recognizes a polymorphic region of HLA-DR (as described in U.S. Patent Application Publication No. US2016-0257762, which is incorporated herein by reference in its entirety). The present disclosure provides, at least in part, MVR CAR polypeptides. A schematic of an exemplary MVR CAR construct according to the present disclosure is shown in FIG. 1. In some embodiments, the extracellular domain of the CAR comprises an antigen-binding domain. In some embodiments, the antigen-binding domain is or comprises an antibody agent. In some embodiments, the antigen-binding domain is or comprises an antibody agent that specifically binds to HLA-DR.

[0096] In some embodiments, the CAR polypeptide comprises a single-chain variable fragment (scFv) form of an anti-MVR antibody agent. In some embodiments, the CAR polypeptide comprises SEQ ID NO: 19. In some embodiments, the CAR comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 19.

[0097] In some embodiments, the CAR polypeptide comprises a single-chain variable fragment (scFv) form of an anti-MVR antibody agent. In some embodiments, the CAR polypeptide comprises SEQ ID NO: 20. In some embodiments, the CAR comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 20.

[0098] SEQ ID NO: 19 - MVRL2H2 (amino acid sequence) TIFF2025118764000013.tif33161

[0099] SEQ ID NO: 20 - MVRL2H2 (DNA sequence) TIFF2025118764000014.tif96161

[0100] CD19 CAR CD19 is a biomarker for normal B cells, neoplastic B cells, and follicular dendritic cells. CD19 is critically involved in establishing the intrinsic signaling threshold of B cells through regulating both B cell receptor-dependent and -independent signals. Furthermore, CD19, together with the complement receptor CD21, the tetraspanin membrane protein CD81 (TAPA-1), and CD225, functions as a key signaling component of a multimolecular complex on the surface of mature B cells. CD19 also plays an essential role in maintaining the balance between humoral antigen-induced responses and tolerance induction.

[0101] The present disclosure provides, at least in part, a CD19 CAR polypeptide. In some embodiments, the extracellular domain of the CAR comprises an antigen-binding domain. In some embodiments, the antigen-binding domain is or comprises an antibody drug. In some embodiments, the antigen-binding domain is or comprises an antibody drug that specifically binds to CD19.

[0102] In some embodiments, the CAR polypeptide comprises a single-chain variable fragment (scFv) form of an anti-CD19 antibody agent. In some embodiments, the CAR polypeptide comprises SEQ ID NO: 21. In some embodiments, the CAR comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 21.

[0103] In some embodiments, the CAR polypeptide comprises a single-chain variable fragment (scFv) form of an anti-CD19 antibody agent. In some embodiments, the CAR polypeptide comprises SEQ ID NO: 22. In some embodiments, the CAR comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 22.

[0104] SEQ ID NO: 21 - CD19 (amino acid sequence) TIFF2025118764000015.tif32160 SEQ ID NO: 22 - CD19 (DNA sequence) TIFF2025118764000016.tif89161

[0105] nucleic acid As used herein, "nucleic acid" is used to include any compound and / or substance, including polynucleotides. Exemplary nucleic acids or polynucleotides include, but are not limited to, ribonucleic acid (RNA) and / or deoxyribonucleic acid (DNA).

[0106] In some embodiments, the nucleic acid construct comprises a region encoding a CAR, wherein the CAR comprises (a) an extracellular domain comprising an antigen-binding domain, (b) a transmembrane domain, and (c) an intracellular domain comprising an intracellular signaling domain derived from 4-1BB / CD137 and five additional amino acids. In some embodiments, the nucleic acid construct can be inserted into an expression vector or viral vector using methods known in the art, and the nucleic acid molecule can be operably linked to an expression control sequence. Non-limiting examples of expression vectors include plasmid vectors, transposon vectors, cosmid vectors, and virus-derived vectors (e.g., any adenovirus-derived vector (AV), cytomegalovirus-derived vector (CMV), simian virus-derived vector (SV40), adeno-associated virus (AAV) vector, lentiviral vector, and retroviral vector). In some embodiments, the expression vector is a viral vector. In some embodiments, the viral vector is a lentiviral vector.

[0107] In some embodiments, the expression vector further comprises a promoter operably linked to the nucleic acid. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the expression vector comprises SEQ ID NO: 23, 24, 25, 26, 27, and / or 28. In some embodiments, the expression vector comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 23, 24, 25, 26, 27, and / or 28.

[0108] SEQ ID NO: 23 - EF1α promoter TIFF2025118764000017.tif145161 SEQ ID NO: 24 - U5 repeat TIFF2025118764000018.tif11160 SEQ ID NO: 25 - Gag / Pol TIFF2025118764000019.tif174161 SEQ ID NO: 26 - cPPT TIFF2025118764000020.tif67161 SEQ ID NO: 27 - Woodchuck / PRE TIFF2025118764000021.tif74160 Sequence number 28 - R / Region TIFF2025118764000022.tif11161

[0109] Lentiviral vectors are derived from lentiviruses. They are based on single-stranded RNA lentiviruses, a subclass of retroviruses. These vectors combine the advantages of moderate cloning capacity and stable gene expression, allowing them to transduce dividing and non-dividing cells, including neural cells. Upon infection, the lentiviral genome integrates the transgene into the host genome, promoting long-term gene expression. Lentiviral vectors, such as HIV-based vectors, are examples of retroviral vectors used for gene delivery. Unlike other retroviruses, HIV-based vectors are known to integrate their passenger genes into non-dividing cells, allowing them to be used to treat persistent forms of disease.

[0110] Additional sequences can be added to such cloning and / or expression sequences to optimize cloning and / or expression function, aid in polynucleotide isolation, improve polynucleotide introduction into cells, etc. The use of cloning vectors, expression vectors, adapters, and linkers is well known in the art.

[0111] In some embodiments, the nucleic acid molecule is inserted into a vector that is capable of expressing the CAR of the present disclosure when introduced into an engineered immune cell. In some embodiments, the engineered immune cell is a T cell.

[0112] CAR-T cell production Provided herein are methods for producing immune cells comprising a CAR. In some embodiments, the immune cells into which a CAR is introduced are human immune cells. In some embodiments, the immune cells are autologous human immune cells. In some embodiments, the immune cells are allogeneic human immune cells. In some embodiments, the immune cells are CD4 + T cells (helper T cells, TH cells), CD8 + In some embodiments, the immune cells are NK cells, including, but not limited to, T cells (cytotoxic T cells, CTLs), memory T cells, regulatory T cells (Treg cells), and apoptotic T cells.

[0113] In some embodiments, viral infection of immune cells can include transfecting host cells (e.g., 293T cells, PBMCs, Plat-GP cells, or PA317) with a CAR expression vector and a packaging plasmid to prepare a recombinant virus, and infecting immune cells with the recombinant virus. Viral infection can be performed by any method known in the art. In some embodiments, transfer of the CAR expression vector into immune cells can be confirmed by examining CAR expression by flow cytometry, Northern blotting, Southern blotting, PCR (e.g., RT-PCR), ELISA, or Western blotting, or by examining the expression of a marker gene inserted into the vector.

[0114] In some embodiments, the disclosure provides a method of producing an engineered immune cell, the method comprising introducing into an immune cell (i) a nucleic acid encoding a CAR, wherein the CAR comprises (a) an extracellular domain comprising an antigen-binding domain, (b) a transmembrane domain, and (c) an intracellular domain comprising a costimulatory endodomain, wherein the costimulatory endodomain comprises an intracellular signaling domain derived from 4-1BB / CD137 and five additional amino acids, or (ii) a vector comprising a nucleic acid encoding a CAR, wherein the CAR comprises (a) an extracellular domain comprising an antigen-binding domain, (b) a transmembrane domain, and (c) an intracellular domain comprising a costimulatory endodomain, wherein the costimulatory endodomain comprises an intracellular signaling domain derived from 4-1BB / CD137 and five additional amino acids.

[0115] In some embodiments, to enhance the immunological efficacy of the 4-1BB cytoplasmic signaling domain, five amino acids are added to the 4-1BB cytoplasmic domain used in producing CARs as a costimulatory signaling factor. In some embodiments, the completed construct comprises an antigen-binding domain that is an scFv, an EFL-α promoter, a human CD8 hinge region and transmembrane domain, and an intracellular signaling domain. Specifically, the intracellular signaling domain comprises a stimulatory domain and a costimulatory signaling domain. In some embodiments, the transmembrane domain may comprise, but is not limited to, the alpha, beta, or zeta chain of the T cell receptor, or one or more of CD28, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. In some embodiments, the transmembrane domain comprises CD8. In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain within the CD3 zeta primary signaling domain selected from CD28, OX40, CD27, ICAM-1, ICOS (CD278), and 4-1BB / CD137. In some embodiments, the costimulatory domain comprises 4-1BB with an additional five consecutive amino acids. In some embodiments, the costimulatory domain is linked to CD3 zeta.

[0116] In some embodiments, the methods of producing engineered immune cells of the present disclosure further comprise culturing the engineered immune cells in vitro for at least 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or 12 days.

[0117] In some embodiments, the method of producing the engineered immune cells further comprises culturing the engineered immune cells after the introducing step. In some embodiments, the method of producing the engineered immune cells further comprises obtaining the immune cells from the subject before the introducing step.

[0118] Any method for expressing CAR in immune cells known in the art can be used in the context of the present disclosure. For example, various expression nucleic acid vectors, such as linear polynucleotides, polynucleotides linked to ionic or amphiphilic compounds, plasmids, or viral vectors, are known in the art, but the present disclosure is not limited thereto. In some embodiments, the vector for expressing CAR in immune cells can be or include a self-replicating plasmid or virus, or a derivative thereof. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, etc. In some embodiments, a lentivirus vector, which is a retrovirus vector, can be used. In some embodiments, the vector is a non-plasmid and non-viral compound (e.g., liposome).

[0119] The present disclosure encompasses the recognition that CAR-T cells generated by the methods described herein are therapeutically useful (e.g., in the treatment of cancer).

[0120] therapeutic use Provided herein is a method for treating a subject with cancer or other malignant tumors, comprising administering to the subject a composition comprising or delivering immune cells comprising CAR. In some embodiments, the cancer is an anti-glypican 3-associated cancer. In some embodiments, the cancer is an anti-CD19-associated cancer. In some embodiments, the cancer is an anti-MVR-associated cancer.

[0121] Cancer can refer to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth leads to the formation of malignant tumors, which can invade adjacent tissues and even metastasize to distant parts of the body via the lymphatic system or bloodstream. Cancer or cancerous tissue can include tumors.

[0122] "Anti-glypican 3-associated cancers" are cancers characterized by the presence of glypican 3 on the surface of cancer cells. GPC3, a membrane-bound heparan sulfate proteoglycan, is overexpressed in approximately 70-80% of hepatocellular carcinomas, but is not generally expressed in healthy tissue. In addition, GPC3 overexpression has been observed in several tumors, including, but not limited to, hepatocellular carcinoma, hepatoblastoma, germ cell tumors (e.g., yolk sac tumor, trophoblastic tumor), Wilms' tumor, gastric cancer, non-small cell lung cancer, and thyroid cancer.

[0123] "Anti-CD19-associated cancers" are cancers characterized by CD19 expression, indicating that CD19 plays an essential role in B-cell development and maturation. CD19 expression is highly conserved in most B-cell malignancies. It is expressed in most acute lymphocytic leukemias (ALL), chronic lymphocytic leukemias (CLL), and B-cell lymphomas.

[0124] "Anti-MVR-associated cancers" are characterized by cancer cells that have increased expression of the HLA-DR antigen compared to non-cancerous cells of a subject. In some embodiments, cancers with high expression of the HLA-DR antigen include, but are not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, hematological cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, gastric cancer, thyroid cancer, pancreatic cancer, and prostate cancer. In some embodiments, diseases associated with HLA-DR expression include, but are not limited to, atypical and / or non-classical cancers, malignancies, precancerous conditions, or proliferative disorders that express HLA-DR, or a combination thereof.

[0125] In some embodiments, cancers to be treated according to the methods of the present disclosure include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. In some embodiments, cancers may include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastric cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer.

[0126] In some embodiments, the cancer suitable for treatment according to the methods of the present disclosure is a blood cancer. In some embodiments, the blood cancer is a leukemia. In some embodiments, the cancer is one or more acute leukemias (including but not limited to, B-cell acute lymphoblastic leukemia ("BALL"), T-cell acute lymphoblastic leukemia ("TALL"), acute lymphoblastic leukemia (ALL); one or more chronic leukemias (including but not limited to, chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL)); additional blood cancers or conditions (including but not limited to, B-cell lymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, and and selected from the group consisting of diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndromes, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, and "preleukemia" (which includes a diverse collection of blood conditions united by the defective production (or dysplasia) of myeloid blood cells).

[0127] In some embodiments, the cancer to be treated by the methods of the present disclosure is B-cell lymphoma (i.e., malignant lymphoma of B-cell origin). Examples of B-cell lymphoma include, but are not limited to, Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, mucosa-associated lymphoid tissue lymphoma (MALT), chronic lymphocytic leukemia, mantle cell lymphoma (MCL), Burkitt's lymphoma, mediastinal large B-cell lymphoma, Waldenstrom's macroglobulinemia, nodal marginal zone B-cell lymphoma (NMZL), splenic marginal zone lymphoma (SMZL), intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, and AIDS-related lymphoma, as long as the lymphoma is derived from B cells.

[0128] The immune cells (e.g., CAR-T cells) can be administered to a patient in need thereof in a therapeutically effective amount. For example, a therapeutically effective amount of immune cells (e.g., CAR-T cells) can be administered in a therapeutically effective amount of at least about 10 4 cells, at least about 10 5 cells, at least about 10 6 cells, at least about 10 7 cells, at least about 10 8 cells, at least about 10 9 cells, or at least about 10 10 In some embodiments, the therapeutically effective amount of T cells can be about 10 4 cells, about 10 5 cells, about 10 6 cells, about 10 7 cells, or approximately 10 8 In some embodiments, the therapeutically effective amount of T cells is about 0.4 x 10 8 ~about 2×10 8 In some embodiments, the therapeutically effective amount of T cells is about 0.4 x 10 8 , about 0.5×10 8 , about 0.6×10 8 , about 0.7×10 8 , about 0.8×10 8 , about 0.9×10 8 , about 1.0×10 8 , about 1.1×108 , about 1.2×10 8 , about 1.3×10 8 , about 1.4×10 8 , about 1.5×10 8 , about 1.6×10 8 , about 1.7×10 8 , about 1.8×10 8 , about 1.9×10 8 , or approximately 2.0 × 10 8 T cells.

[0129] In some embodiments, the therapeutically effective amount of CAR T cells is about 2 x 10 6 cells / kg, approximately 3×10 6 cells / kg, approximately 4×10 6 cells / kg, approximately 5×10 6 cells / kg, approximately 6×10 6 cells / kg, approximately 7×10 6 cells / kg, approximately 8×10 6 cells / kg, approximately 9×10 6 cells / kg, approximately 1×10 7 cells / kg, approximately 2×10 7 cells / kg, approximately 3×10 7 cells / kg, approximately 4×10 7 cells / kg, approximately 5×10 7 cells / kg, approximately 6×10 7 cells / kg, approximately 7×10 7 cells / kg, approximately 8×10 7 cells / kg, or approximately 9 x 10 7 In some embodiments, the therapeutically effective amount of immune cells (e.g., CAR-T cells) is about 1 x 10 cells / kg of body weight. 6 ~about 2×10 6 T cells, with a maximum dose of approximately 1 × 10 8 In some embodiments, the therapeutically effective amount of T cells is about 1 x 10 per kg of body weight. 6 or about 2 x 10 6 T cells, with a maximum dose of approximately 1 × 10 8 T cells.

[0130] The number of cells will depend on the intended end use of the composition and also on the type of cells included in the composition. For example, in some embodiments, a population of T cells comprising a CAR will comprise more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, or more than 35% of such cells. In some embodiments, a population of T cells comprising a CAR will comprise 10%-50%, 15%-45%, 20%-40%, 25%-35%, or 20%-30% of such T cells. In some embodiments, the T cell population for administration is in a volume of 1 liter or less. In some embodiments, the T cells for administration are in a volume of less than 500 ml, less than 250 ml, or less than 100 ml. In some embodiments, the desired T cell density is typically less than 10 6 >10 cells / ml, typically 7 >10 cells / ml, typically 8 A clinically relevant number of immune cells is ≥ 10 7 cells, 10 8 cells, 10 9 cells, 10 10 cells, 10 11 cells, or 10 12 The cells can be distributed over multiple infusions equal to or greater than these.

[0131] In some embodiments, the composition can be administered parenterally to a patient. In some embodiments, the composition comprising or delivering T cells comprising a CAR can be administered parenterally to a patient in one or more doses. In some embodiments, the composition comprising or delivering T cells comprising a CAR can be administered parenterally to a patient once daily, once every 2-7 days, once a week, once every two weeks, once a month, once every three months, or once every six months.

[0132] In some embodiments, the disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject engineered immune cells comprising a CAR, wherein the CAR comprises (a) an extracellular domain comprising an antigen-binding domain, (b) a transmembrane domain, and (c) an intracellular domain comprising a costimulatory endodomain, wherein the costimulatory endodomain comprises an intracellular signaling domain derived from 4-1BB / CD137 and five additional amino acids.

[0133] In some embodiments, the subject has previously been administered one or more additional anti-cancer therapies, wherein the anti-cancer therapies are selected from the group consisting of ionizing radiation, chemotherapeutic agents, therapeutic antibodies, and checkpoint inhibitors. In some embodiments, the subject has been identified or diagnosed as having cancer.

[0134] Pharmaceutical Composition In some embodiments, the present disclosure provides a pharmaceutical composition comprising T cells comprising a CAR, wherein the CAR comprises (a) an extracellular domain comprising an antigen-binding domain, (b) a transmembrane domain, and (c) an intracellular domain comprising a costimulatory endodomain, wherein the costimulatory endodomain comprises an intracellular signaling domain derived from 4-1BB / CD137 and five additional amino acids. In some embodiments, the T cells comprising a CAR are autologous T cells. In some embodiments, the pharmaceutical composition can comprise a buffer, a diluent, a solubilizer, an emulsifier, a preservative, an adjuvant, an excipient, or any combination thereof. In some embodiments, the composition can also comprise one or more additional therapeutically active agents, if desired.

[0135] In some embodiments, the T cells of the present disclosure are formulated by first harvesting them from their culture medium, then washing and concentrating a therapeutically effective amount of the cells in a medium and container system (a "pharmaceutically acceptable" carrier) suitable for administration. A suitable infusion medium can be any isotonic medium formulation, typically saline, Normosol R (Abbott), or Plasma-Lyte A (Baxter), although 5% dextrose in water or lactated Ringer's solution can also be utilized. The infusion medium may also be supplemented with human serum albumin.

[0136] In some embodiments, the composition is formulated for parenteral administration. For example, the pharmaceutical compositions provided herein can be provided in a sterile injectable form (e.g., a form suitable for subcutaneous injection or intravenous infusion). For example, in some embodiments, the pharmaceutical composition is provided in a liquid dosage form suitable for injection. In some embodiments, the pharmaceutical composition is provided as a powder (e.g., a lyophilized and / or sterilized powder), optionally under vacuum, which can be reconstituted with an aqueous diluent (e.g., water, a buffer, a salt solution, etc.) before injection. In some embodiments, the pharmaceutical composition is diluted and / or reconstituted with water, sodium chloride solution, sodium acetate solution, benzyl alcohol solution, phosphate buffered saline, etc. In some embodiments, the powder is gently mixed (e.g., without shaking) with the aqueous diluent.

[0137] In some embodiments, the CAR and / or T cells comprising a nucleic acid encoding a CAR of the present disclosure are formulated with a pharmaceutically acceptable parenteral vehicle. Examples of such vehicles include water, saline, Ringer's solution, dextrose solution, and 1-10% human serum albumin. Liposomes and non-aqueous vehicles (e.g., fixed oils) can also be used. The vehicle or lyophilized powder can contain additives to maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). In some embodiments, the formulation is sterilized by known or suitable techniques. Pharmaceutical compositions may further comprise pharmaceutically acceptable additives; as used herein, such additives include any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersing or suspending aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, appropriate for the particular desired dosage form. Remington's The Science and Practice of Pharmacy, 21st Edition, A.R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006) discloses various additives used in formulating pharmaceutical compositions and known techniques for their preparation. Except insofar as any conventional additive medium is incompatible with the substance or its derivatives, for example, by producing any undesirable biological effects or interacting in a deleterious manner with any other components of the pharmaceutical composition, its use is contemplated as being within the scope of this disclosure.

[0138] In some embodiments, compositions comprising a T cell population comprising a CAR of the present disclosure and / or a nucleic acid encoding a CAR are stably formulated. In some embodiments, stable formulations of a T cell population comprising a CAR of the present disclosure and / or a nucleic acid encoding a CAR can include saline or phosphate buffer with a selected salt, as well as formulations comprising a preservative and a preservative, and multi-purpose preservative formulations suitable for pharmaceutical or veterinary use. The preservative formulations contain at least one known preservative, or optionally, at least one selected from phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrate, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate), alkylparaben (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, thimerosal, or mixtures thereof, in an aqueous diluent. As known in the art, any suitable concentration or mixture can be used, for example, 0.001-5% or any range or value therein, such as, but not limited to, 0.001, 0.003, 0.005, 0.009, 0.01, 0.02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1. 0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.3, 4.5, 4.6, 4.7, 4.8, 4.9, or any range or value therein may be used.Non-limiting examples include no preservatives, 0.1-2% m-cresol (e.g., 0.2, 0.3, 0.4, 0.5, 0.9, 1.0%), 0.1-3% benzyl alcohol (e.g., 0.5, 0.9, 1.1, 1.5, 1.9, 2.0, 2.5%), 0.001-0.5% thimerosal (e.g., 0.005, 0.01%), 0.001-2.0% phenol (e.g., , 0, 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%), 0.0005 to 1.0% alkyl parabens (e.g., 0.00075, 0.0009, 0.001, 0.002, 0.005, 0.0075, 0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5, 0.75, 0.9, 1.0%), and the like.

[0139] In some embodiments, the pharmaceutical composition is provided in a form that can be refrigerated and / or frozen. In some embodiments, the pharmaceutical composition is provided in a form that cannot be refrigerated and / or frozen. In some embodiments, the reconstituted solution and / or liquid dosage form can be stored for a specified period of time after reconstitution (e.g., 2 hours, 12 hours, 24 hours, 2 days, 5 days, 7 days, 10 days, 2 weeks, 1 month, 2 months, or more). In some embodiments, storing a composition containing an antibody drug for longer than the specified time period results in degradation of the antibody drug. The liquid dosage form and / or reconstituted solution may contain particulate matter and / or discoloration prior to administration. In some embodiments, the solution should not be used if discoloration or cloudiness is present and / or if particulate matter remains after filtration. General considerations for the formulation and / or manufacture of pharmaceutical agents are described, for example, in Remington: The Science and Practice of Pharmacy 21 st ed., Lippincott Williams & Wilkins, 2005.

[0140] In some embodiments, a pharmaceutical composition comprising a CAR of the present disclosure and / or T cells comprising a nucleic acid encoding a CAR can be contained in a container for storage or administration, e.g., a vial, a syringe (e.g., an IV syringe), or a bag (e.g., an IV bag). Pharmaceutical compositions according to the present disclosure can be prepared, packaged, and / or sold in bulk as a single unit dose and / or as a plurality of single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition comprising a predetermined amount of an active ingredient. The amount of the active ingredient is approximately equal to the dosage of the active ingredient that would be administered to a subject and / or a convenient fraction of such a dosage (e.g., one-half or one-third of such a dosage).

[0141] kit The present disclosure further provides kits comprising one or more containers filled with at least one CAR and / or CAR-encoding nucleic acid described herein. The kits can be used in any applicable method, including therapeutic methods, diagnostic methods, cell proliferation and / or isolation methods, etc. Optionally, such containers can be accompanied by a notice in a format prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which notice reflects (a) the agency's approval for manufacture, use, or sale for human administration, (b) instructions for use, or both.

[0142] In some embodiments, the kit can include one or more reagents for detection (e.g., detection of a CAR and / or a nucleic acid encoding a CAR). In some embodiments, the kit can include a CAR and / or a nucleic acid encoding a CAR in a detectable form (e.g., covalently linked to a detectable moiety or entity). In some embodiments, one or more CARs and / or nucleic acids encoding a CAR provided herein can be included in a kit for use in treating a subject. In some embodiments, a CAR and / or nucleic acid encoding a CAR provided herein can be included in a kit for use in preparing autologous T cells expressing a CAR.

[0143] In some embodiments, the kits can provide one, two, three, four, or more antigen-specific antibody agents, each suitable for cloning into a CAR construct. In some embodiments, the kits can provide other reagents for measuring the binding affinity of the antibody agents and / or CARs and / or CAR T cells with T cells identified or isolated from a subject. In some embodiments, the kits can provide other reagents for assaying the functional avidity of the antibody agents and / or CARs and / or CAR T cells for T cells of the subject. [Example]

[0144] The present disclosure is further described in the following examples, which do not limit the scope of the disclosure as described in the claims.

[0145] Example 1: GPC3 lentiviral transfer plasmid Standard DNA cloning techniques known in the art were used to join the VH and VL regions to generate a DNA construct encoding a single-chain variable fragment (scFv) form of the humanized anti-GC33 antibody drug (Figure 2). The lentiviral transfer plasmids used herein are listed in Table 1.

[0146] [Table 1]

[0147] The huGC33 VH-VL-scFv was cloned into the lentiviral vector pELPS4-MVRL2H2-euBBz, which contains the costimulatory domain 4-1BB along with five additional amino acids. The lentiviral vector construct pELPS4-huGC33 VH-VL was digested with restriction enzymes. The results of the restriction enzyme digestion are shown in Figure 3.

[0148] To generate a CAR construct lacking the five additional amino acids in the 4-1BB costimulatory domain, the huGC33 VH-VL-scFv was cloned into the lentiviral vector pELPS2-CD19-BBz. pELPS2-CD19-BBz is a lentiviral vector containing the 4-1BB costimulatory domain without the five additional amino acids. The lentiviral vector construct huGC33(VH-VL)-BBz was digested with restriction enzymes. The results of the restriction enzyme digestion are shown in Figure 4.

[0149] Example 2: Pharmaceutical composition of GPC3 CAR-T cells PBMC cryovials (5 x 10 7 PBMCs were thawed and activated by placing 10 mL of CAR-T cell culture medium and 1 mL of PBMCs (1 mL / vial of cells) in a water bath for 2-3 minutes. 10 mL of CAR-T cell culture medium and 1 mL of PBMCs were placed in a 50 mL conical tube and centrifuged at 1500 rpm for 5 minutes. The supernatant was removed, and the CAR-T cells were resuspended in 5 mL of fresh medium before counting. Fresh cell medium was added to the tube until the cell density reached 1 × 10. 6 The T cell activation beads were adjusted to 1 × 10 cells / mL. 6 The medium was supplemented with IL-2, adding 10 μL per cell. The cell culture medium was placed in a T75 flask and cultured in an incubator at 37°C with 5% CO .

[0150] Next, CAR-T cells were generated by spinoculation of activated T cells with CAR-encoding lentivirus. Activated PBMCs were counted from the cell culture and seeded into 24-well plates in the presence of 500 μL of cell culture medium containing lentivirus. After spinoculation transduction, transduced cells from one well were cultured in cell culture medium supplemented with IL-2.

[0151] Cultured CAR-T cells were counted every 2–3 days, and fresh medium and IL-2 were added after each count (Figure 5). On day 12, cultured CAR-T cells were harvested, placed in a freezing container, and stored at −80°C.

[0152] Analysis of CAR expression on day 12 of culture showed no expression in the control group, whereas CAR-T cell groups showed 54–66% CAR expression (Figure 6).

[0153] Target cells (GPC3-positive cell line) were harvested and seeded into 96-well U-bottom plates. Effector cells (CAR-T cells) were then added to the wells at effector cell:target cell ratios of 10:1, 3:1, 1:1, or 0.3:1 and cultured at 37°C for 24 hours. After incubation, CytoTox96 reagent was added to each well, and cytotoxicity was quantified by measuring absorbance at 490 nm (Figure 7).

[0154] Example 3: huGC33(VH-VL)-BBz CAR-T cells and huGC33(VH-VL)-euBBz CAR-T cells in vivo To verify and compare the efficacy of huGC33(VH-VL)-BBz CAR-T cells and huGC33(VH-VL)-euBBz CAR-T cells, NSG mice (6-8 weeks old, male) were transfected with Huh-7_Luf-GFP cells (2 × 10 6 200 μL of cells per mouse was injected, and 35 days after injection, the tumor size was approximately 200 mm 3 The mice were divided into five groups of four. The control group was injected with 5% HSA, while the other groups were injected with CAR-T cells. Tumor growth was monitored twice weekly by measuring tumor size using the TM900 (Figure 8).

[0155] After CAR-T cell administration, mice were bled once weekly from the orbit. 100 μL of each blood sample was centrifuged at 12,000 rpm for 10 minutes to determine the percentage and number of CAR-T cells. 100 μL of blood was placed in a FACS tube and live / dead cell staining was performed using the Zombie NIR™ Fixable Viability Kit. After reaching a concentration of 0.1 μL / 100 μL DPBS / tube, staining was performed for 10 minutes at room temperature. 25 μL of counting beads, 0.5 μL CD45, 0.5 μL CD8, 1.0 μL CD45RO, 1.0 μL CD62L, 1.0 μL PD-1, 1.0 μL Tim-3, 0.5 μL CD4, 0.5 μL CD69, and 0.0125 μL Flag were added to 100 μL of FACS buffer and stained for 30 minutes at room temperature. After 30 minutes, 1X RBC lysis buffer was added and incubated at room temperature for 5 minutes. After centrifugation at 2,000 rpm for 4 minutes, the supernatant was discarded. 2 mL of FACS buffer was added to the tube, followed by centrifugation at 2,000 rpm for 4 minutes. Analysis was performed using a FACSCelesta (Figure 9).

[0156] Six weeks after CAR-T injection, mouse spleens, livers, and bone marrow were harvested to assess the ratios of huGC33(VH-VL)-BBz and huGC33(VH-VL)-euBBz CAR-T cells. Tissue samples were processed, filtered through a 40-μm cell strainer, and then centrifuged at 2,000 rpm for 5 minutes. The supernatant was discarded. 5 μL of 1X ACK buffer was added and incubated for 10 minutes. Next, 10 mL of DPBS was added and centrifuged at 2,000 rpm for 5 minutes. FACS staining was performed as described above (Figure 10).

[0157] Example 4: Construction of CD19-euBBz CAR To enhance the immunological efficacy of the 4-1BB cytoplasmic signaling domain, we constructed a new CAR expression vector (CD19-euBBz CAR) by adding five amino acids to the 4-1BB cytoplasmic signaling domain used in CAR-T as a costimulatory signaling element. The completed construct contains an anti-CD19 scFv under the EF1 alpha promoter, the hinge region and transmembrane domain of human CD8, and an intracellular signaling domain. Specifically, the intracellular signaling domain consists of a stimulatory domain and a costimulatory domain. The intracellular signaling domain is the costimulatory signaling domain 4-1BB, to which five consecutive amino acids have been added and to which CD3 zeta binds. The final CAR gene fragment was ligated into an ELPS lentiviral expression vector that had been digested with BamH I and Sal I. Furthermore, the scFv portion was replaced using BamH I / Nhe I restriction enzymes, and cloning was performed.

[0158] Example 5: CD19-euBBz and CD19-BBz CAR-T cells The 293T cell culture used for recombinant lentivirus production was prepared in high-glucose DMEM (Welgene, LM001-05) containing 10% FBS (Millipore, TMS-013-BKR) and 1x P / S (Gibco, 15140-122). The 293T cells were incubated in DMEM containing 10% FBS for 24 hours and then transduced in a 37°C, 5% CO2 incubator. The following day, for transfection, the transfection reagent and lentiviral plasmid were mixed in the appropriate ratio and incubated for 48 hours. The lentivirus-containing supernatant was then collected and centrifuged at 400 x g for 10 minutes. Additionally, the supernatant was filtered through a 0.45 μm syringe filter using a 50 mL syringe. The resulting supernatant was mixed 3:1 with a lentivirus concentration kit (Clontech, 631231) and incubated at 4°C for 24-48 hours. This was followed by centrifugation at 4°C and 4,000 rpm for 2 hours to obtain virus, which was then resuspended in 0.5 mL of RPMI (Welgene, LM001-01) without FBS to produce lentivirus.

[0159] To determine the transduction efficiency of mammalian cells, transducing units (TU / mL) were measured by analyzing the number of lentiviral particles capable of transducing Jurkat cells. CAR expression can be assayed by FACS. On day 1, Jurkat cells were transduced at 1 × 10 per well. 5 The cells were seeded into a 96-well plate at 100 μL per well. On day 2, the lentivirus was serially diluted 1 / 3 in the 96-well plate and used to transduce Jurkat cells already seeded. At this time, polybrene (Millipore) was added to RPMI medium (10% FBS and 1x P / S) to further enhance lentiviral transduction. After centrifugation at 1200 x g for 2 hours at 32°C, the cells were incubated at 37°C in a 5% CO2 incubator for 3 hours, and 100 μL of RPMI alone was added per well. On day 5, the lentivirus flag was stained with anti-Flag-DYKDDDDK (Biolegend, catalog no. 637310) and the percentage of transduced cells was analyzed using a flow cytometer. Titers were calculated as described in Follenzi and Naldini, 2002 (Follenzi and Naldini, 2002).

[0160] FACS staining was performed to confirm the production rate of the two types of CAR-T cells after 14 days of incubation. 2 × 10 for each CAR-T cell type. 5Cells were collected in FACS tubes (FALCON, catalog no. 352052), then 2 mL of FACS buffer was added and centrifuged at 2,000 rpm for 5 minutes using a Thermo ST16 centrifuge. After discarding the supernatant, 0.5 μL / tube of anti-CD8 APC (SKI, Biolegend, catalog no. 344722), 0.5 μL / tube of anti-CD4 BV650 (RPA-T4, Biolegend, catalog no. 300536), and 0.125 μL / tube of anti-flag PE (L5, Biolegend, catalog no. 637310) were added and stained for 30 minutes at room temperature. 2 mL of FACS buffer was added, centrifuged at 2,000 rpm for 5 minutes, and this process was repeated once more. For live / dead cell staining, 1 μL / tube of 7-AAD (Biolegend, Cat. No. 420404) was added and left at room temperature for 5 minutes, and then analyzed using FACS (BD, FACSCelesta).

[0161] FACS staining was used to confirm the percentage of CD19 CAR-T cells produced. For the improved construct CD19-euBBZ CAR-T cells, the CD4+ / CAR+ ratio was 29.4%, the CD8+ / CAR+ ratio was 50.8%, and the total CAR-T ratio was 80.2%. For the non-improved construct CD19-BBz CAR-T cells, the cell ratios were 42.7%, the CD8+ / CAR+ ratio was 29.3%, and the total CAR-T ratio was 72.0%. Therefore, CD19-euBBz CAR-T cells showed 8.2% higher CAR expression and approximately doubled the CD8+ / CAR+ ratio (21.5%) compared to CD19-BBz CAR-T cells (Figure 11A).

[0162] Example 6: Confirmation of cytotoxicity of generated CD19 CAR-T cells To assess the cytotoxicity of the two CAR-T cells cultured for 14 days, CAR-T(E):LCL(T) ratios of 30:1, 10:1, 3:1, and 1:1 were plated into a 96-well white plate (Corning, Cat. No. 3917). Initially, 6 × 10 CAR-T cells were cultured at 1:1 ratio. 5cells / 50μL, 2×10 5 cells / 50μL, 9×10 4 cells / 50 μL, and 2 x 10 4 Next, the target cell line, i.e., the CBK LCL-Luc cell line, was plated at 2 × 10 cells / 50 μL in a 37°C CO2 incubator (Mammert, INC0153med). 4 After 4 hours, 100 μL of Bright-Glo™ (Promega, Cat. No. E2620) was added to each well, and the relative light units (RLU) were measured 5 minutes later using a luminometer (Thermo, Fluoroskan FL).

[0163] It was found that there was no difference in cytotoxicity between CAR-T cells transfected with conventional 4-1BB and CAR-T cells transfected with euBBz, which has five amino acids added to the 4-1BB domain.

[0164] The results showed that co-incubation of the two CAR-T cells with the CBK LCL-Luc cell line at a 30:1 ratio resulted in approximately 80% cytotoxicity after 4 hours, whereas co-incubation at a 10:1 ratio resulted in approximately 50% cytotoxicity. Reducing the number of CAR-T cells co-incubated with the cancer cells by one-third also reduced cytotoxicity by approximately one-third. Furthermore, adding five amino acids to the 4-1BB domain in vitro confirmed that this did not affect in vitro cytotoxicity (Figure 11B).

[0165] Example 7: Derivation of a subcutaneous animal model and validation of CAR-T with automated calipers and IVIS imaging NSG (NOD-scid IL2rγμL1) mice (The Jackson Laboratory) were used as experimental animals and maintained under constant conditions in an animal enclosure. The temperature was 23±2°C, with a 12-hour light-dark cycle and humidity of 50±10%, and food and drink were provided ad libitum. For efficacy experiments using CD19-euBBz CAR-T, which contains five amino acids added to the 4-1BB domain, CBK LCL-Luc cell lines were cultured at 2×10 6 The cells were prepared in 100 μL DPBS per head and injected subcutaneously into 6-week-old female mice to induce the subcutaneous administration animal model. The tumor size measured using an automatic caliper (Youngbio, TM900) was 50–100 mm. 3 Once the total number of CD19-euBBz CAR-T cells and CD19-BBz CAR-T cells reached 2 × 10 6 cells / 100 μL DPBS / head (dose 1) and 6 × 10 6 To confirm efficacy, cells were administered once via the tail vein at 100 μL DPBS per head (dose 2). Tumor size and survival rates were monitored periodically in all animal experiments.

[0166] More specifically, tumor size and photon counts were measured using automated calipers and an IVIS imaging device (PerkinElmer, Luna III) at intervals of 3 and 4 days after CAR-T administration (Figures 12 and 13). When using the TM900, tumor size was determined after placing the device at the tumor site. To confirm imaging and photon counts using the IVIS imaging device, mice were first intraperitoneally administered 150 mg / kg of XenoLight™ D-luciferin (PerkinElmer, catalog number 122799). After 15 minutes, inhalation anesthesia was induced using isoflurane, and 5 minutes later, the presence of cancer cells was imaged using IVIS. After imaging, normalization was performed, and then luciferase counts (photon counts) were confirmed and graphed. After establishing a subcutaneous administration animal model using the CBK LCL-Luc cell line, the effects of CD19-BBz CAR-T cells and CD19-euBBz CAR-T cells were compared using IVIS imaging. As shown in Figure 12, the efficacy of the two types of CAR-T cells was confirmed within one week of administration.

[0167] 2 × 10 CD19-euBBz CAR-T cells 6 cells / 100 μL DPBS / head and 6 x 10 6 In the experimental group treated with 100 μL DPBS / head, cancer cells were observed by IVIS imaging one week after administration. In addition, in the group treated with CD19-BBz CAR-T, 6 × 10 6 When cells / 100 μL DPBS / head were administered, few cancer cells were observed by imaging within one week of administration, although cancer cells were identified in the experimental group administered CD19-BBz CAR-T one week later.

[0168] One week after CAR-T administration, the luciferase levels measured in each subject were as shown in the imaging images. 6Luciferase activity was only detected in the group receiving 2 x 10 CD19 CAR-T cells per mouse. Over a three-week follow-up period, tumors continued to grow in mice not receiving CAR-T cells, while no tumor cells were detected in the three experimental groups in which tumor cells initially disappeared. However, after 10 days, tumors in mice receiving 2 x 10 CD19 CAR-T cells per mouse were detected. 6 Although luciferase levels decreased in the group receiving 2 × 10 cells / 100 μL DPBS / head, cancer cells were not completely eradicated after 3 weeks. 6 The efficacy of CD19-BBz CAR-T administered at 6 × 10 cells / 100 μL DPBS / head was 6 Experimental imaging of cancer cells revealed that the cytotoxicity of CD19-euBBz CAR-T cells was similar to that of the group treated with 100 μL DPBS per head. While the results showed no difference in cytotoxicity between CD19-euBBz CAR-T cells and CD19-BBz CAR-T cells in vitro, the efficacy of CD19-euBBz CAR-T cells was five times higher in animal models (Figure 12).

[0169] Example 8: Confirmation of CD19-euBBz CAR-T rates in an in vivo animal model After subcutaneous administration of CAR-T cells to verify the efficacy of the improved construct CAR-T cells in an animal model, the presence of CAR-T cells was confirmed in the blood of mice. More specifically, mice were bled via the orbit at intervals of 3 and 4 days after CAR-T administration. At each blood collection, 70 μL of blood was collected, and 60 μL of the blood was used to confirm the percentage and cell count of CAR-T cells. The 60 μL of blood was placed in a 5 mL FACS tube and subjected to live / dead cell staining using Zombie Aqua BV510 (Biolegend, catalog no. 423101). After reaching a concentration of 0.1 μL / 100 μL DPBS / tube, staining was performed at room temperature for 10 minutes. Counting beads (Molecularprobes, catalog no. C36950), anti-CD45 FITC (HI30 (Biolegend, catalog no. 304006)), anti-CD8 BV786 (SK-1 (Biolegend, catalog no. 344740)), anti-CD4 BV650, and anti-flag PE were added and stained for 30 minutes at room temperature. Each antibody was mixed in 0.5 μL / 100 μL of FACS buffer per tube, and 25 μL of counting beads was added. After 30 minutes, 2 mL of 1x RBC lysis buffer (Biolegend, catalog no. 422401) was added and incubated for 5 minutes at room temperature. After centrifugation at 2,000 rpm for 5 minutes, the supernatant was discarded. 2 mL of FACS wash buffer was added to the tube, and the mixture was centrifuged at 2,000 rpm for 5 minutes. This process was repeated once more, after which 50 μL of FACS buffer was added and analyzed using FACS.

[0170] One week after CAR-T cell administration, the CD19-euBBz CAR-T group had 2 × 10 6 cells / 100 μL DPBS / head group and 6 × 10 6 Approximately 20% of CD19-euBBz CAR-T cells were detected in the blood in both the cells / 100 μL DPBS / head groups, but in the CD19-BBz CAR-T group, 6 × 10 6When mice were administered 100 μL of CD19 CAR-T cells per mouse, only 5% of the mice were CD19 CAR-T cells. After 3 days, the number and percentage of CAR-T cells in the mice reached a maximum and then decreased. Within 1 week, 3 groups in which CAR-T cells were identified (CD19-euBBz CAR-T; 2 × 10 6 cells / 100 μL DPBS / head and 6 x 10 6 Cells / 100μL DPBS / Head, CD19-BBz CAR-T;6×10 6 In the case of 2 × 10 CD19-BBz CAR-T cells per mouse, the cancer cells were able to contact a relatively large number of CAR-T cells before they could proliferate in the mouse body, resulting in rapid cell death. 6 In the experimental group administered 2 x 10 cells / 100 μL DPBS / head, the percentage and number of CAR-T cells reached a maximum after 2 weeks, with the CAR-T percentage at approximately 25%, indicating relatively sufficient proliferation of cancer cells. The CD19-euBBz CAR-T was quantitatively more stable than the CD19-BBz CAR-T, with the CAR-T percentage initially increasing and then decreasing. However, in the case of the CD19-BBz CAR-T, the percentage of CAR-T cells increased and decreased at a slower rate, resulting in a longer time for tumors to disappear in the mice. Consequently, similar to the results of this experiment, the CD19-euBBz CAR-T administered at 2 x 10 6 The group receiving 6 × 10 CD19-BBz CAR-T cells / 100 μL DPBS / head received 6 The mice showed similar CAR-T levels and efficacy to those administered with cells / 100 μL DPBS / head, demonstrating that CD19-euBBz CAR-T has superior efficacy (Figure 14).

[0171] Sequence information SEQUENCE LISTING <110> Eutilex Co., Ltd. <120> CHIMERIC ANTIGEN RECEPTOR WITH 4-1BB COSTIMULATORY DOMAIN <150> US 62 / 867,503 <151> 2019-06-27 <150> PCT / KR2019 / 010244 <151> 2019-08-12 <150> US 16 / 715,462 <151> 2019-12-16 <150> US 62 / 991,493 <151> 2020-03-18 <150> US 63 / 004,827 <151> 2020-04-03 <150> US 63 / 043,237 <151> 2020-06-24 <160> 28 <170> PatentIn version 3.5 <210> 1 <211> 15 <212> DNA <213> Artificial <220> <223> synthetic oligonucleotide <400> 1 cgtttctctg ttgtt 15 <210> 2 <211> 141 <212> DNA <213> Artificial <220> <223> synthetic oligonucleotide <400> 2 cgtttctctg ttgttaaacg gggcagaaag aaactcctgt atatattcaa acaaccattt 60 atgagaccag tacaaactac tcaagaggaa gatggctgta gctgccgatt tccagaagaa 120 gagagagg gagtgaact g 141 <210> 3 <211> 5 <212> PRT <213> Artificial <220> <223> synthetic polypeptide <400> 3 Arg Phe Ser Val Val 1 5 <210> 4 <211> 47 <212> PRT <213> Artificial <220> <223> synthetic polyptpdie <400> 4 Arg Phe Ser Val Val Lys Arg Gly Arg Lys Leu Leu Tyr Ile Phe 1 5 10 15 Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly 20 25 30 Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 45 <210> 5 <211> 207 <212> DNA <213> Artificial <220> <223> CD8 / Hinge / Transmembrane <400> 5 accacgacgc cagcgccgcg accaccaca ccggcgccca ccatcgctag ccagcccctg 60 tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120 gactcgcct gtgatatcta catctgggcg cccttggccg ggacttgtgg ggtccttctc 180 ctgtcactgg ttcaccct ttactgc 207 <210> 6 <211> 339 <212> DNA <213> Artificial <220> <223> synthetic oligonucleotide <400> 6 agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60 tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120 cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180 gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240 cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300 tacgacgccc ttcacatgca ggccctgccc cctcgctaa 339 <210> 7 <211> 69 <212> DNA <213> Artificial <220> <223> CD8 alpha leader sequence <400> 7 ggatccatgg ccttaccagt gaccgccttg ctcctgccgc tggccttgct gctccacgcc 60 gccaggccg 69 <210> 8 <211> 24 <212> DNA <213> Artificial <220> <223> Flag-tag sequence <400> 8 gactacaagg acgacgatga caag 24 <210> 9 <211> 16 <212> PRT <213> Artificial <220> <223> Light chain CDR1 <400> 9 Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His 1 5 10 15 <210> 10 <211> 7 <212> PRT <213> Artificial <220> <223> Light chain CDR2 <400> 10 Lys Val Ser Asn Arg Phe Ser 1 5 <210> 11 <211> 9 <212> PRT <213> Artificial <220> <223> Light chain CDR3 <400> 11 Ser Gln Asn Thr His Val Pro Pro Thr 1 5 <210> 12 <211> 5 <212> PRT <213> Artificial <220> <223> Heavy chain CDR1 <400> 12 Asp Tyr Glu Met His 1 5 <210> 13 <211> 17 <212> PRT <213> Artificial <220> <223> Heavy chain CDR2 <400> 13 Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe Lys 1 5 10 15 Gly <210> 14 <211> 6 <212> PRT <213> Artificial <220> <223> Heavy chain CDR3 <400> 14 Phe Tyr Ser Tyr Thr Tyr 1 5 <210> 15 <211> 112 <212> PRT <213> Artificial <220> <223> human GC33 light chain variable region <400> 15 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95 Thr His Val Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 16 <211> 115 <212> PRT <213> Artificial <220> <223> human GC33 heavy chain variable region <400> 16 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe 50 55 60 Lys Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 17 <211> 336 <212> DNA <213> Artificial <220> <223> human GC33 light chain variable region <400> 17 gacgtcgtta tgacacagag tcccctctcc ttgccggtga ccctgggtca gcctgcgtcc 60 atctcttgca gatcctccca gtctctggta cactccaacg gcaacacata cttgcactgg 120 taccaacaaa gacctggtca gtcaccgcga cttctcatat ataaagtttc caataggttc 180 agtggagtgc cagacaggtt cagtggttca ggatcaggca ctgatttcac gcttaaaatc 240 agtcgggttg aggcggagga cgtaggagtt tactattgca gccagaatac gcacgtgccg 300 cctactttg gctctggaac caagttggaa ataaag 336 <210> 18 <211> 345 <212> DNA <213> Artificial <220> <223> human GC33 heavy chain variable region <400> 18 caagtgcaac tcgtacaatc aggtgctgaa gtcaaaaagc cgggagcctc tgttaaagtg 60 tcctgtaaag ccagcggcta cacctttacc gattatgaga tgcactgggt tcggcaggct 120 ccgggccaag gtctggagtg gatcggggct cttgacccaa agacgggcga cacggcttat 180 tcacaaaaat tcaaaggtag ggctactctg actgccgata agtccaccag caccggtat 240 atggagctct ctagcttgcg aagcgaggac acggcggtgt actattgcac acgcttctat 300 agttacacat attggggtca aggcacgctt gtgaccgtgt ctagc 345 <210> 19 <211> 243 <212> PRT <213> Artificial <220> <223> synthetic polypeptide <400> 19 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Asp His Ile Asn Asn Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Lys Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Thr Pro Phe 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu 115 120 125 Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys 130 135 140 Thr Val Ser Gly Phe Ser Leu Ser Arg Tyr Ser Val His Trp Ile Arg 145 150 155 160 Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu Gly Met Ile Trp Gly Gly 165 170 175 Gly Ser Thr Asp Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ser 180 185 190 Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr 195 200 205 Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asn Glu Gly Asp Thr 210 215 220 Thr Ala Gly Thr Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser <210> 20 <211> 729 <212> DNA <213> Artificial <220> <223> synthetic oligonucleotide <400> 20 gatattcaga tgacccagtc cccgagctcc ctgtccgcct ctgtgggcga tagggtcacc 60 atcacctgca aggccagtga ccacatcaac aactggctgg cctggtatca acagaaacca 120 ggaaaagctc cgaaactact gatcagcggc gccacctctc tggaaaccgg agtcccttct 180 cgcttctctg gttccggatc tgggaaggat tacactctga ccatcagcag tctgcagccg 240 gaagacttcg caacttatta ctgtcagcag tactggtcca cccccttcac cttcggacag 300 ggtaccaagg tggagatcaa aggcggaggc ggatctggcg gcggaggaag tggcggaggg 360 ggatctcagg tgcagctgca ggagtcgggc ccaggactgg tgaagccttc ggagaccctg 420 tccctcacct gcactgtctc tggtttctcc ctgagtcggt actctgtgca ttggatccgg 480 cagcccccag ggaagggact ggagtggctg gggatgatct ggggaggcgg cagcaccgac 540 tacaacagcg ccctgaagtc ccgactgacc atatcaaagg acaactccaa gaaccaggtg 600 tccttgaagc tgagctctgt gaccgctgcg gacacggccg tgtattactg tgcgagaaat 660 gagggcgata ccaccgccgg cacttggttt gcctattggg gccagggaac cctggtcacc 720 gtctcctca 729 <210> 21 <211> 242 <212> PRT <213> Artificial <220> <223> synthetic polypeptide <400> 21 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu 115 120 125 Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys 130 135 140 Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg 145 150 155 160 Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser 165 170 175 Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile 180 185 190 Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln 195 200 205 Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly 210 215 220 Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val 225 230 235 240 Ser Ser <210> 22 <211> 726 <212> DNA <213> Artificial <220> <223> synthetic oligonucleotide <400> 22 gacatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60 atcagttgca gggcaagtca ggacattagt aaatatta attggtatca gcagaaacca 120 gatgaactg ttaaactcct gatctaccat acatcagat tacactcagg agtcccatca 180 aggttcagtg gcagtgggtc tggaacagat tattctca ccattagcaa cctggagcaa 240 gaagatattg catcattactt gttcggaggg ggtaatacggc tccgtacac gttcggaggg 300 gggaccaagc tggagatcac aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc 360 ggatctgagg tgaactgca ggagtcagga cctggcctgg tggcgccctc acagagcctg 420 tccgtcacat gcactgtctc aggggtctca taccgact atggtgtaag ctggattcgc 480 cagcctccac gaaagggtct ggagtggctg ggagtatatat ggggtagtga aaccacatac 540 tataattcag ctctcaatc cagactgacc atcatcagg acactccaa gagccaagtt 600 ttcttaaaaa tgaacagtct gcaactgat gatacagcca tttactactg tgccaacat 660 attackacg gtgtagcta tgctatggac tactggggcc aaggaacctc agtcaccgtc 720 tcctca 726 <210> 23 <211> 1184 <212> DNA <213> Artificial <220> <223> synthetic oligonucleotide <400> 23 cgtgaggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt 60 tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg 120 aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa 180 gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtaa 240 gtgccgtgtg tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt 300 gaattacttc cacctggctg cagtacgtga ttcttgatcc cgagcttcgg gttggaagtg 360 ggtgggagag ttcgaggcct tgcgcttaag gagccccttc gcctcgtgct tgagttgagg 420 cctggcctgg gcgctggggc cgccgcgtgc gaatctggtg gcaccttcgc gcctgtctcg 480 ctgctttcga taagtctcta gccatttaaa atttttgatg acctgctgcg acgctttttt 540 tctggcaaga tagtcttgta aatgcgggcc aagatctgca cactggtatt tcggtttttg 600 gggccgcggg cggcgacggg gccctgcgt cccagcgcac atgttcggcg aggcggggcc 660 tgcgagcgg gccaccgaga atcggacggg ggtagtctca agctggccgg cctgctctgg 720 tgcctgcct cgcgccgccg tgtatcgccc cgccctgggc ggcaaggctg gccggtcgg 780 caccagttgc gtgagcggaa agatggccgc ttcccggccc tgctgcaggg agctcaaaat 840 ggaggacgcg gcgctcgggga gagcgggcgg gtgagtcacc cacacaaagg aaaagggcct 900 ttccgtcctc agccgtcgct tcatgtgact ccactgagta ccgggcgccg tccaggcacc 960 tcgattagtt ctcgagcttt tggagtacgt cgtctttagg ttggggggag gggttttatg 1020 cgatggagtt tccccacact gagtgggtgg agactgaagt taggccagct tggcacttga 1080 tgtaattctc cttggaattt gccctttttg agtttggatc ttggttcatt ctcaagcctc 1140 agacagtggt tcaaagtttt tttcttccat ttcaggtgtc gtga 1184 <210> 24 <211> 84 <212> DNA <213> Artificial <220> <223> synthetic oligonucleotide <400> 24 agtagtgtgt gcccgtctgt tgtgtgactc tggtactag agatccctca gacccttta 60 gtcagtgtgg aaatctcta gcag 84 <210> 25 <211> 1377 <212> DNA <213> Artificial <220> <223> synthetic oligonucleotide <400> 25 cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc 60 ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt 120 tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga 180 gatttagatc gcgatggga aaaattcggt taggccagg gggaaagaa aaataataat 240 taaaacatat agtatgggca agcagggagc tegacgatt cgcagttaat cctggcctgt 300 tagaaacatc agaaggctgt agacaatc tgggacagct acaaccatcc cttcagacag 360 gatcagaga acttagatca ttatatata cagtagcaac cctctattgt gtgcatcaa 420 'ggatagagat aaagacacc aaggaagctt gawghagat agaggaag aaaaaaaaaa 480 gtaagaccac cgcacagcaa gcggccgctg atctcagac ctggaggagg agatatgagg 540 gaattga gagtgatt attaataat aaagtagtaaaattgacc attaggta 600 gcacccacca aggcaagg aagagtggtg cagagagaaa aaagagcagt gggaatagga 660 gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg 720 ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg 780 agggctattg aggcgcaca gcatctgttg caaccacag tctggggcat caagcagctc 840 caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg 900 ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggatgctag ttggagtaat 960 aaatctctgg aacagatttg gatcacacg acctggatgg agtgggacag agaattac 1020 aattacaca gcttaataca ctccttaatt gagaatcgc aaaaccagca agaaagaat 1080 gaacagaat tattggaatt agataatgg gcaagttgt ggatttggtt taacataca 1140 aattggctgt ggtatataa attattcata atgatagtag gaggcttggt aggtttaaga 1200 atagttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg 1260 tttcagaccc acctcccaac cccgagggga cccgacaggc ccgaaggaat agagaagaa 1320 ggtggagaga gagacagaga cagatccatt cgattagtga acggatctcg acggtat 1377 <210> 26 <211> 547 <212> DNA <213> Artificial <220> <223> synthetic oligonucleotide <400> 26 tagactgtag cccaggaata tggcagctag attgtacaca tttagaagga aaagttatct 60 tggtagcagt tcatgtagcc agtggatata tagagcaga agtaattcca gcagagacag 120 ggcaagaaac agcatacttc ctcttaaaat tagcaggag atggccagta aaaacagtac 180 atacagacaa tggcagcaat ttcaccagta ctacagttaa ggccgcctgt tggtgggcgg 240 ggatcaagca ggaatttggc attccctaca atccccaag tcaggagta atagaatcta 300 tgaaaaga attaagaa attaggac aggtagaga tcaggctgaa catcttaaga 360 cagcagtaca aatggcagta ttcatccaca attttaaag aaaagggggg attggggggt 420 acagtgcagg ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac 480 aaaaacaaat tacaaaaatt caaaatttc gggtttatta cagggacagc agagatccag 540 tttggct 547 <210> 27 <211> 591 <212> DNA <213> Artificial <220> <223> synthetic oligonucleotide <400> 27 atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc 60 cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta 120 tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt 180 ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg 240 gttggggcat tgccaccacc tgtcagctcc tttccgggac ttcgctttc cccctcccta 300 ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt 360 tgggcactga caattccgtg gtgttgtcgg ggaagctgac gtcctttcca tggctgctcg 420 cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca 480 atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc 540 gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcct g 591 <210> 28 <211> 98 <212> DNA <213> Artificial <220> <223> synthetic oligonucleotide <400> 28 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca 60 ctgcttaagc ctcaataaag cttgccttga gtgcttca 98

Claims

[Claim 1] An immune cell comprising a chimeric antigen receptor (CAR), the CAR comprising: (a) an extracellular domain comprising an antigen-binding domain; (b) a transmembrane domain; and (c) an intracellular domain comprising a costimulatory endodomain, wherein the costimulatory endodomain comprises an intracellular signaling domain derived from 4-1BB / CD137 and five additional amino acids; and The immune cell comprising:

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