Chimeric antigen receptor with a 4-1BB costimulatory domain

Abstract

The present disclosure provides a CAR-T composition directed to a CAR comprising (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 derived from 4-1BB / CD137 and five additional amino acids. The present disclosure also provides vectors, compositions, and therapeutic methods using the antigen-binding molecule and engineered immune cells comprising the CAR. The CAR compositions provided herein can be used to treat certain cancers.

Description

[Technical Field]

[0001] Cross-reference of related applications This application is a PCT application claiming priority and benefits of U.S. Patent Application No. 62 / 867,503 filed on 27 June 2019, International Patent Application PCT / KR2019 / 010244 filed on 12 August 2019, U.S. Patent Application No. 16 / 715,462 filed on 16 December 2019, U.S. Patent Application No. 62 / 991,493 filed on 18 March 2020, U.S. Patent Application No. 63 / 004,827 filed on 3 April 2020, and U.S. Patent Application No. 63 / 043,237 filed on 24 June 2020, the disclosures of each application being incorporated herein by reference in their entirety. [Background technology]

[0002] background Cancer remains one of the leading causes of death worldwide. Recent statistics report 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 cancer cases and 8.2 million cancer deaths worldwide in 2012. Due to population growth and aging, as well as exposure to risk factors such as smoking, unhealthy diets, and lack of exercise, it is projected that by 2030, the number of new cancer cases worldwide will increase to 21.7 million and cancer deaths to 13 million. Furthermore, the pain and medical costs associated with cancer treatment reduce the quality of life for cancer patients and their families.

[0003] T cells modified using chimeric antigen receptors (CAR-T cells) have great therapeutic potential in treating diseases such as cancer. CAR-T therapies confer potent target affinity and signaling function to T cells. However, the remarkable efficacy of CAR-T therapy is often accompanied by serious side effects (e.g., cytokine release syndrome (CRS)). Therefore, there remains an unmet need for the development of CAR-T therapies and strategies that mitigate these side effects. [Overview of the project]

[0004] overview This specification provides immune cells comprising a chimeric antigen receptor (CAR), wherein the CAR comprises (a) an extracellular domain including an antigen-binding domain, (b) a transmembrane domain, and (c) an intracellular domain including a costimulatory endodomain, the costimulatory endodomain comprising 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. In some embodiments, the chimeric antigen receptor is composed of two polypeptides.

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

[0007] In some embodiments, the antigen-binding domain is humanized. In some embodiments, the antigen-binding domain is humanized. In some embodiments, the antigen-binding domain is scFv. In some embodiments, the antigen-binding domain specifically binds to disease-related antigens. In some embodiments, the antigen-binding domain specifically binds to tumor antigens. In some embodiments, the antigen-binding domain specifically binds to antigens selected from the group consisting of glypican 3 (GPC3), malignant tumor variant receptors (MVRs), and CD19.

[0008] In some embodiments, the transmembrane domains include 4-1BB / CD137, activated 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 that specifically binds to CD83, LIGHT, LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1), M The transmembrane domain is selected from proteins 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), signal transduction lymphocyte activating molecules (SLAM proteins), 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 CD8α-derived transmembrane domain. In some embodiments, the intracellular domain further includes a CD3ζ-derived intracellular domain.

[0009] In some embodiments, the chimeric antigen receptor further includes a signal peptide or a leader sequence. In some embodiments, the chimeric antigen receptor further includes a hinge region. In some embodiments, the hinge region is the CD8α hinge. In some embodiments, the chimeric antigen receptor further includes an additional antigen-binding domain. In some embodiments, the additional antigen-binding domain is 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] This specification provides nucleic acids encoding a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor comprises (a) an extracellular domain including an antigen-binding domain, (b) a transmembrane domain, and (c) an intracellular domain including 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. In some embodiments, the chimeric antigen receptor is composed of two polypeptides.

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

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

[0015] In some embodiments, the transmembrane domains include 4-1BB / CD137, activated 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 that specifically binds to CD83, LIGHT, LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1), M The transmembrane domain is selected from proteins 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), signal transduction lymphocyte activating molecules (SLAM proteins), 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 CD8 alpha-derived transmembrane domain.

[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 a 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] This specification provides vectors comprising one of the nucleic acids described herein. In some embodiments, the vector further comprises a promoter operably bound to the nucleic acid. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inductive promoter. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a lentiviral vector.

[0018] This specification provides a method for producing engineered immune cells, comprising introducing one of the nucleic acids or one of the vectors described herein into immune cells to thereby produce engineered immune cells. In some embodiments, the method further comprises culturing the engineered immune cells after the introduction 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 includes obtaining immune cells from a subject prior to the induction step. In some embodiments, the method further includes administering the manipulated immune cells to the subject. In some embodiments, the subject has been diagnosed or identified as having cancer.

[0020] This specification provides engineered immune cells produced by any one of the methods described herein.

[0021] This specification provides a pharmaceutical composition comprising one of the manipulated immune cells described herein and a pharmaceutically acceptable carrier.

[0022] This specification provides a method for treating cancer in a subject, comprising administering to the subject any one of the manipulated immune cells described herein or any one of the pharmaceutical compositions described herein. In some embodiments, the cancer is anti-glypican-3 related cancer, anti-CD19 related cancer, or anti-MVR related cancer. In some embodiments, the cancer is carcinoma, lymphoma (e.g., Hodgkin lymphoma and non-Hodgkin 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 cancers. In some embodiments, the subject has previously received one or more additional anticancer treatments, the anticancer treatment being 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 with cancer. [Invention 1001] Immune cells containing a chimeric antigen receptor (CAR), wherein the CAR is (a) an extracellular domain containing an antigen-binding domain, (b) 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 cells, including the immune cells. [Invention 1002] The immune cell of the present invention 1001, wherein the chimeric antigen receptor is a single polypeptide. [Invention 1003] The immune cell of the present invention 1001, wherein the chimeric antigen receptor is composed of two polypeptides. [Invention 1004] An immune cell according to any of the present invention 1001 to 1003, wherein the aforementioned co-stimulatory endodomain comprises an intracellular signaling domain derived from 4-1BB / CD137 and five additional amino acids, where the five additional amino acids are encoded by Sequence ID No. 1. [Invention 1005] The immune cell of the present invention 1004, wherein the aforementioned co-stimulatory endodomain contains SEQ ID NO: 2. [Invention 1006] An immune cell according to any of the invention 1001 to 1005, wherein the antigen-binding domain is of the humanized type. [Invention 1007] An immune cell according to any of the present invention 1001 to 1006, wherein the antigen-binding domain is of the human type. [Invention 1008] An immune cell according to any of the present invention 1001 to 1007, wherein the antigen-binding domain is scFv. [Invention 1009] An immune cell according to any of the present invention 1001 to 1008, wherein the antigen-binding domain specifically binds to an antigen associated with a disease. [Invention 1010] An immune cell according to any of the present invention 1001 to 1009, wherein the antigen-binding domain specifically binds to a tumor antigen. [Invention 1011] An immune cell according to any of the present invention 1001 to 1010, 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. [Invention 1012] The aforementioned transmembrane domains include 4-1BB / CD137, activated 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 that specifically binds to CD83, LIGHT, LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1), MHC class 1 molecule, NK An immune cell according to any of the invention 1001 to 1011, 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), signal transduction lymphocyte activating 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. [Invention 1013] An immune cell according to any of the invention 1001 to 1012, wherein the transmembrane domain is a transmembrane domain derived from CD8α. [Invention 1014] An immune cell according to any of the present invention 1001 to 1013, wherein the intracellular domain further comprises an intracellular domain derived from CD3ζ. [Invention 1015] An immune cell according to any of items 1001 to 1014 of the present invention, further comprising the chimeric antigen receptor as a signal peptide or a leader sequence. [Invention 1016] An immune cell according to any of items 1001 to 1015 of the present invention, wherein the chimeric antigen receptor further comprises a hinge region. [Invention 1017] The immune cell of the present invention 1016, wherein the hinge region is a CD8α hinge. [Invention 1018] An immune cell according to any of items 1001 to 1017 of the present invention, wherein the chimeric antigen receptor further comprises an additional antigen-binding domain. [Invention 1019] The immune cell of the present invention 1018, wherein the additional antigen-binding domain is scFv. [Invention 1020] The immune cells according to any one of the present invention 1001 to 1019, wherein the immune cells are human immune cells. [Invention 1021] The immune cells of the present invention 1020, wherein the aforementioned human immune cells are autologous human immune cells. [Invention 1022] The immune cells of the present invention 1020, wherein the aforementioned human immune cells are allogeneic human immune cells. [Invention 1023] The immune cell according to any one of the present invention 1001 to 1022, wherein the aforementioned immune cell is a T cell. [Invention 1024] The immune cells according to any of the present invention 1001 to 1023, wherein the aforementioned immune cells are NK cells. [Invention 1025] A nucleic acid encoding a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor is (a) an extracellular domain containing an antigen-binding domain, (b) 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 nucleic acid, including the nucleic acid. [Invention 1026] The nucleic acid of the present invention 1025, wherein the chimeric antigen receptor is a single polypeptide. [Invention 1027] The nucleic acid of the present invention 1025, wherein the chimeric antigen receptor is composed of two polypeptides. [Invention 1028] The nucleic acid of any of the Invention 1025-1027, wherein the aforementioned co-stimulatory endodomain comprises an intracellular signaling domain derived from 4-1BB / CD137 and five additional amino acids, where the five additional amino acids are encoded by the nucleotide sequence of Sequence ID No. 1. [Invention 1029] The nucleic acid of the present invention 1028, wherein the aforementioned co-stimulatory endodomain contains SEQ ID NO: 2. [Invention 1030] A nucleic acid according to any of the present invention 1025 to 1029, wherein the antigen-binding domain is of the humanized type. [Invention 1031] A nucleic acid according to any of the present invention 1025 to 1030, wherein the antigen-binding domain is of the human type. [Invention 1032] A nucleic acid according to any of the invention 1025 to 1031, wherein the antigen-binding domain is scFv. [Invention 1033] The nucleic acid according to any of the present invention 1025 to 1032, wherein the antigen-binding domain specifically binds to an antigen associated with a disease. [Invention 1034] A nucleic acid according to any of the present invention 1025 to 1033, wherein the antigen-binding domain specifically binds to a tumor antigen. [Invention 1035] The nucleic acid according to any of the present invention 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. [Invention 1036] The aforementioned transmembrane domains include 4-1BB / CD137, activated 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 that specifically binds to CD83, LIGHT, LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1), MHC class 1 molecule, N A nucleic acid of any of the present invention 1025-1035, which 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), signal transduction lymphocyte activating 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. [Invention 1037] The nucleic acid according to any of the invention 1025 to 1036, wherein the transmembrane domain is a transmembrane domain derived from CD8α. [Invention 1038] The nucleic acid according to any one of the present invention 1025 to 1037, wherein the intracellular domain further comprises an intracellular domain derived from CD3ζ. [Invention 1039] The nucleic acid according to any of the present invention 1025 to 1038, further comprising a signal peptide or a leader sequence, wherein the chimeric antigen receptor further comprises a signal peptide or a leader sequence. [Invention 1040] A nucleic acid according to any one of the present invention 1025 to 1039, wherein the chimeric antigen receptor further comprises a hinge region. [Invention 1041] The nucleic acid of the present invention 1040, wherein the hinge region is a CD8α hinge. [Invention 1042] A vector comprising any nucleic acid according to invention 1025 to 1041. [Invention 1043] The vector of the present invention 1042 further comprises a promoter operably bound to the nucleic acid. [Invention 1044] The vector of the present invention 1043, wherein the promoter is a constitutive promoter. [Invention 1045] The vector of the present invention 1043, wherein the promoter is an inducible promoter. [Invention 1046] A viral vector, which is any of the vectors described in invention 1042 to 1045. [Invention 1047] The vector according to the present invention 1046, wherein the viral vector is a lentiviral vector. [Invention 1048] A method for producing engineered immune cells, comprising introducing a nucleic acid of any of invention 1025 to 1041 or a vector of any of invention 1042 to 1047 into immune cells, thereby producing the engineered immune cells. [Invention 1049] The method of the present invention 1048, further comprising culturing the manipulated immune cells after the introduction step. [Invention 1050] The method according to any one of the present invention 1048 to 1049, wherein the immune cell is a T cell. [Invention 1051] The method according to any of the present invention 1048 to 1050, wherein the immune cells are NK cells. [Invention 1052] Any method of the present invention 1048 to 1051 further comprising obtaining the immune cells from a subject before the introduction step. [Invention 1053] The method of the present invention 1052, further comprising administering the manipulated immune cells to the subject. [Invention 1054] The method of the present invention 1052 or 1053, wherein the subject has been diagnosed or identified as having cancer. [Invention 1055] Engineered immune cells produced by any of the methods described in 1048 to 1054 of this invention. [Invention 1056] A pharmaceutical composition comprising manipulated immune cells according to the present invention 1055 and a pharmaceutically acceptable carrier. [Invention 1057] A method for treating cancer in a subject, comprising administering manipulated immune cells of the present invention 1055 or a pharmaceutical composition of the present invention 1056 to the subject. [Invention 1058] The method of the present invention 1057, wherein the cancer is an anti-glypican 3-related cancer, an anti-CD19-related cancer, or an anti-MVR-related cancer. [Invention 1059] The method of the present invention 1057, wherein the cancer is carcinoma, lymphoma (e.g., Hodgkin lymphoma and non-Hodgkin 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. [Invention 1060] The method according to any one of items 1057 to 1059 of the present invention, wherein the subject has previously received one or more additional anticancer treatments, and the anticancer treatment is selected from the group consisting of ionizing radiation, chemotherapeutic agents, therapeutic antibodies, and checkpoint inhibitors. [Invention 1061] Any method of the present invention 1057 to 1060, wherein the subject is identified or diagnosed as having the cancer. [Brief explanation of the drawing]

[0023] [Figure 1] A schematic diagram of an exemplary MVR CAR construct is shown. [Figure 2] This shows exemplary enzyme mapping results after cloning MVRL2H2-4-1BB. [Figure 3] The results of restriction enzyme digestion of huGC33(VH-VL)-euBBz are shown, with the predicted size indicated on the DNA ladder and the results shown in gel electrophoresis images. [Figure 4] The results of restriction enzyme digestion of huGC33(VH-VL)-BBz are shown, with the predicted size indicated on the DNA ladder and the results shown in gel electrophoresis images. [Figure 5] Figure 5A is a graph showing the total proliferation rate of CAR-T cells over 11 days in vitro. Figure 5B is a graph comparing the proliferation rates of CAR-T cells in vitro. Figure 5C is a graph showing the cell viability of CAR-T cells in vitro. [Figure 6] This paper shows the 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 the LDH-based cytotoxicity assay using target cells derived from the Huh-7 cell line. Figure 7B is a graph showing the LDH-based cytotoxicity assay using target cells derived from the PLC / PRF / 5 cell line. [Figure 8] This 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 in a mouse model 5 weeks after injection 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 in a mouse model 5 weeks after injection of huGC33(VH-VL)-euBBz and huGC33(VH-VL)-BBz CAR-T cells. [Figure 10] This shows FACS staining analysis of CAR-T cells in the blood, bone marrow, spleen, and liver of mice 5 weeks after injection of huGC33(VH-VL)-euBBz and huGC33(VH-VL)-BBz CAR-T cells. [Figure 11] Figure 11A shows CAR expression in T cells transduced with CD19-BBz and CD19-euBBz. Figure 11B is a graph of luciferase-based cytotoxicity assays showing the killing activity in T cells transduced with CD19-BBz and CD19-euBBz. [Figure 12] This shows the results of IVIS imaging of the effects of CD19-BBz CAR-T cells and CD19-euBBz CAR-T cells using an animal model. [Figure 13] This graph shows the photon values ​​of cancer cells in animal models 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 orbital blood collection from mice at 3-4 day intervals using FACS. Figure 14B is a set of graphs showing the percentage of CD4 / CD8 CAR-T cells present in the blood after orbital blood collection from mice at 3-4 day intervals using FACS. Figure 14C is a graph showing the total number of CD19 CAR-T cells present in the blood after orbital blood collection from mice at 3-4 day intervals using FACS. [Figure 15]Figure 15A shows a schematic diagram of an exemplary GPC3 CAR construction. Figure 15B shows a schematic diagram of an exemplary GPC3 CAR construction. [Modes for carrying out the invention]

[0024] Detailed explanation This disclosure describes a chimeric antigen receptor (CAR) containing a 4-1BB costimulatory endodomain, as well as a method for producing and using the same.

[0025] definition Approximately: When used herein in relation to a value, the term "approximately" refers to a value that is contextually similar to the value mentioned. Generally, a person skilled in the art familiar with the context will understand the appropriate degree of variation that "approximately" encompasses in that context. For example, in some embodiments, the term "approximately" may encompass a range of values ​​within the range of 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less than or equal to the value mentioned.

[0026] Administration: As used herein, the term “administration” typically refers to administering a composition to a subject or system in order to achieve delivery of the drug that is the composition itself or a drug contained in the composition. Those skilled in the art will recognize, in reasonable circumstances, the various routes that may be used for administration to a subject, e.g., a human. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In some specific embodiments, administration may be bronchial (e.g., bronchial infusion), buccal, percutaneous (e.g., one or more of the following, or including, topical, intradermal, interdermal, percutaneous, etc., to the dermis), enteral, intra-arterial, intradermal, gastric, intramedullary, intramuscular, intranasal, intraperitoneal, intramedullary, intravenous, intraventricular, intraspecific organ (e.g., intrahepatic), mucous membrane, intranasal, oral, transrectal, subcutaneous, sublingual, topical, tracheal (e.g., intratracheal infusion), transvaginal, vitreous, etc. In some embodiments, administration may involve single doses, multiple doses, or a fixed number of doses. In some embodiments, administration may involve medication that is intermittent (e.g., multiple doses separated by time) and / or periodic (e.g., individual doses separated by a common time period). In some embodiments, administration may involve continuous medication (e.g., perfusion) for at least a selected time period.

[0027] Affinity: As is 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 various ways. In some embodiments, affinity is measured by quantitative assays. In some embodiments, the binding partner concentration may be fixed above the ligand concentration to mimic physiological conditions. Alternatively or additionally, in some embodiments, the binding partner concentration and / or ligand concentration may be varied. In some such embodiments, affinity can be compared to a standard under equivalent conditions (e.g., concentration).

[0028] Antibody Drugs: As used herein, the term “antibody drug” can refer to a drug that specifically binds to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex containing 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, antibody drugs may include one or more sequence elements that have been humanized, primated, chimeric, etc., as known in the art. In many embodiments, the term “antibody drug” is used to refer to one or more constructs or forms known or developed in the art for utilizing the structural and functional characteristics of an antibody in an alternative presentation. For example, in some embodiments, antibody agents utilized according to the present invention include, but are not limited to, intact IgA, IgG, IgE, or IgM antibodies; bi- or multi-specific antibodies (e.g., Zybodies®); antibody fragments, e.g., 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); camel-like (cameloid) antibodies; mask antibodies (e.g., Probodies®); S mall M odular I mmuno PHarmaceuticals ("SMIP"); single-chain or tandem diabodies (TandAb)); VHH; Anticalins (registered trademark); Nanobodies (registered trademark) minibodies; BiTE (registered trademark); Ankyrin repeat protein or DARPIN (registered trademark); Avimers (registered trademark); DART; TCR-like antibodies; Adnectins (registered trademark); Affilins (registered trademark), Trans-bodies (registered trademark); Affibodies (registered trademark); TrimerX (registered trademark); MicroProteins; Fynomers (registered trademark), Centyrins (registered trademark); and KALBITOR (registered trademark). In some embodiments, antibody drugs may lack covalent modifications (e.g., glycan attachments) that they would have if naturally produced. In some embodiments, antibody drugs may include covalent modifications (e.g., glycan attachments), a payload [e.g., a detectable portion, a therapeutic portion, a catalytic portion, etc.], or other pendant groups [e.g., polyethylene glycol, etc.]. In some embodiments, the antibody drug is a polypeptide having an amino acid sequence containing one or more structural elements recognized by those skilled in the art as complementarity-determining regions (CDRs), or comprising the same. In some embodiments, the antibody drug is a polypeptide containing 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 the reference antibody. In some embodiments, the contained CDR is substantially identical to the reference CDR in that its sequence is identical, or in that it contains 1 to 5 amino acid substitutions compared to the reference CDR. In some embodiments, the contained CDR is substantially identical to the reference CDR in that it exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the reference CDR.In some embodiments, the included CDR is substantially identical to the reference CDR in that it exhibits at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR in that at least one amino acid in the included CDR is deleted, added, or substituted compared to the reference CDR, but otherwise the included CDR has the same amino acid sequence as the reference CDR. In some embodiments, one to five amino acids in the included CDR are deleted, added, or substituted compared to the reference CDR, but otherwise the included CDR has the same amino acid sequence as the reference CDR, so the included CDR is substantially identical to the reference CDR. In some embodiments, at least one amino acid in the included CDR is substituted compared to the reference CDR, but otherwise the included CDR has the same amino acid sequence as the reference CDR, so the included CDR is substantially identical to the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR in that, although one to five amino acids in the included CDR are deleted, added, or substituted compared to the reference CDR, the included CDR has the same amino acid sequence as the reference CDR. In some embodiments, the antibody drug is a polypeptide having an amino acid sequence containing a structural element recognized by those skilled in the art as an immunoglobulin variable domain, or comprises such a polypeptide. 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 at least a part of a chimeric antigen receptor (CAR), or comprises such a CAR.

[0029] Antigen: As used herein, the term “antigen” may refer to an agent that binds to an antibody drug. In some embodiments, an antigen may or may not bind to an antibody drug and induce a specific physiological response in an organism. Generally, an antigen may be or contain any chemical entity (e.g., small molecules, nucleic acids, polypeptides, carbohydrates, lipids, polymers (including biological polymers (e.g., nucleic acid and / or amino acid polymers) and non-biological polymers (e.g., polymers other than nucleic acid or amino acid polymers))). In some embodiments, an antigen may be or contain a polypeptide. In some embodiments, an antigen may be or contain a glycan. Those skilled in the art will understand that, generally, an antigen may be obtained in an isolated or pure form, or alternatively in a crude form (e.g., together with other materials (e.g., a relatively crude preparation of a source containing an extract or antigen, such as a cell extract)). In some specific embodiments, the antigen exists in a cellular context (e.g., the antigen is expressed on the surface of a cell or inside 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 a 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 may be any chemical class, including, for example, carbohydrates, lipids, nucleic acids, metals, polypeptides, or small molecules. In some embodiments, the antigen-binding domain may be or contain 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] Related to: The term “related” between two events or entities is used herein when the presence, level, and / or form of one event or entity is related to the other. For example, a particular entity (e.g., polypeptide, gene signature, metabolite, microorganism, etc.) is considered related to a particular disease, disorder, or condition if its presence, level, and / or form is related to the incidence and / or susceptibility of that disease, disorder, or condition (e.g., in the entire population concerned). In some embodiments, two or more entities are physically related if they interact directly or indirectly to each other so that they are physically close to and / or in a state of being physically close to each other. In some embodiments, two or more entities that are physically related to each other are covalently bonded to each other. In some embodiments, two or more entities that are physically related to each other are not covalently bonded to each other, but non-covalently, for example, by hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0032] Bonding: As used herein, the term “bonding” will be understood to typically refer to a non-covalent association between or among two or more entities. A “direct” bond involves physical contact between entities or parts. An indirect bond involves physical interaction through physical contact with one or more intermediate entities. Bonding between two or more entities can typically be evaluated in a variety of arbitrary contexts (including when the interacting entities or parts are studied individually or in the context of a more complex system (e.g., covalently or otherwise in relation to a carrier entity and / or in a biological system or cell)).

[0033] Cancer: The terms “cancer,” “malignant tumor,” “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 marked loss of control over cell proliferation. In some embodiments, a tumor may be or include cells that are precancerous (e.g., benign), malignant, premetastatic, metastatic, and / or nonmetastatic. This disclosure explicitly identifies certain cancers to which teaching may be particularly appropriate. In some embodiments, a suitable cancer may be characterized by a solid tumor. In some embodiments, a suitable cancer may be characterized by a hematological malignancy. Generally, various types of cancer known in the art include, for example, hematopoietic cancers including leukemia, lymphomas (Hodgkin and non-Hodgkin), myeloma and myeloproliferative disorders, sarcomas, melanomas, adenomas, solid tissue cancers, squamous cell carcinomas of the oral cavity, throat, pharynx, and lung, liver cancer, genitourinary cancers (e.g., prostate, cervix, bladder, uterus, and endometrial cancers), as well as renal cell carcinoma, bone cancer, pancreatic cancer, skin cancer, melanoma of the skin and eye, endocrine cancers, thyroid cancer, parathyroid cancer, head and neck cancers, breast cancer, gastrointestinal cancers, as well as nervous system cancers and benign lesions (e.g., papillomas).

[0034] CDR: As used herein, “CDR” may refer to a complementarity-determining region within the variable region of an antibody drug. Each variable region of the heavy chain and light chain has three CDRs, each variable region referred to as CDR1, CDR2, and CDR3. A “set of CDRs” or “CDR set” refers to a group of three or six CDRs that reside in either a single antigen-binding variable region or an antigen-binding CDR of the same type of heavy chain and light chain variable region. In the art, certain systems (e.g., Rabat, Chothia) have been established for defining CDR boundaries. Those skilled in the art are aware of the differences between these systems and can understand CDR boundaries to the extent necessary to understand and practice the claimed invention.

[0035] Chemotherapy Agents: As used herein, the term “chemotherapy agent” has the meaning understood in the art and refers to one or more apoptosis-promoting agents, cell stabilizers, and / or cytotoxic agents, for example, specifically, agents available or recommended for use in the treatment of one or more diseases, disorders, or conditions associated with undesirable cell proliferation. In many embodiments, chemotherapy agents are useful in the treatment of cancer. In some embodiments, chemotherapy agents are one or more alkylating agents, one or more anthracyclines, one or more cytoskeletal disruptors (e.g., microtubule targeting agents (e.g., taxanes, mytansines, and their analogs)), one or more epothilons, one or more histone deacetylase inhibitors HDACs, one or more topoisomerase inhibitors (e.g., topoisomerase I and / or topoisomerase II inhibitors), one or more kinase inhibitors, one or more nucleotide analogs or nucleotide precursor analogs, one or more peptide antibiotics, one or more platinum-based drugs, one or more retinoids, one or more vinca alkaloids, and / or one or more analogs of the following (i.e., those sharing relevant antiproliferative activity), or may include these: 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, epotilon, etoposide, fluorouracil, gemcitabine, Hydroxyurea, idarubicin, imatinib, irinotecan, mytansine and / or its analogs (e.g., DM1), mechloretamine, mercaptopurine, methotrexate, mitoxantrone, mytansinoids, oxaliplatin, paclitaxel, pemetrexed, teniposide, thioguanine, topotecan, barbicin, vinblastine, vincristine, vindesine, vinorelbine, and one or more of these in combination.In some embodiments, chemotherapeutic agents can be used in the context of antibody-drug conjugates. In some embodiments, chemotherapeutic agents include 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, and P 4 / D10-Doxorubicin, gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, glenbatumumab 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 This chemotherapeutic agent is found in antibody-drug conjugates 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 lorbotuzumab meltansine.

[0036] Engineered: Generally, the term "engineered" can refer to a state in which something has been processed by human hands. For example, if a polypeptide sequence has been processed by human hands, that polypeptide is considered "engineered." For example, in some embodiments of the present invention, an engineered polypeptide includes a sequence that contains one or more amino acid mutations, deletions, and / or insertions introduced into a reference polypeptide sequence by human hands. In some embodiments, an engineered polypeptide includes a polypeptide that has been fused (i.e., covalently bonded) with one or more additional polypeptides by human hands to form a fusion polypeptide that does not exist naturally in vivo. Similarly, a cell or organism is considered "engineered" if it has been processed in such a way that its genetic information is altered (e.g., new genetic material that was not previously present has been introduced, or previously present genetic material has been altered or removed, by substitution or deletion mutations, or by a mating protocol, through transformation, crossbreeding, somatic cell hybridization, transfusion, transduction, or other mechanisms). It is customary and understood by those skilled in the art that a manipulated polypeptide or cell derivative or progeny is typically still referred to as “manipulated,” even if the actual processing was carried out on the original entity.

[0037] Host Cells: As used herein, the term “host cells” can refer to cells of an organism that have been selected, modified, transformed, grown, used, or processed in any way for the production of material by the cells, such as the expression of genes, DNA or RNA sequences, proteins or enzymes by the cells. Host cells include, but are not limited to, lymphocytes (e.g., T cells, B cells, and NK cells), neutrophils, and immune cells, including monocytes / macrophages.

[0038] In vitro: As used herein, the term “in vitro” refers to events occurring in an artificial environment, such as a test tube or reaction vessel, or cell culture, rather than within a multicellular organism.

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

[0040] Isolated: As used herein, the term “isolated” can mean (1) a substance and / or entity separated from at least a portion of the components to which it was initially formed (whether in nature and / or in an experimental environment), and / or (2) a substance and / or entity designed, produced, prepared and / or manufactured by human hands. Isolated substances and / or entities may 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 to which they were initially formed. In some embodiments, the 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 about 99% ultrapure. As used herein, a substance is “pure” if it is substantially free of other components. In some embodiments, as will be understood by those skilled in the art, a substance can still be considered “isolated” or even “pure” after being combined with other components, such as one or more carriers or excipients (buffers, solvents, water, etc.), and in such embodiments, the isolation percentage or purity of the substance is calculated without including such the substance alone or excipients. To give just one example, in some embodiments, a biological polymer (e.g., naturally occurring polypeptides or polynucleotides) is considered “isolated” if: a) for reasons of its origin or source it is not related to some or all of the components that accompany it in its native state in nature; b) it substantially does not contain other polypeptides or nucleic acids of the same species from which it thrives in nature; or c) it is expressed by, or otherwise related to, components from a cell or other expression system that is not the species that thrives in nature. Thus, in some embodiments, for example, a chemically synthesized polypeptide, or a polypeptide synthesized in a cell system different from the cell system that thrives in nature it thrives in nature, is considered an “isolated” polypeptide.Alternatively or additionally, a polypeptide subjected to one or more purification techniques may be considered an “isolated” polypeptide to the extent that it has been separated from a) other naturally occurring components and / or b) other components that were associated with it when it was first produced.

[0041] Functionally coupled: As used herein, the term “functionally coupled” can refer to a parallel relationship in which the components described are made possible to function in the intended manner. A “functionally coupled” control element to a functional element is related in such a way that the expression and / or activation of the functional element is achieved under conditions adapted to the control element. In some embodiments, a “functionally coupled” control element is sequential (e.g., covalently coupled) to an objective code element. In some embodiments, the control element acts on the objective functional element transversely or otherwise.

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

[0043] Polypeptide: As used herein, the term “polypeptide” has the meaning generally recognized in the art and refers to a polymer of at least three amino acids. Those skilled in the art will understand that the term “polypeptide” is sufficiently common and is 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 skilled in the art will understand that protein sequences generally tolerate some degree of substitution without disrupting activity. Thus, polypeptides that retain activity and share at least 30–40% overall sequence identity, often about 50%, 60%, 70%, or more than 80% sequence identity, with other polypeptides of the same class, and further include at least one region with much higher identity, often more than 90%, or even 95%, 96%, 97%, 98%, or 99%, within a single highly conserved region (usually a region containing at least 3–4 amino acids, often more than 20), are included in the related term “polypeptide” as used herein. Polypeptides comprise L-amino acids, D-amino acids, or both, and may include any various amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, and methylation. In some embodiments, proteins may include native amino acids, non-native amino acids, synthetic amino acids, and combinations thereof. The term “peptide” is generally used to refer to polypeptides with a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, proteins may be antibody drugs, antibody fragments, their biologically active portions, and / or characteristic portions thereof.

[0044] Preventing or Preventing: As used herein, the terms “prevention” or “prevention,” when used in relation to the development of a disease, disorder, and / or condition, may mean a reduction in the risk of developing a disease, disorder, and / or condition, and / or a delay in the development and / or worsening of one or more characteristics or symptoms of the disease, disorder, and / or condition. In some embodiments, prevention is evaluated on a population basis, and a drug is considered to “prevent” a particular disease, disorder, or condition if, in a population susceptible to the disease, disorder, or condition, the incidence, frequency, and / or intensity of one or more symptoms of the disease, disorder, or condition is statistically significant.

[0045] Recombinant: As used herein, the term “recombinant” means polypeptides designed, manipulated, prepared, produced, manufactured, and / or isolated by recombinant means, e.g., polypeptides expressed using recombinant expression vectors transfected into host cells; polypeptides isolated from recombinant combinatorial human polypeptide libraries; polypeptides isolated from or otherwise manipulated from animals (e.g., mice, rabbits, sheep, fish, etc.) that are transgenic to a gene(s) or genetic component that codes for and / or directly expresses one or more components, parts, elements, or domains of the polypeptide or itself; and / or polypeptides prepared, expressed, produced, or isolated by any other means involving splicing or ligating selected nucleic acid sequence elements with each other, chemically synthesizing selected sequence elements, and / or otherwise producing nucleic acids that code for and / or direct the expression of the polypeptide or one or more components, parts, elements, or domains of the polypeptide or itself. 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 arise from mutagenesis (e.g., in vivo or in vitro) of known sequence elements derived from natural or synthetic sources (e.g., the germline of the source organism of interest (e.g., human, mouse, etc.)).

[0046] Specific Binding: As used herein, the term “specific binding” may refer to the ability to identify a promising binding partner within an environment in which binding may occur. A binding substance that interacts with one particular target in the presence of other potential targets is said to “specifically bind” to the target it interacts with. In some embodiments, specific binding is assessed by detecting or measuring the degree of association between the binding substance and its partner. In some embodiments, specific binding is assessed by detecting or measuring the degree of dissociation of the binding substance-partner complex. In some embodiments, specific binding is assessed by detecting or measuring the ability of the binding substance to compete for alternative interactions between its partner and another entity. In some embodiments, specific binding is assessed by performing such detection or measurement over a certain concentration range.

[0047] Subject: As used herein, the term “Subject” means an organism, typically a mammal (e.g., a human, and in some embodiments, a prenatal human form). In some embodiments, the Subject has a relevant disease, disorder, or condition. In some embodiments, the Subject is susceptible to a disease, disorder, or condition. In some embodiments, the Subject exhibits one or more symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the Subject exhibits no symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the Subject is a person who has one or more characteristics specific to susceptibility to or risk of a disease, disorder, or condition. In some embodiments, the Subject is a patient. In some embodiments, the Subject is an individual whose diagnosis and / or treatment is controlled and / or has been controlled.

[0048] Therapeutic Agent: As used herein, the term “therapeutic agent” generally refers to any agent that, when administered to an organism, induces a desired pharmacological effect. In some embodiments, an agent is considered a therapeutic agent if it exhibits a statistically significant effect in a suitable population. In some embodiments, a suitable population may be a population of model organisms. In some embodiments, a suitable population may be defined by various criteria, such as a particular age group, gender, genetic background, or pre-existing clinical condition. In some embodiments, a therapeutic agent is a substance that can be used to alleviate, improve, reduce, suppress, prevent, delay the onset, reduce the severity, 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 an agent that has been approved or is subject to approval by a government agency before being marketed for administration to humans. In some embodiments, a “therapeutic agent” is an agent that requires a medical prescription for administration to humans.

[0049] Therapeutic Dose: As used herein, the term “therapeutic dose” means an amount sufficient to treat a disease, disorder, and / or condition when administered to a population suffering from or susceptible to such disease, disorder, and / or condition. In some embodiments, the therapeutic dose is an amount that reduces the incidence and / or severity of one or more symptoms of a disease, disorder, and / or condition, stabilizes one or more of its characteristics, and / or delays its onset. Those skilled in the art will understand that the term “therapeutic dose” does not actually require that a favorable therapeutic outcome be achieved in a particular individual. Rather, the therapeutic dose is considered to be an amount that, when administered to patients in need of such treatment, produces a specific desired pharmacological response in a meaningful number of subjects. For example, in some embodiments, the term “therapeutic dose” in the context of the treatment of the present invention refers to an amount that, when administered to an individual in need, blocks, stabilizes, attenuates, or reverses cancer-supporting processes in the aforementioned individual, or enhances or increases cancer-suppressing processes in the aforementioned individual. In the context of cancer treatment, a “therapeutic dose” is the amount administered to an individual diagnosed with cancer that, when administered, prevents, stabilizes, inhibits, or reduces the further development of cancer in that individual. Particularly preferred “therapeutic doses” in the compositions described herein are those that reverse the development of malignant tumors, such as pancreatic cancer, (through therapeutic treatment) or aid in achieving or prolonging remission of malignant tumors. The therapeutic dose administered to an individual to treat cancer may be the same as, or different from, the therapeutic dose administered to promote remission or inhibit metastasis. As with most cancer treatments, the therapeutic methods described herein are not to be interpreted, limited to, or otherwise limited as a “cure” of cancer, but rather are directed towards “treating” cancer using the compositions described, i.e., to bring about a desirable or beneficial change in the health of an individual with cancer.Such benefits are recognized by experienced healthcare providers in the field of oncology and include, but are not limited to, stabilization of the patient's condition, reduction in tumor size (tumor regression), improvement in functional capacity (e.g., improved function of cancerous tissue or organ), reduction or suppression of further metastasis, reduction of opportunistic infections, increased survival rates, reduced pain, improved motor function, improved cognitive function, improved energy (vitality) (reduced fatigue), improved well-being, restoration of normal appetite, restoration of healthy weight gain, and combinations thereof. In addition, regression of a particular tumor in an individual (e.g., as a result of the treatment described herein) can also be evaluated by taking samples of cancer cells from the tumor site, such as pancreatic adenocarcinoma (e.g., throughout the course of treatment), examining the cancer cells for levels of metabolic and signaling markers to monitor the state of the cancer cells, and confirming at the molecular level that the cancer cells have regressed to a less malignant phenotype. For example, tumor regression induced by the method of the present invention is indicated by finding 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 a state observed in normal individuals without cancer) of metabolic pathways, intercellular signaling pathways, or intracellular signaling pathways that exhibit abnormal activity in individuals diagnosed with cancer. Those skilled in the art will understand that in some embodiments, a therapeutically effective dose can be formulated and / or administered as a single dose. In some embodiments, a therapeutically effective dose can be formulated and / or administered in multiple doses, for example, as part of an administration regimen.

[0050] Transfusion: As used herein, the term “transfusion” may refer to the introduction of foreign nucleic acids into cells using recombinant DNA technology. As used herein, the term “transformation” may refer to the introduction of foreign gene, DNA, or RNA sequences into host cells, thereby causing the host cells to express the introduced gene or sequence and produce the encoded protein or enzyme.

[0051] Transduction. As used herein, "transduction" may refer to the introduction of foreign nucleic acids 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 is structurally different from the reference molecule, for example, a molecule in which one or more chemical parts are present or absent or at different levels than the reference entity. In some embodiments, a variant may also be functionally different from its reference molecule. Generally, whether a particular molecule can be appropriately considered a “variant” of a reference molecule depends on the degree of structural identity with the reference molecule. As those skilled in the art will understand, a biological or chemical reference molecule has certain characteristic structural elements. A variant, by definition, is a distinct molecule that shares one or more characteristic structural elements but differs from the reference molecule in at least one respect. For example, to give just a few examples, a polypeptide may have characteristic sequence elements composed of multiple amino acids whose relative positions 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 relative positions 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 the amino acid sequence or nucleotide sequence. In some embodiments, the 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 with respect to the reference polypeptide or nucleic acid. In some embodiments, the 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, the variant polypeptide or nucleic acid shares one or more of the biological activities of the reference polypeptide or nucleic acid.

[0053] Vector: As used herein, the term “vector” may refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is bound. Vectors may encompass 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 in order to amplify that fragment. The term "replicon" refers to any genetic element (e.g., plasmid, phage, cosmid, chromosome, or virus) that can act as an autonomous unit of in vivo DNA replication. Many vectors known in the art can be used to manipulate nucleic acids and to incorporate response elements and promoters into genes. Preferred vectors include, but are not limited to, plasmids (e.g., PBR322 or pUC plasmid derivatives), modified viruses (e.g., adenoviruses, retroviruses, adeno-associated viruses, or herpesviruses), or Bluescript vectors. For example, DNA fragments corresponding to response elements and promoters can be inserted into a suitable vector by combining them with a selected vector having complementary sticky ends. In some embodiments, the ends of a DNA molecule may be enzymatically modified, or arbitrary sites may be generated by attaching nucleotide sequences to the DNA ends via linkers. In some embodiments, the vector can be engineered to contain a selection marker gene for screening cells into which markers have been incorporated into the cellular genome. Such markers enable the identification and / or screening of host cells that express the protein encoded by that marker.

[0055] One type of vector, a "plasmid," refers to a circular double-stranded DNA loop that can ligate additional DNA segments. Another type of vector, a viral vector, can ligate additional DNA segments into the viral genome. Certain vectors are self-replicating within the host cell into which they are introduced (e.g., bacterial vectors with bacterial replication origins and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors), upon introduction into a host cell, can integrate into the host cell's genome and replicate in conjunction with the host genome. Furthermore, certain vectors can direct the expression of functionally bound genes. 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 regulatory sequences (e.g., transcription and translation elements) necessary to regulate the expression of the fusion protein in a suitable host cell. These regulatory sequences may include a promoter region, an enhancer region, a transcription termination site, a ribosome binding site, a start codon, a splice signal, an intron, a polyadenylation signal, a Shine / Dalgarno translation sequence, and a Kozak consensus sequence. The regulatory sequences are selected considering the host cell in which the fusion protein will be produced. In some embodiments, preferred 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., α-fetal protein, amylase, cathepsin E, M1 muscarinic receptor, γ-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 nucleic acids, the vector may also include one or more regulatory regions and / or selection markers useful for selecting, measuring, and monitoring the results of nucleic acid delivery (e.g., delivery to a particular tissue or duration of expression).

[0058] The vector can be introduced using methods known in the art, such as injection, translocation, electroporation, microinjection, transduction, cell fusion, lipofection, calcium phosphate precipitation (Graham, F. Let al., Virology, 52:456 (1973); Chen and Okayama, Mol. Cell. Biol. 7:2745-2752 (1987)), liposome-mediated textured salt method (Wong, T. Ket al., Gene, 10:87 (1980); Nicolau and Sene, Biochim. Biophys. Acta, 721:185-190 (1982); Nicolau et al., Methods Enzymol., 149:157-176 (1987)), and DEAE-dextran treatment (Gopal, Mol. Cell. The gene can be introduced into desired host cells by means of Biol., 5:1188-1190 (1985), gene bombardment (Yang et al., Proc. Natl. Acad. Sci., 87:9568-9572 (1990)), or by using a gene species or DNA vector transporter (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 are used in a wide range of gene transfer applications in cells and living animals. Possible viral vectors include, but are not limited to, adenoviruses, retroviruses, vaccinia viruses, poxviruses, adeno-associated viruses, herpes simplex viruses, lentiviruses, baculoviruses, Sendai viruses, measles viruses, 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 nucleic acids, vectors may also include one or more regulatory regions and / or select markers useful for screening, measuring, and monitoring the results of nucleic acid delivery (e.g., delivery to tissues or persistence of expression).

[0060] In some embodiments, polynucleotides can be introduced in vivo by lipofection. The use of liposomes for in vitro encapsulation and translocation of nucleic acids is increasing. In some embodiments, liposomes for in vivo gene translocation can be prepared using synthetic cationic lipids designed to limit the difficulties and risks encountered in liposome-mediated translocation (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 may facilitate the encapsulation of negatively charged nucleic acids and 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 WO95 / 18863, WO96 / 17823, and U.S. 5,459,127, which are incorporated herein by reference in their entirety. In some embodiments, direct translocation to specific cell types is obviously particularly desirable in tissues with 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 and oligonucleotide synthesis, as well as tissue culture and transformation (e.g., electroporation, lipofection). Enzyme reactions and purification techniques can be carried out according to the manufacturer's specifications, as commonly performed in the art, or as described herein. These techniques and procedures can generally be carried out according to conventional methods well known in the art, and as described in the various general and more specific references cited and discussed throughout this specification. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual 2. nd See ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) (incorporated herein by reference for all purposes).

[0062] Manipulated immune cells As used herein, “immune cells” refers to cells of the immune system 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 they are genetically modified to express non-native proteins (e.g., chimeric antigen receptors) or to contain exogenous nucleic acids.

[0063] Immune cells (e.g., T cells) can be modified in one or more ways. Immune cells (e.g., T cells) can express at least one non-native molecule that is a receptor for antigens present on the surface of one or more types of cells. In some embodiments, immune cells are designed to contain or express at least one synthetic molecule not found in nature, thus including immune cells (e.g., T cells) not found in nature. In certain embodiments, immune cells (e.g., T cells) are designed to express at least one chimeric antigen receptor (CAR), the CAR includes a CAR that targets a specific tumor antigen (e.g., glypican 3 (GPC3), malignant tumor variant receptor (MVR), HLA-DR (human leukocyte antigen D-associated), or CD19). In certain embodiments, immune cells are T cells, e.g., CD4 + T cells, CD8 +This can be a population of T cells including T cells, Treg cells, Thl T cells, Th2 T cells, Thl7 T cells, nonspecific T cells, or any combination thereof. Immune cells (e.g., T cells) engineered with chimeric antigen receptors have great potential for cancer treatment. In CARs, the receptor recognizes an antigen, and when this antigen binds, it can be programmed to activate immune cells and kill the cells expressing the antigen. Therefore, immune cells expressing CARs against antigens expressed on tumor cells can target and kill tumor cells. For example, recent clinical trials of CD19-targeted CAR transduction T cells (CD19-CAR T cells) for hematological malignancies have demonstrated the potent effects 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 Ts is, at least in part, due to the fusion structure of CARs created by artificially combining a high-affinity antigen-binding domain with multiple signaling domains (Maus, M.V. et al. (2014) Blood 123:2625-2635; van der Stegen, S.J. et al. (2015) Nat. Rev. Drug Discov. 14:499-509).

[0064] A CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the extracellular antigen-binding domain includes a single-chain variable fragment (scFv) capable of recognizing tumor-associated antigens, 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 of an immune receptor tyrosine-based activation motif (e.g., CD3ζ) and a co-stimulatory signaling molecule (e.g., CD28 and CD137(4-1BB)).

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

[0066] In some embodiments, the transmembrane domains include 4-1BB / CD137, activated 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 that specifically binds to CD83, LIGHT, LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA -1) Transmembrane domains derived from proteins selected from MHC class 1 molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signal transduction lymphocyte activating molecules (SLAM proteins), SLAM (SLAMF1), SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor proteins, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, and VLA-6.

[0067] In some embodiments, the intracellular signaling domains include 4-1BB / CD137, activated 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, ligand that specifically binds to CD83, LIGHT, LTBR, ​​Ly9 (CD229), Lyl08 lymphocyte function-associated antigen-1 (LFA-1), MHC class 1 The molecule contains intracellular signaling domains derived from proteins selected from NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activating molecules (SLAM proteins), SLAM (SLAMF1), SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor proteins, TNFR2, TNFSF14, Toll ligand receptors, TRANCE / RANKL, VLA1, and VLA-6, or any combination thereof.

[0068] In some embodiments, the chimeric antigen receptor includes an additional antigen-binding domain. In some embodiments, the chimeric antigen receptor is a bispecific CAR (i.e., targets two antigen-binding domains). In some embodiments, the chimeric antigen receptor is polyvalent (i.e., 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, thymic tissue, tissue from an infection site, ascites, pleural fluid, splenic tissue, or tumors. In addition, immune (e.g., T) cells can be derived from one or more immune cell lines available in the art. In some embodiments, T cells can be obtained from blood collected from a subject using any technique known to those skilled in the art (e.g., FICOLL® isolation and / or apheresis). Further methods for isolating T cells for T cell therapy are disclosed in U.S. Patent Publication 2013 / 0287748. Other non-limiting examples can be found in international patent applications PCT / US2015 / 014520 (published as WO2015 / 120096) and PCT / US2016 / 057983 (published as WO2017 / 070395), each of which is incorporated herein by reference in whole.

[0070] In some embodiments, the immune cells are autologous T cells. In some embodiments, the immune cells are obtained from a subject that is not a patient. In some embodiments, the T cells used in the therapeutic method are syngenic (different donor and recipient but identical twins). In some embodiments, the T cells used in the therapeutic method are allogeneic (same species but different donor) as the recipient. 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 with cancer or a tumor.

[0071] T cells can be engineered to express, for example, a chimeric antigen receptor (CAR). In some embodiments, CAR-T cells can be engineered to express an extracellular single-chain variable fragment (scFv). In some embodiments, the CAR is engineered to express the co-stimulatory domain as separate polypeptide chains. Exemplary CAR-T cell therapies and constructs are described in U.S. Patent Publications 2013 / 0287748, 2014 / 0227237, 2014 / 0099309, and 2014 / 0050708, which are incorporated herein by reference in their entirety.

[0072] CAR Construction This 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 is capable of providing specificity to immunoeffector cells for a particular antigen. In some embodiments, a 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 proliferation, function, persistence, and antitumor activity of immune (e.g., CAR-T) cells, costimulatory signals can be provided by incorporating intracellular signaling domains from costimulatory molecules of immune (e.g., T cell) cells into the CAR construct. In some embodiments, the selection and placement of costimulatory domains within the CAR construct can influence immune (e.g., CAR-T) cell function and cell fate, and may have different effects on immune (e.g., CAR-T) cell dynamics, cytotoxic function, and potentially safety profile. Non-limiting examples of costimulatory molecules include CD28, ICOS, CD27, 4-1BB / CD137, 0X40, and CD40L.

[0074] As used herein, 4-1BB / CD137 is an activation-inducible T cell costimulatory molecule expressed in 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 receptor described herein comprises a five-amino acid sequence following the 4-1BB signaling domain, which can further be 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 containing an antigen-binding domain, (b) a transmembrane domain, and (c) an intracellular domain containing a costimulatory endodomain, wherein the costimulatory endodomain contains an intracellular signaling domain derived from 4-1BB / CD137 and five additional amino acids. As intended herein, the CAR construct may include an extracellular domain targeting any desired antigen-binding domain. In some embodiments, the costimulatory endodomain contains an intracellular signaling domain derived from 4-1BB / CD137 and five additional amino acids, where the five additional amino acids are encoded by SEQ ID NO: 1. In some embodiments, the costimulatory endodomain contains SEQ ID NO: 2. In some embodiments, the costimulatory endodomain contains 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: 2 or 4.

[0076] Sequence ID 1 - 5 additional amino acids (DNA sequence) CGTTTCTCTGTTGTT Sequence ID 2 - 4-1BB co-stimulatory domain and 5 additional amino acids (DNA sequence) TIFF0007829911000001.tif18161 Sequence ID 3 - 5 additional amino acids (amino acid sequence) ITOSVV Sequence ID 4 - 4-4-1BB co-stimulatory domain and 5 additional amino acids (amino acid sequence) TIFF0007829911000002.tif4139

[0077] In some embodiments, the extracellular binding domain of the CAR includes an antigen-binding domain. In some embodiments, the antigen-binding domain specifically binds to disease-related antigens. In some embodiments, the antigen-binding domain specifically binds to tumor antigens. In some embodiments, the antigen-binding domain specifically binds to any number of targets, including surface antigens, cytoplasmic, or nuclear antigens. For example, antigen-binding domains include 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 (erbl, erb2, erb3, erb4, and their variants), 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), and members of the vascular endothelial growth factor family (VEGFR1-3). The antigen-binding domain can bind to the 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, ligands for the α-folate receptor and 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 antibodies or fragments thereof that bind to cytoplasmic or nuclear antigens such as La / SSB antigen, members of the Rho family of GTPases, and members of high-mobility group proteins. Similarly, the antigen-binding domain can bind to the alpha and beta chains or gamma and delta chains of T cell receptors (TCRs), or fragments thereof.In some embodiments, the antigen-binding domain can bind to peptides presented by human leukocyte antigen class (HLA) I and II protein complexes. Examples, but not limited to, include the EGFR family, Survivin, sry-like high-mobility box (SOX) protein family, melanoma-related antigens (e.g., autoimmune cancer / testicular antigen NY-ESO-1, melanoma antigen family A member MAGEA, melanoma antigens preferentially expressed in melanoma PRAME, gp100, MART-1), and leukemia-related antigens (e.g., AMLI-ETO, DEK-CAN, PML-RAR alpha, Flt3-ITD, NPM1, AurA, Bcl-2, These 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, NuSAPl, 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 cytokine receptors (e.g., IL-13 receptor, IL-22 receptor), NKG2D receptors (e.g., ULBP1, ULBP2), EGFR family members, or autoreactive TCRs. In some embodiments, the antigen-binding domain specifically binds to tumor antigens. 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, mesoserine, SAP-1, Survivin, NY-ESO-1 / LAGE-1, PRAME, SSX-2, BRCA1 / 2, CDK4, CML66, or CD19. In some embodiments, the antigen-binding domain is or contains an antibody drug.In some embodiments, the antigen-binding domain is or comprises an antibody drug that specifically binds to GPC3, MVR, HLA-DR, or CD19.

[0078] In some embodiments, the transmembrane domains include 4-1BB / CD137, activated 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 that specifically binds to CD83, LIGHT, LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA -1) The transmembrane domain is derived from a protein selected from MHC class 1 molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activating molecules (SLAM proteins), 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 transmembrane domain includes SEQ ID NO: 5.In some embodiments, the transmembrane domain includes 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] Sequence ID 5 - CD8 / Hinge / Transmembrane TIFF0007829911000003.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] Sequence ID 6 - CD3ζ TIFF0007829911000004.tif46161

[0082] In some embodiments, CAR further comprises a T2A self-cleaving peptide. In some embodiments, CAR further comprises a signal peptide or a reader sequence. In some embodiments, CAR further comprises a CD8α reader sequence. In some embodiments, CAR further comprises a flag tag sequence. In some embodiments, CAR further comprises a hinge region. In some embodiments, the hinge region is a CD8α hinge. In some embodiments, CAR further comprises SEQ ID NO: 7. In some embodiments, 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] Sequence ID 7 - CD8α Reader Sequence TIFF0007829911000005.tif10159 Sequence ID 8 - Flag-Tag Array TIFF0007829911000006.tif3128

[0084] GPC3 CAR Glypican 3 (GPC3) is a cell surface protein encoded by the human GPC3 gene and is an oncofetal antigen that is frequently reexpressed in neoplastic hepatocytes (Vidali, et al, 2008, J hepatol 48:399-406). GPC3 is highly expressed in the fetal liver and not expressed in normal adult liver tissue. However, its expression is reactivated in hepatocellular carcinoma and is closely associated with the development of liver cancer. The detection rate of GPC3 expression is relatively high in the early stages of liver cancer and increases with the development of liver cancer. Furthermore, GPC3 is also expressed in tumors such as melanoma, ovarian clear cell carcinoma, yolk sac tumor, and neuroblastoma. Considering that GPC3 is specifically highly expressed in hepatocellular carcinoma, melanoma, and other tumors, it is emerging as a useful immunohistochemical diagnostic test (Anatelli, et al., 2008, Am J Clin Path 130:219-223) and a promising 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 in organogenesis or as a receptor for cell growth factors. The protein core of GPC3 contains two subunits: an N-terminal subunit and a C-terminal subunit. A glycosylphosphatidylinositol (GPI) anchor is attached to the serine at position 560, located on the carboxyl (C) terminal side of GPC3. The GPI anchor plays a role in localizing GPC3 on the cell surface through covalent bonding with cell membrane lipids. In addition, the serine at positions 495 and 509 of GPC3 are modified with heparan sulfate chains (HS chains), which are known to regulate multiple growth signaling pathways, including Wnt signaling, FGF signaling, and BMP signaling. The growth signaling pathways involved are known to differ depending on the type of cancer. For example, in hepatocellular carcinoma (HCC), cells grow in response to stimulation of the Wnt signaling pathway.

[0086] This disclosure provides, at least in part, GPC3 CAR polypeptides. In some embodiments, the extracellular binding domain of the GPC3 CAR includes an antigen-binding domain. In some embodiments, the antigen-binding domain is or includes an antibody drug. In some embodiments, the antigen-binding domain is or includes 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 including a light chain CDR1 containing SEQ ID NO: 9, a light chain CDR2 containing SEQ ID NO: 10, and a light chain CDR3 containing SEQ ID NO: 11, and a heavy chain variable domain including a heavy chain CDR1 containing SEQ ID NO: 12, a heavy chain CDR2 containing SEQ ID NO: 13, and a heavy chain CDR3 containing SEQ ID NO: 14; ii) a transmembrane domain; and iii) an intracellular signaling domain that activates T cells when the antigen is bound to an antibody drug.

[0088] TIFF0007829911000007.tif52161

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

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

[0091] Sequence ID 15 - Human GC33 light chain variable region (amino acid sequence) TIFF0007829911000008.tif11160 Sequence ID 16 - Human GC33 heavy chain variable region (amino acid sequence) TIFF0007829911000009.tif18159 Sequence ID 17 - Human GC33 light chain variable region (DNA sequence) TIFF0007829911000010.tif39161 Sequence ID 18 - Human GC33 heavy chain variable region (DNA sequence) TIFF0007829911000011.tif46160

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

[0093] The primary function of HLA-DRs is to present potentially exogenous peptide antigens to the immune system, inducing or suppressing the response of T (helper) cells, ultimately leading to the production of antibodies against the same peptide antigen. HLA-DRs are αβ heterodimers, cell surface receptors, with each subunit containing two extracellular domains, a transmembrane domain, and a cytoplasmic tail. Both the α and β chains are fixed to the membrane. The N-terminal domain of the mature protein forms an alpha-helix, constituting the exposed portion of the binding groove, while the C-terminal cytoplasmic region interacts with the other chains beneath the binding groove to form a β-sheet that spans the cell membrane. The majority of peptide contact sites are located in the first 80 residues of each chain.

[0094] HLA-DRs are expressed selectively on antigen-presenting cells (e.g., dendritic cells, macrophages, monocytes, and B cells). Since the amount of HLA-DR "antigens" on the cell surface often increases in response to stimulation, HLA-DRs are also markers of immune stimulation. In B-cell malignancies, HLA-DR expression levels are high, while in normal cells, the expression spectrum is limited. Therefore, 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). In the Phase I / II trial, although toxicity was not serious, efficacy was limited, and further trials were discontinued (Lin, TS, et al. (2009) Leuk. Lymphoma 50:1958-1963).

[0095] As used herein, malignant tumor variant receptor (MVR) antibody agents recognize the polymorphic region of HLA-DR (as described in U.S. Patent Application Publication US2016-0257762, which is incorporated herein by reference in its entirety). This disclosure provides, at least in part, MVR CAR polypeptides. A schematic diagram of an exemplary MVR CAR construct according to this disclosure is shown in Figure 1. In some embodiments, the extracellular domain of the CAR includes an antigen-binding domain. In some embodiments, the antigen-binding domain is or includes an antibody agent. In some embodiments, the antigen-binding domain is or includes an antibody agent that specifically binds to HLA-DR.

[0096] In some embodiments, the CAR polypeptide comprises a single-stranded variable fragment (scFv) form of an anti-MVR antibody drug. 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-stranded variable fragment (scFv) form of an anti-MVR antibody drug. 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] Sequence ID 19 - MVRL2H2 (amino acid sequence) TIFF0007829911000012.tif33161

[0099] Sequence ID 20 - MVRL2H2 (DNA sequence) TIFF0007829911000013.tif96161

[0100] CD19 CAR CD19 is a biomarker for normal B cells, neoplastic B cells, and follicular dendritic cells. CD19 plays a crucial role in establishing the intrinsic signaling threshold of B cells by regulating both B cell receptor-dependent and B cell-independent signaling. Furthermore, along with the complement receptor CD21, tetraspanin membrane protein CD81 (TAPA-1), and CD225, CD19 functions as a major signaling component of multimolecular complexes 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] This disclosure provides CD19 CAR polypeptides, at least in part. In some embodiments, the extracellular domain of the CAR includes an antigen-binding domain. In some embodiments, the antigen-binding domain is or includes an antibody drug. In some embodiments, the antigen-binding domain is or includes an antibody drug that specifically binds to CD19.

[0102] In some embodiments, the CAR polypeptide comprises a single-stranded variable fragment (scFv) form of an anti-CD19 antibody drug. 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-stranded variable fragment (scFv) form of an anti-CD19 antibody drug. 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] Sequence ID 21 - CD19 (amino acid sequence) TIFF0007829911000014.tif32160 Sequence ID 22 - CD19 (DNA sequence) TIFF0007829911000015.tif89161

[0105] nucleic acid As used herein, “nucleic acid” is used to include any compound and / or substance containing 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 includes a region encoding a CAR, where the CAR includes (a) an extracellular domain containing an antigen-binding domain, (b) a transmembrane domain, and (c) an intracellular domain containing 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 in a manner known in the art, and the nucleic acid molecule can be functionally bound to an expression regulatory 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 includes a promoter operably bound to the nucleic acid. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inductive promoter. In some embodiments, the expression vector includes SEQ ID NOs. 23, 24, 25, 26, 27, and / or 28. In some embodiments, the expression vector includes sequences that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs. 23, 24, 25, 26, 27, and / or 28.

[0108] Sequence ID 23 - EF1α promoter TIFF0007829911000016.tif145161 Sequence ID 24 - U5 Repeat TIFF0007829911000017.tif11160 Sequence ID 25 - Gag / Pol TIFF0007829911000018.tif174161 Sequence ID 26 - cPPT TIFF0007829911000019.tif67161 Sequence ID 27 - Woodchuck / PRE TIFF0007829911000020.tif74160 Sequence ID 28 - R / region TIFF0007829911000021.tif11161

[0109] Lentiviral vectors are derived from lentiviruses. They are based on single-stranded RNA lentiviruses, a subclass of retroviruses. These vectors offer the advantages of moderate cloning ability and stable gene expression, and can transduce both dividing and non-dividing cells, including nerve 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, and therefore can be used to treat persistent forms of disease.

[0110] By adding additional sequences to such cloning and / or expression sequences, the function of cloning and / or expression can be optimized, polynucleotide isolation can be facilitated, and the introduction of polynucleotides into cells can be improved. The use of cloning vectors, expression vectors, adapters, and linkers is well known in the art.

[0111] In some embodiments, a nucleic acid molecule is inserted into a vector capable of expressing the CAR of this disclosure upon introduction into engineered immune cells. In some embodiments, the engineered immune cells are T cells.

[0112] Production of CAR-T cells This specification provides a method for producing immune cells containing CARs. In some embodiments, the CAR-transformed immune cells 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 + These include, but are not limited to, T cells (cytotoxic T cells, CTLs), memory T cells, regulatory T cells (Treg cells), and apoptotic T cells. In some embodiments, the immune cells are NK cells.

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

[0114] In some embodiments, the Disclosure provides a method for producing engineered immune cells, comprising introducing into immune cells (i) a nucleic acid encoding a CAR, wherein the CAR comprises (a) an extracellular domain including an antigen-binding domain, (b) a transmembrane domain, and (c) an intracellular domain including 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 including an antigen-binding domain, (b) a transmembrane domain, and (c) an intracellular domain including 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, five amino acids are added to the 4-1BB cytoplasmic domain used to produce CAR as a co-stimulatory signaling factor in order to enhance the immunological efficacy of the cytoplasmic signaling domain 4-1BB. In some embodiments, the completed construct includes an antigen-binding domain which is scFv, an EFL-α promoter, a hinge region and transmembrane domain of human CD8, and an intracellular signaling domain. Specifically, the intracellular signaling domain includes a stimulatory domain and a co-stimulatory signaling domain. In some embodiments, the transmembrane domain may, 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 includes CD8. In some embodiments, the intracellular signaling domain includes a co-stimulatory 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 co-stimulatory domain includes 4-1BB with five consecutive amino acids. In some embodiments, the co-stimulatory domain is bound to CD3 zeta.

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

[0117] In some embodiments, the method for producing engineered immune cells further includes culturing the engineered immune cells after the introduction step. In some embodiments, the method for producing engineered immune cells further includes obtaining immune cells from a subject before the introduction step.

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

[0119] This disclosure includes the recognition that CAR-T cells generated by the methods described herein are therapeutically useful (for example, in the treatment of cancer).

[0120] therapeutic use This specification provides a method for treating a subject having cancer or other malignant tumor, comprising administering to the subject a composition comprising or delivering immune cells containing CARs. In some embodiments, the cancer is anti-glypican 3-related cancer. In some embodiments, the cancer is anti-CD19-related cancer. In some embodiments, the cancer is anti-MVR-related cancer.

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

[0122] "Anti-glypican-3 related 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 generally not expressed in healthy tissues. In addition, GPC3 overexpression is found in several tumors (for example, but not limited to, hepatocellular carcinoma, hepatoblastoma, germ cell tumors (e.g., yolk sac tumors, trophoblastic tumors), Wilms' tumor, gastric cancer, non-small cell lung cancer, and thyroid cancer).

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

[0124] "Anti-MVR-associated cancers" are characterized by cancer cells that express increased HLA-DR antigens compared to non-cancerous cells. In some embodiments, cancers with high HLA-DR antigen expression 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.

[0125] In some embodiments, cancers treatable according to the methods of this disclosure may include, but are not limited to, carcinomas, lymphomas (e.g., Hodgkin lymphoma and non-Hodgkin lymphoma), blastomas, sarcomas, and leukemias. 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, as well as various types of head and neck cancers.

[0126] In some embodiments, the cancer suitable for treatment according to the methods of this disclosure is hematological cancer. In some embodiments, hematological cancer is leukemia. In some embodiments, 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)); further hematological cancers or hematological conditions (including, but not limited to, B-cell lymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, and The following are 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 states, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndromes, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasms, Waldenström macroglobulinemia, and "preleukemia" (a collection of diverse blood conditions combined by myeloid blood cell production failure (or dysplasia)).

[0127] In some embodiments, the cancer to be treated by the method of the present disclosure is B-cell lymphoma (i.e., malignant lymphoma of B-cell origin). Examples of B-cell lymphoma include Hodgkin lymphoma, non-Hodgkin lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, mucosa-associated lymphoid tissue lymphoma (MALT), chronic lymphocytic leukemia, mantle cell lymphoma (MCL), Burkitt lymphoma, mediastinal large B-cell lymphoma, Waldenström macroglobulinemia, nodular 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. However, it is not particularly limited to these as long as it is a lymphoma of B-cell origin.

[0128] Immune cells (e.g., CAR-T cells) can be administered to a patient who needs them in a therapeutically effective amount. For example, the therapeutically effective amount of immune cells (e.g., CAR-T cells) is 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 cells. In some embodiments, the therapeutically effective amount of T cells is about 10 4 cells, about 10 5 cells, about 10 6 cells, about 10 7 cells, or about 10 8 cells. In some embodiments, the therapeutically effective amount of T cells is about 0.4×10 <00(...) 8 ~ about 2×10 8 T cells. In some embodiments, the therapeutically effective amount of T cells is about 0.4×10 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 These are T cells.

[0129] In some embodiments, the therapeutically effective dose of CAR T cells is approximately 2 × 10⁻⁶ 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 10⁶ 7 The value is cells / kg. In some embodiments, the therapeutically effective dose of immune cells (e.g., CAR-T cells) is approximately 1 × 10⁶ cells per kg of body weight. 6 ~about 2×10 6 These are T cells, and the maximum dose is approximately 1 × 10⁶ 8 These are T cells. In some embodiments, the therapeutically effective dose of T cells is approximately 1 × 10⁶ cells per kg of body weight. 6 Or approximately 2 × 10 6 These are T cells, and the maximum dose is approximately 1 × 10⁶ 8 These are T cells.

[0130] The number of cells depends on the intended end use of the composition and also on the type of cells contained in the composition. For example, in some embodiments, a population of T cells containing CARs will contain 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 containing CARs will contain 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 less than 1 liter. 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 10 6 Cells / ml greater than 10 7 Cells / ml greater than 10 8 The number of cells / ml is greater than 10. A clinically appropriate number of immune cells is 10 in total. 7 cells, 10 8 cells, 10 9 cells, 10 10 cells, 10 11 cells, or 10 12 It can be distributed into multiple intravenous infusions equal to or exceeding the number of cells.

[0131] In some embodiments, the composition can be administered to the patient parenterally. In some embodiments, the composition containing or delivering CAR-containing T cells can be administered to the patient parenterally in one or more doses. In some embodiments, the composition containing or delivering CAR-containing T cells can be administered to the patient parenterally once daily, once every 2 to 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 for treating cancer in a subject requiring such treatment, comprising administering engineered immune cells containing a CAR to the subject, wherein the CAR comprises (a) an extracellular domain containing an antigen-binding domain, (b) a transmembrane domain, and (c) an intracellular domain containing a costimulatory endodomain, the costimulatory endodomain comprising an intracellular signaling domain derived from 4-1BB / CD137 and five additional amino acids.

[0133] In some embodiments, the subject has previously received one or more additional anticancer therapies, the anticancer therapies being 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 with cancer.

[0134] Pharmaceutical composition In some embodiments, the disclosure provides a pharmaceutical composition comprising a CAR-containing T cell, 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, the costimulatory endodomain comprising an intracellular signaling domain derived from 4-1BB / CD137 and five additional amino acids. In some embodiments, the CAR-containing T cell is an autologous T cell. In some embodiments, the pharmaceutical composition may include a buffer, diluent, solubilizer, emulsifier, preservative, adjuvant, excipient, or any combination thereof. In some embodiments, the composition may also include one or more additional therapeutic agents, if desired.

[0135] In some embodiments, the T cells of this disclosure are first harvested from their culture medium and then formulated by washing and concentrating a therapeutically effective amount of cells in a medium and container system suitable for administration ("pharmaceutically acceptable" carrier). Suitable infusion media can be any isotonic media formulation, typically physiological saline, Normosol R (Abbott), or Plasma-Lyte A (Baxter), but 5% glucose in water or Ringer's lactate solution may also be used. Human serum albumin may be added to the infusion medium.

[0136] In some embodiments, the composition is formulated for parenteral administration. For example, the pharmaceutical compositions provided herein may be provided in a sterile injectable form (e.g., a form suitable for subcutaneous or intravenous injection). 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 optionally provided as a powder (e.g., lyophilized and / or sterile powder) under vacuum, which can be reconstituted with an aqueous diluent (e.g., water, buffer, 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 with the aqueous diluent (e.g., without shaking).

[0137] In some embodiments, T cells containing the CAR and / or nucleic acids encoding the CAR of this disclosure are formulated with a pharmaceutically acceptable parenteral vehicle. Examples of such vehicles include water, saline, Ringer's solution, glucose solution, and 1-10% human serum albumin. Liposomes and non-aqueous vehicles (e.g., non-volatile oils) can also be used. The vehicle or lyophilized powder may contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and additives that maintain chemical stability (e.g., buffers and preservatives). In some embodiments, the formulation is sterilized by known or preferred techniques. The pharmaceutical composition may further contain pharmaceutically acceptable additives, which, as used herein, include any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersions or suspension aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, etc., suitable for a particular desired dosage form. Remington's *The Science and Practice of Pharmacy*, 21st Edition, ARGennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006) discloses various additives used in the formulation of pharmaceutical compositions and known techniques for their preparation. Unless any conventional additive medium is incompatible with the substance or its derivatives, for example, by causing any undesirable biological effects or interacting with any other pharmaceutical composition components in a harmful manner, its use is considered to be within the scope of this disclosure.

[0138] In some embodiments, compositions comprising a T cell population containing the CAR and / or CAR-encoding nucleic acid of the present disclosure are stably formulated. In some embodiments, stable formulations of the T cell population containing the CAR and / or CAR-encoding nucleic acid of the present disclosure may include formulations comprising a phosphate buffer containing physiological saline or a selected salt, as well as a preservative solution and preservative, and a multipurpose preservative formulation suitable for pharmaceutical or veterinary use. The preservative formulation may contain at least one known preservative, or optionally, at least one phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercury nitrate, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate), alkylparabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, thimerosal, or a mixture thereof in an aqueous diluent. As is known in the art, any suitable concentration or mixture can be used, for example, 0.001 to 5% or any range or value within this range, for example, 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. It can be used with 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 within this range.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., Examples include 0, 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%, and alkylparabens in concentrations of 0.0005-1.0% (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%).

[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 specific period 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 longer). In some embodiments, if a composition containing an antibody drug is stored for longer than the specified time, degradation of the antibody drug will occur. The liquid dosage form and / or reconstituted solution may contain particulate matter and / or discoloration before administration. In some embodiments, the solution should not be used if there is discoloration or cloudiness, and / or if particulate matter remains after filtration. General considerations regarding the formulation and / or manufacture of pharmaceutical drugs can be found, for example, Remington: The Science and Practice of Pharmacy 21. st This can be found in Lippincott Williams & Wilkins, ed., 2005.

[0140] In some embodiments, pharmaceutical compositions comprising T cells containing the CAR and / or nucleic acids encoding the CAR of this disclosure may be contained in a container for storage or administration, e.g., a vial, syringe (e.g., an IV syringe), or bag (e.g., an IV bag). Pharmaceutical compositions according to this disclosure may be prepared, packaged, and / or sold in large quantities as single unit doses and / or as multiple single unit doses. As used herein, “unit dose” is an individual amount of a pharmaceutical composition containing a predetermined amount of the active ingredient. The amount of the active ingredient is approximately equal to the dose of the active ingredient that is expected to be administered to a subject and / or a convenient fraction of such a dose (e.g., half or one-third of such a dose).

[0141] kit This disclosure further provides a kit comprising one or more containers filled with at least one CAR and / or nucleic acid encoding a CAR as described herein. The kit can be used in any applicable method, including therapeutic, diagnostic, cell proliferation and / or isolation methods. Optionally, such containers may be accompanied by a notice in the form prescribed by a government agency that regulates the manufacture, use and sale of pharmaceutical or biological products. This notice may reflect (a) government agency approval for manufacture, use and sale for human administration, (b) instructions for use, or both.

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

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

[0144] The following embodiments further illustrate the present disclosure, but these embodiments do not limit the scope of the present disclosure as set forth in the claims.

[0145] Example 1: GPC3 lentivirus transfer plasmid Using standard DNA cloning techniques known in the art, a DNA construct encoding a single-stranded variable fragment (scFv) form (Figure 2) of a humanized anti-GC33 antibody drug was generated by ligating the VH and VL regions. The lentiviral transfer plasmids used herein are shown in Table 1.

[0146] [Table 1]

[0147] huGC33 VH-VL-scFv was cloned into the lentiviral vector pELPS4-MVRL2H2-euBBz. pELPS4-MVRL2H2-euBBz is a lentiviral vector containing the co-stimulatory 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 restriction enzyme digestion are shown in Figure 3.

[0148] To create a CAR construct without five additional amino acids in the 4-1BB co-stimulatory domain, huGC33 VH-VL-scFv was cloned into the lentiviral vector pELPS2-CD19-BBz. pELPS2-CD19-BBz is a lentiviral vector containing the co-stimulatory domain 4-1BB without 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 (5×10 7 cells / 1 mL / vial) were thawed and activated by placing them in a water bath for 2 - 3 minutes. 10 mL of CAR-T cell culture medium and 1 mL of PBMC 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 and then counted. Fresh cell medium was added to adjust the cell density to 1×10 6 cells / mL.T cell activation beads were added at 1×10 6 10 μL per cell, and IL-2 was supplemented in the medium. The cell culture medium was placed in a T75 flask and cultured in an incubator at 25% CO2 and 37°C.

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

[0151] [[ID=二十]]The cultured CAR-T cells were counted every 2 - 3 days, and fresh culture solution and IL-2 were added after each count (Figure 5). On the 12th day, the cultured CAR-T cells were collected, placed in a freezing container, and stored at -80°C. <C

[0152] Analysis of CAR expression on day 12 of culture revealed that while no CAR expression was observed in the control group, CAR expression was observed in 54-66% of the CAR-T cell group (Figure 6).

[0153] Target cells (GPC3-positive cell line) were harvested and seeded in a 96-well U-bottom plate. Next, effector cells (CAR-T cells) were added to the wells in effector cell:target cell ratios of 10:1, 3:1, 1:1, and 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 the absorbance at 490 nm (Figure 7).

[0154] Example 3: In vivo study of huGC33(VH-VL)-BBz CAR-T cells and huGC33(VH-VL)-euBBz CAR-T cells 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 inoculated with Huh-7_Luf-GFP cells (2×10⁶). 6 Cells (200 μL / head) were 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 observed by measuring the size of the tumors twice a week using TM900 (Figure 8).

[0155] After CAR-T cell administration, orbital blood was collected from mice once a week, and 100 μL of each blood sample was centrifuged at 12,000 rpm for 10 minutes to confirm 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® Fixed Viability Kit. After reaching a concentration of 0.1 μL / 100 μL DPBS / tube, staining was performed at room temperature for 10 minutes. 25 μL of count beads, 0.5 μL of CD45, 0.5 μL of CD8, 1.0 μL of CD45RO, 1.0 μL of CD62L, 1.0 μL of PD-1, 1.0 μL of Tim-3, 0.5 μL of CD4, 0.5 μL of CD69, and 0.0125 μL of Flag were added to 100 μL of FACS buffer and stained at room temperature for 30 minutes. After 30 minutes, 1X RBC lysis buffer was added and the mixture was reacted at room temperature for 5 minutes. After centrifugation at 2,000 rpm for 4 minutes, all supernatant was discarded. 2 mL of FACS buffer was added to the tube, and centrifugation was performed at 2,000 rpm for 4 minutes. Analysis was then performed using FACSCelesta (Figure 9).

[0156] Six weeks after CAR-T injection, the spleen, liver, and bone marrow of mice were collected, and the ratio of huGC33(VH-VL)-BBz CAR-T cells to huGC33(VH-VL)-euBBz CAR-T cells was evaluated. Tissue samples were processed, filtered through a 40 pm cell strainer, then centrifuged at 2,000 rpm for 5 minutes, and all supernatant was discarded. 5 μL of 1X ACK buffer was added and allowed to react 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 cytoplasmic signaling domain 4-1BB, a novel CAR expression vector (CD19-euBBz CAR) was constructed by adding five amino acids as a co-stimulatory signaling factor to the 4-1BB cytoplasmic signaling domain used in CAR-T. The completed construct includes an anti-CD19 scFv containing 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 co-stimulatory signaling domain. The intracellular signaling domain is the co-stimulatory signaling domain 4-1BB, to which five consecutive amino acids are added and CD3 zeta is bound. The finally produced CAR gene fragment was bound to an ELPS lentiviral expression vector cleaved with BarnH I and Sal I. In addition, cloning was performed by substituting only the scFv portion using BarnH I / Nhe I restriction enzymes.

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

[0159] To determine the transduction efficiency of mammalian cells, the transformation units (TU / mL) were measured by analyzing the number of lentiviral particles that could actually be transduced using Jurkat cells. CAR expression can be assayed by FACS. On day 1, 1 × 10⁶ Jurkat cells were used per well. 5 Cells were seeded in 96-well plates at 100 μL / 100 μL. On day 2, lentivirus was serially diluted to 1 / 3 in the 96-well plates, and lentiviral transduction was performed on already seeded Jurkat cells. At this time, lentiviral transduction was further increased by introducing polyblen (Millipore) into RPMI medium (10% FBS and 1×P / S). After centrifugation at 1200xg, 32°C for 2 hours, the cells were incubated in a 37°C 5% CO2 incubator for 3 hours, and only 100 μL of RPMI per well was added. On day 5, the flag of the lentivirus that infected the cells was stained with anti-Flag-DYKDDDDK (Biolegend, catalog number 637310), and the percentage of transduced cells was analyzed by flow cytometry. The titer was calculated using this data as described in Follenzi and Naldini, 2002.

[0160] FACS staining was performed, and the production ratio of two types of CAR-T cells was confirmed after incubation for 14 days. 2 × 10⁶ cells were produced for each CAR-T cell type. 5Cells were collected in FACS tubes (FALCON, catalog number 352052), then 2 mL of FACS buffer was added, and the cells were 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 number 344722)), 0.5 μL / tube of anti-CD4 BV650 (RPA-T4 (Biolegend, catalog number 300536)), and 0.125 μL / tube of anti-flag PE (L5 (Biolegend, catalog number 637310)) were added, and staining was performed at room temperature for 30 minutes. 2 mL of FACS buffer was added, and the cells were centrifuged at 2,000 rpm for 5 minutes, after which this process was repeated. For staining viable / dead cells, 1 μL / tube of 7-AAD (Biolegend, catalog number 420404) was added and left at room temperature for 5 minutes, after which analysis was performed using FACS (BD, FACSCelesta).

[0161] When the proportion of CD19 CAR-T cells produced was examined using FACS staining, the improved construct CD19-euBBZ CAR-T cells showed that 29.4% were CD4+ / CAR+, 50.8% were CD8+ / CAR+, and 80.2% were total CAR-T cells. In the case of the unimproved construct CD19-BBz CAR-T cells, the cell ratios were confirmed to be 42.7% CD4+ / CAR+, 29.3% CD8+ / CAR+, and 72.0% total CAR-T cells. Therefore, CD19-euBBz CAR-T cells showed 8.2% higher CAR expression and approximately twice the proportion of CD8+ / CAR+ cells (21.5%) compared to CD19-BBz CAR-T cells (Figure 11A).

[0162] Example 6: Confirmation of cytotoxicity of produced CD19 CAR-T cells To determine the cytotoxicity of two CAR-T cells cultured for 14 days, CAR-T(E):LCL(T) ratios were set to 30:1, 10:1, 3:1, and 1:1, and placed in 96-well white plates (Corning, catalog number 3917). First, 6 × 10⁶ CAR-T cells were placed in each plate. 5cells / 50μL, 2×10 5 cells / 50μL, 9×10 4 Cells / 50 μL, and 2 × 10 4 Cells were placed in wells at a rate of 50 μL / cell. Next, the target cell line, namely the CBK LCL-Luc cell line, was placed in a 37°C CO2 incubator (Mammert, INC0153med) at a rate of 2 × 10⁻¹⁶. 4 Cells were added to a volume of 50 μL, and the mixture was allowed to react for 4 hours. After 4 hours, 100 μL of Bright-Glo® (Promega, catalog number E2620) was added to each well, and the relative light units (RLU) were measured using a Luminometer (Thermo, Fluoroskan FL) after 5 minutes.

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

[0164] The results showed that when two CAR-T cells and CBK LCL-Luc cell lines were incubated together in a 30:1 ratio, the cytotoxicity after 4 hours was approximately 80%, while when incubated in a 10:1 ratio, the cytotoxicity was approximately 50%. When the number of each type of CAR-T cell incubated with cancer cells was reduced to one-third, the cytotoxicity also decreased to approximately one-third. Furthermore, it was confirmed that adding five amino acids to the 4-1BB domain in vitro did not affect the in vitro cytotoxicity (Figure 11B).

[0165] Example 7: Induction of subcutaneous administration animal models and validation of CAR-T using automated calipers and IVIS imaging. For the experimental animals, NSG (NOD-scid IL2rγμL1) mice (The Jackson Laboratory) were used and managed under controlled conditions in an animal breeding facility. The temperature was 23±2℃, with a 12-hour light-dark cycle and humidity of 50±10%, and feed and water were provided freely. In efficacy experiments using CD19-euBBz CAR-T, which has five amino acids added to the 4-1BB domain, CBK LCL-Luc cell lines were used in 2 × 10⁶ experiments. 6 The solution was prepared in a cell / 100 μL DPBS / head ratio and subcutaneously injected into 6-week-old female mice to induce a subcutaneous administration animal model. The tumor size was measured using an automated caliper (Youngbio, TM900) and was 50-100 mm. 3 Once this is reached, CD19-euBBz CAR-T cells and CD19-BBz CAR-T cells are divided into 2 × 10⁻¹⁰ cells. 6 Cells / 100 μL DPBS / head (dose 1) and 6 × 10 6 Efficacy was confirmed by administering 100 μL of DPBS per head (dose 2) of cells once via the tail vein. In all animal studies, tumor size and survival rate were regularly monitored.

[0166] More specifically, tumor size and photon values ​​were measured at intervals of 3 and 4 days after CAR-T administration using an automated caliper and an IVIS imaging device (PerkinElmer, Luna III) (Figures 12, 13). When using TM900, the tumor size was determined after placing the device at the tumor site. When imaging and confirming photon values ​​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 after 5 minutes, the presence of cancer cells was imaged using IVIS. After imaging, normalization was performed, and then luciferase values ​​(photon values) were confirmed and graphed. After constructing 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 effects were observed within one week of administering the two types of CAR-T cells.

[0167] 2 × 10⁶ CD19-euBBz CAR-T cells 6 Cells / 100 μL DPBS / head and 6 × 10 6 In the experimental group administered cells / 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 were administered at a dose of 100 μL / head of DPBS, almost no cancer cells were observed on imaging within one week of administration. However, cancer cells were identified in the experimental group administered CD19-BBz CAR-T after one week.

[0168] One week after CAR-T administration, as shown in the imaging, luciferase levels were measured in each subject, and CD19 CAR-T was 2 × 10⁶ 6Luciferase values were confirmed only in the group administered with cells / 100 μL DPBS / intracranially. After observation for more than 3 weeks, tumors continued to grow in mice not administered with CAR-T cells, and cancer cells were not identified in the three experimental groups where cancer cells had disappeared initially. However, 10 days later, although the luciferase level decreased in the group administered with 2×10 6 cells / 100 μL DPBS / intracranially, cancer cells did not completely disappear after 3 weeks. When 2×10 6 cells / 100 μL DPBS / intracranially were administered with CD19-euBBz CAR-T, the efficacy was found to be the same as that of the group treated with CD19-BBz CAR-T at 6×10 6 cells / 1**00 μL DPBS / intracranially by experimental imaging of cancer cells. From the results, although no difference in cytotoxicity between CD19-euBBz CAR-T and CD19-BBz CAR-T was observed in vitro, it was confirmed that the efficacy of CD19-euBBz CAR-T was 5 times higher in the animal model. (Figure 12)

[0169] Example 8: Confirmation of the proportion of CD19-euBBz CAR-T in an in vivo animal model After verifying the efficacy of improved construct-CAR-T cells by administering CAR-T cells in a subcutaneous animal model, the presence of CAR-T cells was confirmed in the blood of mice. More specifically, orbital blood was collected from mice at intervals of 3 and 4 days after CAR-T administration. 70 μL of blood was collected at each collection, and 60 μL of the blood was used to confirm the percentage and number of CAR-T cells. 60 μL of blood was placed in a 5 mL FACS tube, and live / dead cell staining was performed using Zombie Aqua BV510 (Biolegend, catalog number 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 number C36950), anti-CD45 FITC (HI30 (Biolegend, catalog number 304006)), anti-CD8 BV786 (SK-1 (Biolegend, catalog number 344740)), anti-CD4 BV650, and anti-flag PE were added and stained at room temperature for 30 minutes. Each antibody was mixed in 0.5 μL / 100 μL of FACS buffer in a tube, and 25 μL of counting beads was added. After 30 minutes, 2 mL of 1× RBC lysis buffer (Biolegend, catalog number 422401) was added and the mixture was reacted at room temperature for 5 minutes. After centrifugation at 2,000 rpm for 5 minutes using a centrifuge, all supernatant was discarded. 2 mL of FACS wash buffer was added to this tube, and centrifugation was performed at 2,000 rpm for 5 minutes. After repeating this process once more, 50 μL of FACS buffer was added, and the data was analyzed using FACS.

[0170] One week after CAR-T cell administration, the CD19-euBBz CAR-T cell administration group showed 2 × 10⁶ cells. 6 Cells / 100 μL DPBS / head group and 6 × 10 6 In both the cell / 100μL DPBS / head group, approximately 20% of CD19-euBBz CAR-T cells were detected in the blood. However, in the CD19-BBz CAR-T administration group, 6 × 10⁶ CD19-euBBz CAR-T cells were detected. 6When cells / 100 μL DPBS / head were administered, only 5% of CD19 CAR-T was confirmed. Three days later, the number and proportion of CAR-T cells in the mouse body reached the maximum value and then decreased. Within one week, in the three experimental groups where CAR-T cells were confirmed (CD19-euBBz CAR-T; 2×10 6 cells / 100 μL DPBS / head and 6×10 6 cells / DBPS / head, CD19-BBz CAR-T; 6×10 6 cells / 100 μL DPBS / head), since relatively many CAR-T cells could contact the cancer cells before the cancer cells proliferated in the mouse body, the cancer cells died rapidly. However, in the experimental group administered with CD19-BBz CAR-T at 2×10 6 cells / 100 μL DPBS / head, the proportion and number of CAR-T cells reached the maximum value two weeks later, the proportion of CAR-T was about 25%, and the cancer cells proliferated relatively sufficiently. CD19-euBBz CAR-T was more quantitatively stable than CD19-BBz CAR-T, and the proportion of CAR-T increased first and then decreased. However, in the case of CD19-BBz CAR-T, the proportion of CAR-T cells increased and decreased at a later timing, and as a result, it took longer for the tumor to disappear in the mouse body. As a result, similar to this experimental result, the group administered with CD19-euBBz CAR-T at 2×10 6 cells / 100 μL DPBS / head showed the same CAR-T level and effect in mice as the group administered with CD19-BBz CAR-T at 6×10 6 cells / 100 μL DPBS / head, indicating that CD19-euBBz CAR-T has excellent effects (Figure 14).

Claims

1. Immune cells containing a chimeric antigen receptor (CAR), wherein the CAR is (a) an extracellular domain containing an antigen-binding domain that specifically binds to glypican 3 (GPC3), (b) Transmembrane domain and (c) an intracellular domain comprising a co-stimulatory endodomain, wherein the co-stimulatory endodomain comprises an intracellular signaling domain derived from 4-1BB / CD137 and five additional amino acids, and Includes, The aforementioned co-stimulatory endodomain includes Sequence ID No. 4, The antigen-binding domain includes a heavy chain variable region consisting of the amino acid sequence shown in SEQ ID NO: 16, and The antigen-binding domain includes a light chain variable region consisting of the amino acid sequence shown in Sequence ID No.

15. The aforementioned immune cells.

2. The immune cell according to claim 1, wherein the chimeric antigen receptor is a single polypeptide or is composed of two polypeptides.

3. The immune cell according to claim 1 or 2, wherein the antigen-binding domain is humanized or human-type.

4. The immune cell according to any one of claims 1 to 3, wherein the antigen-binding domain is scFv.

5. The transmembrane domains include 4-1BB / CD137, activated 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 that specifically binds to CD83, LIGHT, LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1), MHC class I molecule, NKG2C, NKG2D, An immune cell according to any one of claims 1 to 4, wherein the transmembrane domain is selected from a protein selected from the group consisting of NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signal transduction lymphocyte activating 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 is a transmembrane domain derived from CD8α.

6. The immune cell according to any one of claims 1 to 5, wherein the intracellular domain further comprises an intracellular domain derived from CD3ζ.

7. The immune cell according to any one of claims 1 to 6, wherein the chimeric antigen receptor further comprises a signal peptide, a leader sequence, or a hinge region.

8. The immune cell according to claim 7, wherein the hinge region is a CD8α hinge.

9. The immune cell according to any one of claims 1 to 8, wherein the chimeric antigen receptor further comprises an additional antigen-binding domain.

10. The immune cell according to claim 9, wherein the additional antigen-binding domain is scFv.

11. The immune cell according to any one of claims 1 to 10, wherein the immune cell is a human immune cell.

12. The immune cell according to claim 11, wherein the human immune cell is an autologous human immune cell or an allogeneic human immune cell.

13. The immune cell according to any one of claims 1 to 12, wherein the immune cell is a T cell or an NK cell.

14. A nucleic acid encoding a chimeric antigen receptor (CAR) as defined in any one of claims 1 to 13.

15. A vector comprising the nucleic acid described in claim 14.

16. The vector according to claim 15, further comprising a promoter operably bound to the nucleic acid.

17. The vector according to claim 16, wherein the promoter is a constitutive promoter or an inducible promoter.

18. A viral vector, as described in any one of claims 15 to 17.

19. The vector according to claim 18, wherein the viral vector is a lentiviral vector.

20. A method for producing engineered immune cells in vitro, comprising introducing the nucleic acid described in claim 14 or the vector described in any one of claims 15 to 19 into immune cells in vitro to produce the engineered immune cells.

21. The method according to claim 20, further comprising culturing the manipulated immune cells after the introduction step.

22. The method according to claim 21, wherein the immune cells are T cells or NK cells.

23. Engineered immune cells produced by the method according to any one of claims 20 to 22.

24. A pharmaceutical composition comprising manipulated immune cells according to any one of claims 1 to 13, nucleic acid according to claim 14, or a vector according to any one of claims 15 to 19, and a pharmaceutically acceptable carrier.

25. A pharmaceutical composition according to claim 24 for preventing or treating cancer.

26. The pharmaceutical composition according to claim 25, wherein the cancer is carcinoma, 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, or head and neck cancer.

27. The pharmaceutical composition according to claim 25 or 26, wherein the cancer is a target cancer that has previously received one or more additional anticancer treatments, and the anticancer treatment is selected from the group consisting of ionizing radiation, chemotherapeutic agents, therapeutic antibodies, and checkpoint inhibitors.

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