IL-18-secreting GPC3 CAR-T cells and methods of making and using same

Abstract

Provided herein are CAR-T compositions directed against GPC3, including chimeric receptors, and engineered immune cells against GPC3. The present disclosure also provides vectors, compositions, and therapeutic methods using GPC3 antigen-binding molecules and engineered immune cells, optionally in combination with expression of IL-18. The GPC3 CAR compositions provided herein can be used to treat certain cancers.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 991,497, filed March 18, 2020, U.S. Provisional Patent Application No. 63 / 004,912, filed April 3, 2020, and U.S. Provisional Patent Application No. 63 / 043,401, filed June 24, 2020, each of which is incorporated by reference herein in its entirety. [Background technology]

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

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

[0004] overview An extracellular antigen-binding domain that specifically binds to glypican-3 (GPC3), the extracellular antigen-binding domain comprises a light chain variable domain comprising VL CDR1, 2, and 3, and a heavy chain variable domain comprising VH CDR1, 2, and 3, wherein: the VL CDR1, 2, and 3 comprise SEQ ID NOs: 1, 2, and 3; and the VH CDR1, 2, and 3 comprise SEQ ID NOs: 4, 5, and 6; the extracellular antigen-binding domain, a transmembrane domain, and Intracellular signaling domains a chimeric antigen receptor (CAR) comprising: an exogenous nucleic acid comprising a sequence encoding interleukin-18; Provided herein is an immune cell comprising:

[0005] In some embodiments, the light chain variable domain comprises a sequence at least 80% identical to SEQ ID NO: 10. In some embodiments, the light chain variable domain comprises a sequence at least 90% identical to SEQ ID NO: 10. In some embodiments, the light chain variable domain comprises a sequence at least 96% identical to SEQ ID NO: 10. In some embodiments, the heavy chain variable domain comprises a sequence at least 80% identical to SEQ ID NO: 8. In some embodiments, the heavy chain variable domain comprises a sequence at least 90% identical to SEQ ID NO: 8. In some embodiments, the heavy chain variable domain comprises a sequence at least 96% identical to SEQ ID NO: 8. In some embodiments, the interleukin-18 is human interleukin-18. In some embodiments, the human interleukin-18 comprises a sequence at least 80% identical to SEQ ID NO: 11 or 12. In some embodiments, the human interleukin-18 comprises a sequence at least 90% identical to SEQ ID NO: 11 or 12. In some embodiments, the human interleukin-18 comprises a sequence at least 96% identical to SEQ ID NO: 11 or 12.

[0006] In some embodiments, the sequence encoding interleukin-18 further comprises a sequence encoding a secretory signal sequence. In some embodiments, the secretory signal sequence is an interleukin-2 secretory signal sequence. In some embodiments, the interleukin-2 secretory signal sequence comprises the sequence of SEQ ID NO: 13 or 14.

[0007] In some embodiments, the exogenous nucleic acid further comprises a promoter operably linked to the sequence encoding interleukin-18. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is an NFAT promoter.

[0008] In some embodiments, the antigen-binding domain is humanized. In some embodiments, the antigen-binding domain is human. In some embodiments, the antigen-binding domain is an scFv.

[0009] In some embodiments, the transmembrane domain is selected from the group consisting of 4-1BB / CD137, activating NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8 alpha, CD8 beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, ligand specifically binding to CD83, LIGHT, LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1), M The transmembrane domain is selected from a protein selected from the group consisting of HC class 1 molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1), SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, and VLA-6. In some embodiments, the transmembrane domain is a transmembrane domain derived from CD8 alpha.

[0010] In some embodiments, the intracellular signaling domain is selected from the group consisting of 4-1BB / CD137, activating NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD30, CD4, CD40, C D49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8 alpha, CD8 beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRTAM, 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 cells Co-stimulatory factors (ICOS), integrins, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, ligands that specifically bind to CD83, LIGHT, LTBR, ​​Ly9 (CD229), Ly108, lymphocyte function-associated antigen-1 (LFA-1), MHC class 1 molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, and programs The intracellular signaling domain comprises an intracellular signaling domain derived from a protein selected from the group consisting of PD-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1), SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, and VLA-6, or any combination thereof. In some embodiments, the intracellular signaling domain is derived from 4-1BB and CD3 zeta.

[0011] 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. In some embodiments, the immune cells secrete IL-18 encoded by an exogenous nucleic acid.

[0012] Provided herein are pharmaceutical compositions comprising any of the immune cells described herein and a pharmaceutically acceptable carrier.

[0013] Provided herein are kits comprising any of the pharmaceutical compositions described herein.

[0014] Provided herein is a method of treating a subject having a glypican-3 associated cancer, the method comprising administering to the subject any of the immune cells described herein, or any of the pharmaceutical compositions described herein. [The present invention 1001] An extracellular antigen-binding domain that specifically binds to glypican-3 (GPC3), the extracellular antigen-binding domain comprises a light chain variable domain comprising VL CDR1, 2, and 3, and a heavy chain variable domain comprising VH CDR1, 2, and 3, wherein: a. the VL CDR1, 2, and 3 comprise SEQ ID NOs: 1, 2, and 3; and b. the VH CDR1, 2, and 3 comprise SEQ ID NOs: 4, 5, and 6; the extracellular antigen-binding domain, a transmembrane domain, and Intracellular signaling domains a chimeric antigen receptor (CAR) comprising: an exogenous nucleic acid comprising a sequence encoding interleukin-18; including immune cells. [The present invention 1002] 1001. The immune cell of the present invention, wherein said light chain variable domain comprises a sequence that is at least 80% identical to SEQ ID NO:10. [The present invention 1003] 1002. The immune cell of the present invention, wherein said light chain variable domain comprises a sequence that is at least 90% identical to SEQ ID NO:10. [The present invention 1004] 1003. The immune cell of the present invention, wherein said light chain variable domain comprises a sequence that is at least 96% identical to SEQ ID NO:10. [The present invention 1005] The immune cell of any of claims 1001 to 1004, wherein the heavy chain variable domain comprises a sequence that is at least 80% identical to SEQ ID NO:8. [The present invention 1006] 1005. The immune cell of the present invention, wherein said heavy chain variable domain comprises a sequence that is at least 90% identical to SEQ ID NO:8. [The present invention 1007] 1006. The immune cell of the present invention, wherein said heavy chain variable domain comprises a sequence that is at least 96% identical to SEQ ID NO:8. [The present invention 1008] The immune cell of any of claims 1001 to 1007, wherein the interleukin-18 is human interleukin-18. [The present invention 1009] 1008. The immune cell of the present invention, wherein said human interleukin-18 comprises a sequence that is at least 80% identical to SEQ ID NO: 11 or 12. [The present invention 1010] 1009. The immune cell of the present invention, wherein said human interleukin-18 comprises a sequence that is at least 90% identical to SEQ ID NO: 11 or 12. [The present invention 1011] 10. The immune cell of the present invention, wherein said human interleukin-18 comprises a sequence that is at least 96% identical to SEQ ID NO: 11 or 12. [The present invention 1012] The immune cell of any of claims 1001 to 1011, wherein the sequence encoding interleukin-18 further comprises a sequence encoding a secretory signal sequence. [The present invention 1013] The immune cell of the present invention, wherein the secretory signal sequence is an interleukin-2 secretory signal sequence. [The present invention 1014] 1013. The immune cell of the present invention, wherein the interleukin-2 secretory signal sequence comprises the sequence of SEQ ID NO: 13 or 14. [The present invention 1015] The immune cell of any of claims 1001 to 1014, wherein the exogenous nucleic acid further comprises a promoter operably linked to the sequence encoding interleukin-18. [The present invention 1016] 1015. The immune cell of the present invention, wherein the promoter is a constitutive promoter. [The present invention 1017] 1015. The immune cell of the present invention, wherein the promoter is an inducible promoter. [The present invention 1018] 1015. The immune cell of the present invention, wherein the promoter is an NFAT promoter. [The present invention 1019] The immune cell of any of claims 1001 to 1018, wherein the antigen-binding domain is humanized. [The present invention 1020] The immune cell of any of claims 1001 to 1018, wherein the antigen-binding domain is human. [The present invention 1021] The immune cell of any of claims 1001 to 1020, wherein the antigen-binding domain is an scFv. [The present invention 1022] The transmembrane domain is selected from the group consisting of 4-1BB / CD137, activating NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8 alpha, CD8 beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, and CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, ligand specifically binding to CD83, LIGHT, LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1), MHC class 1 molecule, NK The immune cell of any of claims 1001 to 1021, wherein the transmembrane domain is selected from a protein selected from the group consisting of G2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1), SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, and VLA-6. [The present invention 1023] 1022. The immune cell of the present invention, wherein the transmembrane domain is a transmembrane domain derived from CD8 alpha. [The present invention 1024] The intracellular signaling domain is selected from the group consisting of 4-1BB / CD137, activating NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD30, CD4, CD40, CD49a, CD49D, and CD49 f, CD69, CD7, CD84, CD8, CD8 alpha, CD8 beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRTAM, 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, ligands that specifically bind to CD83, LIGHT, LTBR, ​​Ly9 (CD229), Ly108, lymphocyte function-associated antigen-1 (LFA-1), MHC class 1 molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELP An immune cell according to any one of claims 1001 to 1023, comprising an intracellular signaling domain derived from a protein selected from the group consisting of LG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1), SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, and VLA-6, or any combination thereof. [The present invention 1025] The immune cell of the present invention 1024, wherein the intracellular signaling domain is derived from 4-1BB and CD3 zeta. [The present invention 1026] The immune cell of any one of 1001 to 1025 of the present invention, which is a human immune cell. [The present invention 1027] The immune cell of the present invention, wherein the human immune cell is an autologous human immune cell. [The present invention 1028] The immune cell of the present invention, wherein the human immune cell is an allogeneic human immune cell. [The present invention 1029] The immune cell of any one of 1001 to 1028 of the present invention, which is a T cell. [The present invention 1030] The immune cell of any one of 1001 to 1028 of the present invention, which is an NK cell. [The present invention 1031] The immune cell of any of claims 1001 to 1030, which secretes the IL-18 encoded by the exogenous nucleic acid. [The present invention 1032] A pharmaceutical composition comprising the immune cells of any one of the present inventions 1001 to 1031 and a pharmaceutically acceptable carrier. [The present invention 1033] A kit comprising the pharmaceutical composition of the present invention. [The present invention 1034] A method for treating a subject having a glypican-3-associated cancer, comprising the step of administering to the subject an immune cell of any one of claims 1001 to 1031 or a pharmaceutical composition of claim 1032. [Brief explanation of the drawings]

[0015] [Figure 1] 1 shows the structure of the GPC3 CAR lentiviral plasmid co-expressing IL-18. [Figure 2] 1 shows the results of enzyme mapping after cloning of pELPS-huGC33-P2A-IL18. [Figure 3] 1 shows the results of enzyme mapping after cloning of pELPS-huGC33-NFAT-IL18 and pELPS-huGC33-polyA-IL18-NFAT. [Figure 4A] Graph showing cell growth by comparing the total fold expansion of untreated PBMC cells, huGC33 VHVL, huGC33-VHVL-P2A-IL18, and huGC33-VHVL-NFAT-IL18 CAR-T cells. [Figure 4B]1 is a set of bar graphs comparing the fold proliferation of untreated PBMC cells, huGC33 VHVL, huGC33-VHVL-P2A-IL18, and huGC33-VHVL-NFAT-IL18 CAR-T cells in vitro on days 7, 9, 11, and 14 of cell culture. [Figure 4C] 1 is a set of bar graphs comparing cell viability of untreated PBMC cells, huGC33 VHVL, huGC33-VHVL-P2A-IL18, and huGC33-VHVL-NFAT-IL18 CAR-T cells in vitro on days 7, 9, 11, and 14 of cell culture. [Figure 4D] Figure 1 shows results from a luciferase-based cytotoxicity assay using naive PBMC cells, huGC33 VHVL, huGC33-VHVL-P2A-IL18, and huGC33-VHVL-NFAT-IL18 CAR-T cells in vitro, where the effector (E):target (T) cell ratio (E:T) may be 10:1, 3:1, 1:1, or 0.3:1. The results show that the different CAR-T cell groups exhibited similar in vitro killing activity. [Figure 4E] FACS analysis of huGC33 VHVL, huGC33-VHVL-P2A-IL18, and huGC33-VHVL-NFAT-IL18 transduced T cells for CAR expression on day 7 is shown. [Figure 4F] FACS analysis of huGC33 VHVL, huGC33-VHVL-P2A-IL18, and huGC33-VHVL-NFAT-IL18 transduced T cells for CAR expression on day 9 is shown. [Figure 4G] FACS analysis of huGC33 VHVL, huGC33-VHVL-P2A-IL18, and huGC33-VHVL-NFAT-IL18 transduced T cells for CAR expression on day 11 is shown. [Figure 4H] FACS analysis of huGC33 VHVL, huGC33-VHVL-P2A-IL18, and huGC33-VHVL-NFAT-IL18 transduced T cells for CAR expression on day 14 is shown. [Figure 5A-1] Figure 5A is a set of graphs showing tumor growth in animal models after injection of huGC33-VHVL-P2A-IL18 and huGC33-VHVL-NFAT-IL18 CAR-T cells. [Figure 5A-2] See legend to Figure 5A-1. [Figure 5B-1] Figure 5B is a set of graphs showing the change in CAR-T cell rates in the blood of animals after injection of huGC33-VHVL-P2A-IL18 and huGC33-VHVL-NFAT-IL18 CAR-T cells. The results show that the group of mice injected with huGC33-VHVL-NFAT-IL18 showed the greatest increase in CAR-T cell levels in the blood. [Figure 5B-2] See legend to Figure 5B-1. [Figure 6]

[0033] Figure 1 shows the change in IL-18 concentration in the blood of animals over 14 days after injection of GPC3 VHVL NFAT IL-18 CAR-T cells. The upper panel shows the results for each group, and the lower panel shows the results for each animal that received the injection. The results show that the concentration of IL-18 in the serum reached approximately 20 pg / mL only in mice that received huGC33-VHVL-NFAT-IL18 CAR-T cells. [Figure 7A]Figure 10 is a graph comparing the in vitro cell growth of huGC33-VHVL-NFAT-IL18 and huGC33-VHVL-IL18-NFAT CAR-T cells by measuring the fold expansion for each cell group, untreated PBMC cells, CD19 CAR-T, huGC33 VHVL, huGC33-VHVL-NFAT-IL18, and huGC33-VHVL-IL18-NFAT CAR-T cells over 12 days of cell culture. The huGC33-VHVL-NFAT-IL18 CAR-T cell group produced a maximum fold expansion of 714.0-fold at day 12. The huGC33-VHVL-IL18-NFAT and huGC33-VHVL groups produced similar expansion, just short of 600-fold at day 12. The untreated PBMC cell group showed a total fold expansion of approximately 490-fold. The CD19-CAR-T cell group resulted in approximately 338-fold expansion at day 12. [Figure 7B] 1 is a set of bar graphs comparing fold expansion for untreated PBMC cells, CD19 CAR-T, huGC33 VHVL, huGC33-VHVL-NFAT-IL18, and huGC33-VHVL-IL18-NFAT CAR-T cells on days 5, 7, 9, and 12. The results show no significant difference in cell growth for each group of CAR-T cells. [Figure 7C] 1 is a set of bar graphs comparing cell viability of each group of untreated PBMC cells, CD19 CAR-T, huGC33 VHVL, huGC33-VHVL-NFAT-IL18, and huGC33-VHVL-IL18-NFAT CAR-T cells on days 5, 7, 9, and 12. The results show no significant difference in cell viability of each group of CAR-T cells. [Figure 7D]Figure 1 shows an LDH-based cytotoxicity assay using naive PBMC cells, CD19 CAR-T, huGC33 VHVL, huGC33-VHVL-NFAT-IL18, and huGC33-VHVL-IL18-NFAT CAR-T cells, where the effector (E):target (T) cell ratio (E:T) may be 10:1, 3:1, 1:1, or 0.3:1. The results show that huGC33 VHVL, huGC33 VHVL-NFAT-IL18, and huGC33-VHVL-IL18-NFAT CAR-T cells exhibited similar in vitro killing activity, while naive PBMC cells and CD19 CAR-T cells did not exhibit significant in vitro killing activity. [Figure 7E-1] Figure 7E shows the results from FACS analysis of naive PBMC cells, CD19 CAR-T, huGC33 VHVL, huGC33-VHVL-NFAT-IL18, and huGC33-VHVL-IL18-NFAT CAR-T cells on day 7. [Figure 7E-2] See legend to Figure 7E-1. [Figure 7F-1] Figure 7F shows the results from FACS analysis of naive PBMC cells, CD19 CAR-T, huGC33 VHVL, huGC33-VHVL-NFAT-IL18, and huGC33-VHVL-IL18-NFAT CAR-T cells on day 9. [Figure 7F-2] See legend to Figure 7F-1. [Figure 7G-1] Figure 7G shows the results from FACS analysis of naive PBMC cells, CD19 CAR-T, huGC33 VHVL, huGC33-VHVL-NFAT-IL18, and huGC33-VHVL-IL18-NFAT CAR-T cells on day 12. [Figure 7G-2] See legend to Figure 7G-1. [Figure 7H] IL18 concentrations are shown using naive PBMC cells, CD19 CAR-T, huGC33 VHVL, huGC33-VHVL-NFAT-IL18, and huGC33-VHVL-IL18-NFAT CAR-T cells. [Figure 8A]

[0023] Figure 1 is a set of graphs showing tumor growth in animal models after injection of untreated PBMC cells, CD19 CAR-T, huGC33 VHVL, huGC33-VHVL-NFAT-IL18, and huGC33-VHVL-IL18-NFAT CAR-T cells. Administration of huGC33-VHVL-NFAT-IL18 at 250,000 cells suppressed tumor size in all five treated mice. [Figure 8B] 1 is a set of graphs showing the change in CAR-T cell percentage in the blood of animals after injection of naive PBMC cells, CD19 CAR-T, huGC33 VHVL, huGC33-VHVL-NFAT-IL18, and huGC33-VHVL-IL18-NFAT CAR-T cells. Administration of 250,000 cells of huGC33-VHVL-NFAT-IL18 shows the expansion of huGC33-VHVL-NFAT-IL18 CAR-T cells during tumor growth and the decrease of huGC33-VHVL-NFAT-IL18 CAR-T cells after tumor suppression. [Figure 9A]

[0023] Figure 1 is a set of graphs showing tumor growth in animal models after injection of huGC33-VHVL-NFAT-IL18 and huGC33-VHVL-IL18-NFAT CAR-T cells. Administration of 250,000 cells of huGC33-VHVL-NFAT-IL18 suppressed tumor size in all three treated mice. [Figure 9B] 1 is a set of graphs showing the change in CAR-T cell percentage in the blood of animals after injection of huGC33-VHVL-NFAT-IL18 and huGC33-VHVL-IL18-NFAT CAR-T cells. Administration of 250,000 cells of huGC33-VHVL-NFAT-IL18 shows the expansion of huGC33-VHVL-NFAT-IL18 CAR-T cells during the tumor growth period and the decrease of huGC33-VHVL-NFAT-IL18 CAR-T cells after tumor suppression. [Figure 10A]Figure 1 shows tumor growth after huGC33-VHVL-NFAT-IL18 injection in animals using the GPC3-positive cell line PLC / PRF / 5-GL. Administration of 1 million cells of huGC33-VHVL-NFAT-IL18 in PLC / PRF / 5-GL cells suppressed tumor size at least 28 days after huGC33-VHVL-NFAT-IL18 CAR-T administration. [Figure 10B] 1 is a graph showing tumor growth after injection of huGC33-VHVL-NFAT-IL18 in animals using the GPC3-negative cell line SK-HEP-1. Administration of 1 million cells of huGC33-VHVL-NFAT-IL18 in SK-Hep-1 cells suppressed tumor size at least 21 days after administration of huGC33-VHVL-NFAT-IL18 CAR-T. DETAILED DESCRIPTION OF THE INVENTION

[0016] Detailed Description This disclosure describes chimeric antigen receptor (CAR-T) engineered T cells comprising a GPC3 antigen-binding domain and an IL-18 coding sequence, and methods of making and using them.

[0017] Definition: About: The term "about," when used herein in connection with a value, refers to a value that is similar in the context of the referenced value. Generally, a person of ordinary skill in the art familiar with the context will recognize the degree of relevant variation encompassed by "about" in that context. For example, in some embodiments, the term "about" can encompass values ​​within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or a range of less than the reference value.

[0018] Administration: As used herein, the term "administration" typically refers to the administration of a composition to a subject or system to achieve delivery of an agent that is or is contained in the composition. Those skilled in the art will recognize the various routes that may be utilized for administration to a subject, e.g., a human, in the appropriate circumstances. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In some particular embodiments, administration may be bronchial (e.g., by intrabronchial instillation), buccal, transdermal (e.g., topical administration as opposed to dermal, intradermal, interdermal, transdermal, etc.), enteral, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, intraspecific organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, bronchial (e.g., by intrabronchial instillation), vaginal, intravitreal, etc. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve the application of a fixed number of doses. In some embodiments, administration may involve intermittent dosing (e.g., multiple doses separated in time) and / or periodic dosing (e.g., individual doses separated by a common period of time). In some embodiments, administration may involve continuous dosing (e.g., perfusion) over at least a selected period of time.

[0019] Affinity: As known in the art, "affinity" is a measure of how tightly a particular ligand binds to its partner. Affinity can be measured in different ways. In some embodiments, affinity is measured by a quantitative assay. In some such embodiments, the binding partner concentration can be fixed so that the ligand concentration is in excess, mimicking physiological conditions. Alternatively, or additionally, in some embodiments, the binding partner concentration and / or the ligand concentration can be varied. In some such embodiments, affinity can be compared to a reference under equivalent conditions (e.g., concentrations).

[0020] Antibody agent: As used herein, the term "antibody agent" refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex that contains sufficient immunoglobulin structural elements to confer specific binding. Exemplary antibody agents include, but are not limited to, monoclonal antibodies, polyclonal antibodies, and fragments thereof. In some embodiments, an antibody agent may contain one or more sequence elements that are humanized, primatized, chimeric, etc., as known in the art. In many embodiments, the term "antibody agent" is used to refer to one or more of the constructs or formats known or developed in the art for utilizing antibody structure and functional characteristics in alternative presentations. For example, antibody agents utilized in accordance with the present invention, which are embodiments, include, but are not limited to, intact IgA, IgG, IgE, or IgM antibodies; bi- or multispecific antibodies (e.g., Zybodies®, etc.); antibody fragments such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fv; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR, or fragments thereof); camelid antibodies; masked antibodies (e.g., Probodies®); S mall M odd I mmuno Pand a format selected from harmaceuticals ("SMIPs™"); single-chain or tandem diabodies (TandAb®); VHH; Anticalins®; Nanobodies® minibodies; BiTE®; ankyrin repeat proteins or DARPIN®; Avimers®; DART; TCR-like antibodies; Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®, and KALBITOR®. In some embodiments, antibody agents may lack covalent modifications (e.g., glycan attachment) that they may have when produced in nature. In some embodiments, an antibody agent may contain a covalent modification (e.g., a glycan, a payload (e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.), or other pendant group (e.g., polyethylene glycol, etc.). In many embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence comprises one or more structural elements recognized by those skilled in the art as complementarity determining regions (CDRs). In some embodiments, an antibody agent comprises at least one CDR (e.g., at least one heavy chain CDR and / or is or comprises a polypeptide comprising at least one light chain CDR). In some embodiments, the included CDRs are substantially identical to the reference CDRs in that they are either identical in sequence or contain 1 to 5 amino acid substitutions compared to the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that they exhibit at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR.In some embodiments, the included CDRs are substantially identical to the reference CDRs in that they exhibit at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDRs. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that at least one amino acid within the included CDRs has been deleted, added, or substituted relative to the reference CDRs, but the included CDRs otherwise have an amino acid sequence identical to the amino acid sequence of the reference CDRs. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that 1 to 5 amino acids within the included CDRs have been deleted, added, or substituted relative to the reference CDRs, but the included CDRs otherwise have an amino acid sequence identical to the amino acid sequence of the reference CDRs. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that at least one amino acid within the included CDRs has been substituted relative to the reference CDRs, but the included CDRs otherwise have an amino acid sequence identical to the amino acid sequence of the reference CDRs. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that one to five amino acids within the included CDRs are deleted, added, or substituted relative to the reference CDR, but the included CDRs have an amino acid sequence that is otherwise identical to the amino acid sequence of the reference CDR. In some embodiments, the antibody agent is or comprises a polypeptide whose amino acid sequence comprises structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, the antibody agent is a polypeptide protein having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain. In some embodiments, the antibody agent is or comprises at least a portion of a chimeric antigen receptor (CAR).

[0021] Antigen: The term "antigen," as used herein, refers to an agent that binds to an antibody agent. In some embodiments, an antigen binds to an antibody agent and may or may not induce a specific physiological response in an organism. Generally, an antigen can be or include any chemical entity, such as, for example, a small molecule, a nucleic acid, a polypeptide, a carbohydrate, a lipid, or a polymer (including biological polymers [e.g., nucleic acid and / or amino acid polymers] and polymers other than biological polymers [e.g., polymers other than nucleic acids or amino acid polymers]). In some embodiments, an antigen is or includes a polypeptide. In some embodiments, an antigen is or includes a glycan. Those skilled in the art will recognize that, in general, antigens may be provided in an isolated or pure form, or alternatively, may be provided in a crude form (e.g., together with other materials, e.g., an extract such as a cell extract or other relatively crude preparation of an antigen-containing source). In some specific embodiments, the antigen is present in a cellular context (e.g., the antigen is expressed on the surface of a cell or is expressed intracellularly). In some embodiments, the antigen is a recombinant antigen.

[0022] Antigen-binding domain: As used herein, refers to an antibody agent or portion thereof that specifically binds to a targeting moiety or entity. Typically, the interaction between the antigen-binding domain and its target is non-covalent. In some embodiments, the targeting moiety or entity can be of any chemical class, including, for example, carbohydrates, lipids, nucleic acids, metals, polypeptides, or small molecules. In some embodiments, the antigen-binding domain can be or include 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).

[0023] Associated with: Two events or entities are "associated" with one another, as the term is used herein, if the presence, level, and / or form of one correlates with the presence, level, and / or form of the other. For example, a particular entity (e.g., a polypeptide, gene signature, metabolite, microorganism, etc.) is considered to be associated with a particular disease, disorder, or condition if its presence, level, and / or form correlates with the incidence and / or susceptibility of the particular disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically "associated" with one another if they interact directly or indirectly, such that they are in physical proximity to one another and / or remain in physical proximity to one another. In some embodiments, two or more entities that are physically associated with one another are covalently bound to one another. In some embodiments, two or more entities that are physically associated with one another are not covalently bound to one another, but are non-covalently associated by, for example, hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0024] Binding: The term "binding," as used herein, will be understood to typically refer to a non-covalent association between or within two or more entities. "Direct" binding involves physical contact between the entities or moieties, while indirect binding involves a physical interaction through physical contact with one or more intermediate entities. Binding between two or more entities can typically be assessed in any of a variety of contexts, including when the interacting entities or moieties are studied in isolation (with a carrier entity and / or covalently or otherwise associated in a biological system or cell) or in the context of a more complex system.

[0025] Cancer: The terms "cancer," "malignancy," "neoplasm," "tumor," and "carcinoma" are used herein to refer to cells that exhibit relatively abnormal, uncontrolled, and / or autonomous growth, resulting in an abnormal growth phenotype characterized by a marked loss of control of cell proliferation. In some embodiments, a tumor may be or include cells that are precancerous (e.g., benign), malignant, premetastatic, metastatic, and / or non-metastatic. The present disclosure specifically identifies particular cancers to which its teachings may be particularly relevant. In some embodiments, the relevant cancers may be characterized as solid tumors. In some embodiments, the relevant cancers may be characterized as hematological tumors. In general, examples of different types of cancer known in the art include, for example, hematopoietic cancers including leukemia, lymphoma (Hodgkin's and non-Hodgkin's), myeloma and myeloproliferative disorders; sarcoma, melanoma, adenoma, carcinoma of solid tissue, squamous cell carcinoma of the oral cavity, throat, larynx, and lung, liver cancer, genitourinary tract cancers such as prostate, cervix, bladder, uterus, and endometrial cancer, and benign lesions such as renal cell carcinoma, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, cancers of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, head and neck cancer, breast cancer, gastrointestinal cancer, and cancers of the nervous system, benign lesions such as papilloma, etc.

[0026] CDR: As used herein, refers to a complementarity-determining region within the variable region of an antibody agent. There are three CDRs in each of the heavy and light chain variable regions, designated CDR1, CDR2, and CDR3 for each variable region. A "set of CDRs" or "CDR set" refers to the three or six CDRs occurring in a single variable region capable of binding to an antigen, or a group of CDRs from cognate heavy and light chain variable regions capable of binding to an antigen. Specific systems have been established in the art for defining CDR boundaries (e.g., Kabat, Chothia, etc.). Those skilled in the art will recognize the differences between and among these systems and will be able to understand CDR boundaries to the extent necessary to understand and practice the claimed invention.

[0027] Chemotherapeutic Agent: The term "chemotherapeutic agent" is used herein and has its art-recognized meaning to refer to one or more pro-apoptotic, cytostatic, and / or cytotoxic agents, including, for example, specifically, agents available and / or recommended for use in treating one or more diseases, disorders, or conditions associated with unwanted cell proliferation. In many embodiments, chemotherapeutic agents are useful in the treatment of cancer. In some embodiments, the chemotherapeutic agent may be or may include one or more alkylating agents, one or more anthracyclines, one or more cytoskeletal disruptors (microtubule-targeting agents such as taxanes, maytansine and its analogs), one or more epothilones, one or more histone deacetylase inhibitors (HDACs), one or more topoisomerase inhibitors (e.g., inhibitors of topoisomerase I and / or topoisomerase II), one or more kinase inhibitors, one or more nucleotide analogs or nucleotide precursor analogs, one or more peptide antibiotics, one or more platinum-based agents, one or more retinoids, one or more vinca alkaloids, and / or one or more analogs of the following (i.e., sharing related antiproliferative activity): In some specific embodiments, the chemotherapeutic agent is actinomycin, all-trans retinoic acid, auristatin, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, curcumin, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, gemcitabine, hydroxybenzoates, The chemotherapeutic agent may be or may include one or more of urea, idarubicin, imatinib, irinotecan, maytansine and / or its analogs (e.g., DM1), mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, maytansinoids, oxaliplatin, paclitaxel, pemetrexed, teniposide, thioguanine, topotecan, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, and combinations thereof. In some embodiments, the chemotherapeutic agent may be utilized in the context of an antibody-drug conjugate.In some embodiments, the chemotherapeutic agent is hLL1-doxorubicin, hRS7-SN-38, hMN-14-SN-38, hLL2-SN-38, hA20-SN-38, hPAM4-SN-38, hLL1-SN-38, hRS7-Pro-2-P-Dox, hMN-14-Pro-2-P-Dox, hLL2-Pro-2-P-Dox, hA20-Pro-2-P-Dox, hPAM4-Pro-2-P-Dox, hLL1-Pro-2-P-Dox, P 4 / D10-doxorubicin, gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, glembatumumab vedotin, SAR3419, SAR566658, BIIB015, BT062, SGN-75, SGN-CD19A, AMG-172, AMG-595, BAY-94-9343, ASG-5ME, ASG-22ME, ASG-16M8F, MDX-1203, MLN-0264, anti-PSMA ADC, RG-7450, RG-7458, RG-7593, RG-7596, RG-7598, RG-7599, RG-7600, RG-7636, ABT-414, IMGN-853, IMGN-529, borsetuzumab mafodotin, and lorvotuzumab mertansine.

[0028] Engineered: In general, the term "engineered" refers to the aspect of being manipulated by the hand of man. For example, a polypeptide is considered "engineered" if its sequence has been manipulated by the hand of man. For example, in some embodiments of the present invention, an engineered polypeptide includes a sequence containing one or more amino acid mutations, deletions, and / or insertions introduced by the hand of man into a reference polypeptide sequence. In some embodiments, an engineered polypeptide includes a polypeptide that has been fused (i.e., covalently linked) to one or more additional polypeptides by the hand of man, forming a fusion polypeptide that does not naturally occur in vivo. In comparison, a cell or organism is considered "engineered" if it has been manipulated such that its genetic information has been altered (e.g., new genetic material not previously present has been introduced, e.g., by transformation, mating, somatic hybridization, transfection, transduction, or other mechanisms, or pre-existing genetic material has been altered or removed, e.g., by substitution or deletion mutations or by mating protocols). As is common practice and understood by those skilled in the art, derivatives and / or progeny of engineered polypeptides or cells are typically referred to as "engineered," even if the actual manipulation was performed on an earlier entity.

[0029] In vitro: As used herein, the term "in vitro" refers to events that take place not in a multicellular organism but in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc.

[0030] In vivo: As used herein, refers to events that occur within multicellular organisms, such as humans and non-human animals. In the context of cell-based systems, the term can be used to refer to events that occur within living cells (as opposed to, for example, in vitro systems).

[0031] Isolated: As used herein, refers to a substance and / or entity that (1) has been separated from at least some of the components with which it was associated when originally produced (in nature and / or in an experimental setting) and / or (2) has been designed, produced, prepared, and / or manufactured by the hand of man. 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 with which they were originally associated. In some embodiments, an isolated agent is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. In some embodiments, as will be understood by those of skill in the art, a substance may still be considered "isolated" or even "pure" after combination with certain other components, for example, one or more carriers or excipients (e.g., buffers, solvents, water, etc.), and in such embodiments, the isolation or purity of the substance is calculated without including such carriers or excipients. For example, in some embodiments, a biological polymer, such as a naturally occurring polypeptide or polynucleotide, is considered to be "isolated" if: a) its source of origin or derivatization is unrelated to some or all of the components that accompany it in its natural state; b) it is substantially free from other polypeptides or nucleic acids of the same species as the species in which it naturally occurs; or c) it is expressed by or otherwise associated with components from a cell or other expression system other than the species in which it naturally occurs. Thus, for example, in some embodiments, a polypeptide that is chemically synthesized or synthesized in a cellular system other than the one that naturally produces it is considered to be an "isolated" polypeptide.Alternatively, or additionally, in some embodiments, a polypeptide that has been subjected to one or more purification techniques may be considered to be an "isolated" polypeptide to the extent that it has been separated from other components a) with which it is associated in nature, and / or b) with which it was associated when originally produced.

[0032] Operably linked: As used herein, refers to a juxtaposition wherein the described components are in a relationship permitting them to function in their intended manner. A control element "operably linked" to a functional element is associated in such a way that expression and / or activity of the functional element is achieved under conditions compatible with the control elements. In some embodiments, an "operably linked" control element is contiguous (e.g., covalently linked) with a coding element of interest. In some embodiments, the control element acts in trans on or otherwise from the functional element of interest.

[0033] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to a composition in which an active agent is formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the composition is suitable for administration to a human or animal subject. In some embodiments, the active agent is present in an amount of a unit dose suitable for administration in a treatment regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.

[0034] Polypeptide: As used herein, the term "polypeptide" generally has its art-recognized meaning of a polymer of at least three amino acids. Those of skill in the art will recognize that the term "polypeptide" is intended to be general enough to encompass not only polypeptides having the complete sequences recited herein, but also polypeptides representing functional fragments of such complete polypeptides (i.e., fragments that retain at least one activity). Moreover, those of skill in the art will understand that protein sequences generally tolerate some substitutions without destroying activity. Thus, any polypeptide that retains activity and shares at least about 30-40%, often greater than about 50%, 60%, 70%, or 80% overall sequence identity with another polypeptide of the same class, and more usually contains at least one region of considerable identity, often greater than 90%, or even 95%, 96%, 97%, 98%, or 99% identity within one or more highly conserved regions, and usually encompasses at least 3-4, and often up to 20 or more amino acids, is encompassed within the related term "polypeptide" as used herein. Polypeptides may contain L-amino acids, D-amino acids, or both, and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, etc. In some embodiments, proteins may contain natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to polypeptides having 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, the protein is an antibody agent, an antibody fragment, a biologically active portion thereof, and / or a characteristic portion thereof.

[0035] Prevent or prophylaxis: As used herein, when used in reference to the occurrence of a disease, disorder, and / or condition, refers to reducing the risk of the disease, disorder, and / or condition progressing and / or delaying the onset and / or severity of one or more characteristics or symptoms of the disease, disorder, and / or condition. In some embodiments, prevention is assessed on a population basis, such that an agent is considered to "prevent" a particular disease, disorder, or condition if a statistically significant reduction in the progression, frequency, and / or intensity of one or more symptoms of the disease, disorder, or condition is observed in a population susceptible to that disease, disorder, or condition.

[0036] Recombinant: As used herein, is intended to refer to a polypeptide that is designed, engineered, prepared, expressed, made, manufactured, and / or isolated by recombinant means, e.g., a polypeptide expressed using a recombinant expression vector transfected into a host cell; a polypeptide isolated from a recombinant combinatorial human polypeptide library; a polypeptide isolated from an animal (e.g., mouse, rabbit, sheep, fish, etc.) that is transgenic or that has been otherwise engineered to express a gene or genes or genetic components that encode and / or direct the expression of the polypeptide or one or more components, portions, elements, or domains thereof; and / or a polypeptide prepared, expressed, made, or isolated by any other means by splicing or ligating selected nucleic acid sequence elements together, chemically synthesizing selected sequence elements, and / or otherwise generating nucleic acids that encode and / or direct the expression of the polypeptide or one or more components, portions, elements, or domains thereof. In some embodiments, one or more of such selected sequence elements are found in nature. In some embodiments, one or more of such selected sequence elements are designed in silico. In some embodiments, one or more of such selected sequence elements arise from mutagenesis (e.g., in vivo or in vitro) of known sequence elements, e.g., from natural or synthetic sources, such as the germline of a source organism of interest (e.g., human, mouse, etc.).

[0037] Specific binding: As used herein, the term "specific binding" refers to the ability to distinguish between potential binding partners in an environment in which binding occurs. A binding agent that interacts with one specific target in the presence of other potential targets is said to "specifically bind" to the target with which it interacts. In some embodiments, specific binding is assessed by detecting or determining the degree of association between the binding agent and its partner. In some embodiments, specific binding is assessed by detecting or determining the extent of dissociation of the binding agent-partner complex. In some embodiments, specific binding is assessed by detecting or determining the ability of a binding agent to compete for an alternative interaction between its partner and another entity. In some embodiments, specific binding is assessed by performing such detection or determination over a range of concentrations.

[0038] Subject: As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human, in some embodiments, including prenatal human forms). In some embodiments, the subject is suffering from an associated disease, disorder, or condition. In some embodiments, the subject is predisposed 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 does not exhibit any symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject is a person possessing one or more characteristics characteristic of susceptibility to, or risk for, a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual to whom and / or to whom a diagnosis and / or therapy is administered.

[0039] Therapeutic Agent: As used herein, the phrase "therapeutic agent" generally refers to any agent that induces a desired pharmacological effect when administered to an organism. In some embodiments, an agent is considered a therapeutic agent if it exhibits a statistically significant effect across an appropriate population. In some embodiments, the appropriate population may be a population of model organisms. In some embodiments, the appropriate population may be defined by various criteria, such as a specific age group, sex, genetic background, pre-existing clinical condition, etc. In some embodiments, a therapeutic agent is a substance that can be used to alleviate, ameliorate, relieve, suppress, prevent, delay the onset of, reduce the severity of, and / or reduce the occurrence of one or more symptoms or characteristics of a disease, disorder, and / or condition. In some embodiments, a "therapeutic agent" is a drug that has already been approved, or needs to be approved, by a government agency before it can be marketed for administration to humans. In some embodiments, a "therapeutic agent" is a drug that requires a pharmaceutical formulation for administration to humans.

[0040] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" refers to an amount sufficient to treat a disease, disorder, and / or condition when administered to a population suffering from or susceptible to the disease, disorder, and / or condition according to a therapeutic dosing regimen. In some embodiments, a therapeutically effective amount is an amount that reduces the occurrence and / or severity of one or more symptoms of, stabilizes one or more characteristics of, and / or delays the onset of, a disease, disorder, and / or condition. Those skilled in the art will recognize that the term "therapeutically effective amount" does not, in fact, require successful treatment to be achieved in a particular individual. Rather, a therapeutically effective amount may be an amount that provides a particular desired pharmacological response in a substantial number of subjects when administered to patients in need of such treatment. For example, in some embodiments, the term "therapeutically effective amount" refers to an amount that, when administered to an individual in need thereof in the context of a therapy of the present invention, blocks, stabilizes, attenuates, or reverses processes that support cancer occurring in that individual, or enhances or increases processes that suppress cancer in that individual. In the context of cancer treatment, a "therapeutically effective amount" is an amount that, when administered to an individual diagnosed with cancer, prevents, stabilizes, inhibits, or reduces further progression of the cancer in that individual. Particularly preferred "therapeutically effective amounts" of the compositions described herein serve to reverse the progression of, or achieve or prolong the remission of, a malignant tumor, such as pancreatic carcinoma (in a therapeutic treatment). A therapeutically effective amount administered to an individual to treat cancer in that individual may be the same as or different from a therapeutically effective amount administered to promote remission or inhibit metastasis. As with most cancer therapies, the therapeutic methods described herein should not be construed as, limited to, or otherwise restricted to "curing" cancer; rather, the therapeutic methods are directed to the use of the described compositions to "treat" cancer, i.e., to effect a desired or beneficial change in the health of an individual with cancer.Such benefits are recognized by healthcare providers skilled in the field of oncology and include, but are not limited to, stabilization of a patient's condition, reduction in tumor size (tumor regression), improved vital functions (e.g., improved function of cancerous tissue or organs), reduction or inhibition of further metastasis, reduction in opportunistic infections, increased chances of survival, reduced pain, improved motor function, improved cognitive function, improved sense of energy (vitality, less discomfort), improved sense of 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 a treatment described herein) may be assessed by obtaining samples of cancer cells from the site of the tumor, such as a pancreatic adenocarcinoma (e.g., over the course of treatment), and testing the cancer cells for levels of metabolic and signaling markers to monitor the state of the cancer cells and verify, at the molecular level, regression of the cancer cells to a less aggressive phenotype. For example, tumor regression induced by using the methods of the present invention would be indicated by finding a decrease in any of the pro-angiogenic markers discussed above, an increase in the anti-angiogenic markers described herein, or normalization of a metabolic pathway, intercellular signaling pathway, or intracellular signaling pathway that exhibits aberrant activity in an individual diagnosed with cancer (i.e., a change to a state found in a normal individual without cancer). One of skill in the art will recognize that in some embodiments, a therapeutically effective amount can be formulated and / or administered in a single dose. In some embodiments, a therapeutically effective amount can be formulated and / or administered in multiple doses, for example, as part of a dosing regimen.

[0041] Variant: As used herein in the context of a molecule, e.g., a nucleic acid, protein, or small molecule, the term "variant" refers to a molecule that exhibits significant structural identity with a reference molecule but differs structurally from the reference molecule, e.g., by the presence or absence of, or at the level of, one or more chemical moieties compared to the reference entity. In some embodiments, a variant also differs functionally from its reference molecule. Generally, whether a particular molecule is appropriately considered a "variant" of a reference molecule is based on the degree of structural identity with the reference molecule. As will be recognized by those skilled in the art, any biological or chemical reference molecule possesses certain characteristic structural elements. A variant, by definition, is a distinct molecule that shares one or more such characteristic structural elements but differs from the reference molecule in at least one aspect. To give some examples, a polypeptide may have characteristic sequence elements composed of multiple amino acids that have designated positions relative to each other in linear or three-dimensional space 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 that have designated positions relative to other amino acids in linear or three-dimensional space. In some embodiments, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence. In some embodiments, a variant polypeptide or nucleic acid exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% overall sequence identity with a reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid. In some embodiments, a reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, a variant polypeptide or nucleic acid shares one or more biological activities of a reference polypeptide or nucleic acid.

[0042] Vector: As used herein, refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes operably linked to them. Such vectors are referred to herein as "expression vectors." Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications, as commonly accomplished in the art, or as described herein. The foregoing techniques and procedures may generally be performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification, see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 2, incorporated herein by reference for all purposes. nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).

[0043] Engineered immune cells As used herein, "immune cell" refers to a cell of the immune system, which can be classified as a lymphocyte (e.g., a T cell, a B cell, and a NK cell), a neutrophil, and a monocyte / macrophage. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a NK cell. In some embodiments, the immune cell is an engineered immune cell, by which is meant an immune cell that has been genetically modified to express a non-naturally occurring protein (e.g., a chimeric antigen receptor) or to contain an exogenous nucleic acid.

[0044] Immune cells (e.g., T cells) may be modified in one or more ways. Immune cells (e.g., T cells) may express at least one non-natural molecule that is a receptor for an antigen present on the surface of one or more types of cells. In some embodiments, the immune cells include immune cells (e.g., T cells) that are not found in nature because they contain or are engineered to express at least one synthetic molecule not found in nature. In certain embodiments, the immune cells (e.g., T cells) are engineered to express at least one chimeric antigen receptor (CAR), including a CAR that targets a specific tumor antigen, such as glypican-3 (GPC3). In certain embodiments, the immune cells may be T cells, e.g., a population of T cells including CD4+ T cells, CD8+ T cells, Treg cells, Th1 T cells, Th2 T cells, Th17 T cells, nonspecific T cells, or any combination of the above. Immune cells (e.g., T cells) engineered with chimeric antigen receptors (CAR T cells) have great therapeutic potential for treating cancer. With CARs, upon antigen recognition and binding, the receptor can activate immune cells and program them to kill cells expressing that antigen. Thus, immune cells expressing a CAR against an antigen expressed on tumor cells can target and kill tumor cells. For example, recent clinical trials of T cells transduced with a CD19-targeting CAR (CD19-CAR T cells) for hematological malignancies demonstrated the powerful efficacy of CAR T technology. (Kochenderfer,JNet al.(2010)Blood 116:4099-4102, Porter,DL,et al.(2011)N.Engl.J.Med.365:725-733, Grupp,SAet al.(2013)N.Engl.J.Med.368:1509-1518, Kochenderfer,JNet al. (2015) J. Clin. Oncol. 33:540-549, Brown, CE et al. (2016) N. Engl. J. Med. 375: 2561-2569).The clinical success of CAR T is due, at least in part, to the fusion structure of the CAR, which artificially combines a high-affinity antigen-binding domain with multiple signaling domains (Maus, MV et al. (2014) Blood 123:2625-2635; van der Stegen, SJ et al. (2015) Nat. Rev. Drug Discov. 14:499-509).

[0045] CARs comprise an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the extracellular antigen-binding domain comprises a single-chain variable fragment (scFv) capable of recognizing a tumor-associated antigen, the transmembrane domain uses transmembrane domains derived from molecules such as CD8 and CD28, and the extracellular signaling domain uses immunoreceptor tyrosine-based activation motifs (e.g., CD3ζ) and intracellular signaling domains of costimulatory signaling molecules (e.g., CD28, CD137, and CD137(4-1BB)).

[0046] As used herein, "single-chain variable fragment, scFv" refers to a fragment of an antibody defined as a recombinant protein comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) connected by a linker, the two domains associating together to form an antigen-binding site.

[0047] In some embodiments, the transmembrane domain is selected from the group consisting of 4-1BB / CD137, activating NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8 alpha, CD8 beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, ligand specifically binding to CD83, LIGHT, LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA) -1), MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1), SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, and VLA-6.

[0048] In some embodiments, the intracellular signaling domain is selected from the group consisting of 4-1BB / CD137, activating NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8 alpha, CD8 beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRTAM, 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 Intercellular co-stimulatory factor (ICOS), integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, ligands that specifically bind to CD83, LIGHT, LTBR, ​​Ly9 (CD229), Ly108, lymphocyte function-associated antigen-1 (LFA-1), MHC class 1 molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, protease inhibitors (PAG / Cbp), and protease inhibitors (PAG / Cbp). The antibody comprises an intracellular signaling domain derived from a protein selected from: program death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1), SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, and VLA-6, or any combination thereof.In some embodiments, the intracellular signaling domain of the chimeric receptors described herein comprises a 4-1BB signaling domain followed by a five amino acid sequence, which may be further combined with any other desired extracellular, transmembrane, and / or intracellular domains useful in the context of the chimeric receptor. In some embodiments, the 4-1BB signaling domain followed by a five amino acid sequence is referred to as euBBz. In some embodiments, the chimeric antigen receptor further comprises an additional antigen-binding domain. In some embodiments, the additional antigen-binding domain is an scFv.

[0049] Immune cells (e.g., T cells) can be derived 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, spinal cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, or a tumor. 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 number of techniques known to those skilled in the art, such as FICOLL™ separation and / or apheresis. Additional methods for isolating T cells for T cell therapy are disclosed in U.S. Patent Publication No. 2013 / 0287748, which is incorporated herein by reference in its entirety. Other non-limiting examples can be found in International Application No. PCT / US2015 / 014520 (published as WO2015 / 120096) and International Application No. PCT / US2016 / 057983 (published as WO2017 / 070395), each of which is incorporated herein by reference in its entirety.

[0050] In some embodiments, the immune cells are autologous T cells. In some embodiments, the immune cells are obtained from a subject other than the patient. In some embodiments, the T cells for use in the therapeutic methods are syngeneic (donor and recipient are different but identical twins). In some embodiments, the T cells for use in the therapeutic methods are allogeneic (same species but from a different donor) to the recipient subject. In some embodiments, the T cells are autologous stem cells (for 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 suffering from cancer or a tumor.

[0051] T cells can be engineered to express, for example, chimeric antigen receptors (CARs). In some embodiments, CAR-T cells can be engineered to express an extracellular single-chain variable fragment (scFv). In some embodiments, CARs are engineered to express a costimulatory domain as a separate polypeptide chain. Exemplary CAR-T cell therapies and constructs are described in U.S. Patent Publication Nos. 2013 / 0287748, 2014 / 0227237, 2014 / 0099309, and 2014 / 0050708, each of which is incorporated by reference in its entirety.

[0052] GPC3 Glypican-3 (GPC3) is a cell surface protein encoded by the GPC3 gene in humans and is an oncofetal antigen frequently re-expressed in neoplastic hepatocytes. GPC3 is highly expressed in fetal liver and not in normal adult liver tissue. However, its expression is reactivated in hepatocellular carcinoma (HCC), closely associated with the progression of HCC. The detection rate of GPC3 expression is relatively high during the early stages of HCC and increases with progression. Furthermore, GPC3 is also expressed in tumors such as melanoma, ovarian clear cell carcinoma, yolk sac tumor, neuroblastoma, and other tumors. Given its specifically high expression in HCC, melanoma, and other tumors, GPC3 has emerged as a useful immunohistochemical diagnostic test and potential biomarker.

[0053] GPC3 is a member of the proteoglycan family that functions as an extracellular matrix for cell adhesion during organogenesis and as a receptor for cell growth factors. The GPC3 protein core contains two subunits: an N-terminal subunit and a C-terminal subunit. A glycosylphosphatidylinositol (GPI) anchor is attached to serine at position 560 on the carboxyl (C)-terminal side of GPC3. The GPI anchor localizes GPC3 to the cell surface via covalent attachment to plasma membrane lipids. Furthermore, serine at positions 495 and 509 of GPC3 are modified with heparan sulfate (HS) chains. HS chains are known to regulate multiple growth signaling pathways, including Wnt signaling, FGF signaling, and BMP signaling. The growth signaling pathways involved vary depending on the type of cancer. For example, in hepatocellular carcinoma (HCC), stimulation of the Wnt signaling pathway promotes cell growth.

[0054] GPC3 CAR The present disclosure provides, at least in part, a GPC3 CAR polypeptide. As used herein, "chimeric antigen receptor (CAR)" refers to a receptor that does not exist in nature and can provide immune effector cells with specificity for a particular antigen. Typically, a CAR refers to a receptor used to deliver the specificity of a monoclonal antibody agent to T cells. Generally, a CAR comprises an extracellular binding domain (ectodomain), a transmembrane domain, and an intracellular signaling domain (endodomain). In some embodiments, the extracellular binding domain of a CAR comprises an antigen-binding domain. In some embodiments, the antigen-binding domain is or comprises an antibody agent. In some embodiments, the antigen-binding domain is or comprises an antibody agent that specifically binds to GPC3.

[0055] In some embodiments, the chimeric antigen receptor (CAR) polypeptide comprises i) an extracellular antigen-binding domain comprising a light chain variable domain comprising a light chain CDR1 comprising SEQ ID NO: 1, a light chain CDR2 comprising SEQ ID NO: 2, and a light chain CDR3 comprising SEQ ID NO: 3, and a heavy chain variable domain comprising a heavy chain CDR1 comprising SEQ ID NO: 4, a heavy chain CDR2 comprising SEQ ID NO: 5, and a heavy chain CDR3 comprising SEQ ID NO: 6; ii) a transmembrane domain; and iii) an intracellular signaling domain, wherein binding of an antigen to the antibody agent results in T cell activation.

[0056] [Table 1]

[0057] In some embodiments, the CAR polypeptide comprises i) an extracellular antigen-binding domain comprising a light chain variable domain comprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10, and a heavy chain variable domain comprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8; ii) a transmembrane domain; and iii) an intracellular signaling domain, wherein binding of the antigen to the antibody agent results in T cell activation.

[0058] In some embodiments, the CAR polypeptide comprises i) an extracellular antigen-binding domain comprising a light chain variable domain comprising SEQ ID NO: 10 and a heavy chain variable domain comprising SEQ ID NO: 8, ii) a transmembrane domain, and iii) an intracellular signaling domain, wherein binding of an antigen to the antibody agent results in T cell activation.

[0059] [Table 2]

[0060] IL-18 Interleukin-18 (IL-18) has been characterized as an inducer of interferon-γ (IFN-γ) expression in T cells and has been shown to activate lymphocytes and monocytes without inducing severe dose-limiting toxicity in clinical trials. IL-18 is a pro-inflammatory cytokine encoded by the IL18 gene in humans. Furthermore, IL-18 is known to have various functions in addition to its ability to induce IFN-γ. IL-18 functions include NF-κB activation, Fas ligand expression, induction of both CC and CXC chemokines, and increased production of competent human immunodeficiency virus. Due to its ability to induce IFN-γ production in T cells and macrophages, IL-18 plays an important role in Th1-type immune responses and is involved in both innate and adaptive immunity. IL-18 is related to the IL-1 family in terms of both structure and function.

[0061] In some embodiments, IL-18-secreting CAR-T cells exhibit enhanced CAR-T cell proliferation in vivo and significantly increase long-term survival in mouse models of hematological and solid malignancies. In some embodiments, IL-18-secreting CAR-T cells can enhance effective endogenous anti-tumor immune responses. In some embodiments, GPC3 CAR-T cells can co-express IL-18, thereby enhancing CAR-T cell proliferation and increasing the efficacy of the anti-tumor immune response.

[0062] SEQ ID NO: 11 Human interleukin-18 TIFF0007778081000003.tif61162 SEQ ID NO: 12 Human interleukin-18 (reverse complement) TIFF0007778081000004.tif62162 SEQ ID NO: 13 Interleukin-2 secretory signal sequence TIFF0007778081000005.tif11161 SEQ ID NO: 14 Interleukin-2 secretory signal sequence (reverse complement) TIFF0007778081000006.tif12163

[0063] nucleic acid As used herein, "nucleic acid" is used to include any compound and / or substance comprising a polymer of nucleotides. In some embodiments, a polymer of nucleotides is referred to as a polynucleotide. Exemplary nucleic acids or polynucleotides may include, but are not limited to, ribonucleic acid (RNA) and / or deoxyribonucleic acid (DNA). Provided herein is a nucleic acid comprising a nucleotide sequence encoding a GPC3 CAR, wherein the GPC3 CAR comprises an extracellular antigen-binding domain that specifically binds to glypican-3 (GPC3), a transmembrane domain, and an intracellular signaling domain.

[0064] In some embodiments, the nucleic acid construct comprises a region encoding GPC3 CAR. In some embodiments, the nucleic acid construct may be inserted into an expression vector or viral vector by methods known in the art, and the nucleic acid molecule may be operably linked to an expression control sequence. Non-limiting examples of expression vectors include plasmid vectors, transposon vectors, cosmid vectors, and viral vectors (e.g., any adenovirus vector (AV), cytomegalovirus (CMV) vector, simian virus (SV40) vector, adeno-associated virus (AAV) vector, lentivirus vector, and retrovirus vector). In some embodiments, the expression vector is a viral vector. In some embodiments, the viral vector is a lentivirus vector.

[0065] In some embodiments, the nucleic acid construct comprises a region encoding a GPC3 CAR and a sequence encoding interleukin-18. In some embodiments, the GPC3 CAR polypeptide comprises i) an extracellular antigen-binding domain comprising a light chain variable domain encoded by a nucleic acid comprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9, and a heavy chain variable domain encoded by a nucleic acid comprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7, ii) a transmembrane domain, and iii) an intracellular signaling domain, wherein binding of an antigen to the antibody agent results in T cell activation.

[0066] In some embodiments, the CAR polypeptide comprises i) an extracellular antigen-binding domain comprising a light chain variable domain encoded by a nucleic acid comprising SEQ ID NO: 9 and a heavy chain variable domain encoded by a nucleic acid comprising SEQ ID NO: 7, ii) a transmembrane domain, and iii) an intracellular signaling domain, wherein binding of an antigen to the antibody agent results in T cell activation.

[0067] In some embodiments, the IL-18 is human interleukin-18. In some embodiments, the IL-18 is humanized IL-18. In some embodiments, the IL-18 is encoded by a nucleic acid comprising a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 11 or 12. In some embodiments, the IL-18 is encoded by a nucleic acid comprising SEQ ID NO: 11 or 12.

[0068] In some embodiments, the sequence encoding IL-18 further comprises a sequence encoding a secretory signal sequence. As used herein, "secretory signal sequence" refers to a short peptide (e.g., 16-30 amino acids) present at the N-terminus of most newly synthesized proteins destined for the secretory pathway. These proteins include those present inside cellular organelles (e.g., the endoplasmic reticulum, Golgi, or endosomes), those secreted from the cell, or those inserted into most cell membranes. In some embodiments, the secretory signal sequence is an interleukin-2 secretory signal sequence. In some embodiments, the interleukin-2 secretory signal sequence comprises a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13 or 14. In some embodiments, the IL-2 secretory signal sequence comprises the sequence of SEQ ID NO: 13 or 14.

[0069] In some embodiments, the nucleic acid construct further comprises a promoter operably linked to the sequence encoding IL-18, wherein the promoter is positioned between the sequence encoding CAR and the sequence encoding interleukin-18. As used herein, "promoter" refers to a region of DNA that directs the initiation of transcription of a particular gene. The promoter is located upstream of the DNA (e.g., toward the 5' region of the sense strand), near the transcription start site of the gene. In some embodiments, the promoter is about 100-1000 base pairs in length. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is an NFAT promoter.

[0070] In some embodiments, the nucleic acid construct further comprises a sequence encoding a self-cleaving protein, which sequence is disposed between the sequence encoding the CAR and the sequence encoding IL-18. In some embodiments, the self-cleaving protein sequence is a P2 self-cleaving protein sequence.

[0071] Lentiviral vectors are derived from lentiviruses. They are based on single-stranded RNA lentiviruses, a subclass of retroviruses. They combine the advantages of medium-range cloning capacity and stable gene expression, allowing them to transduce dividing and non-dividing cells, including neurons. 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 exemplary retroviral vectors used for gene delivery. Unlike other retroviruses, HIV-based vectors are known to integrate passenger genes into non-dividing cells and may therefore be useful for treating persistent forms of disease.

[0072] Additional sequences can be added to such cloning and / or expression sequences to optimize their function in cloning and / or expression, to aid in the isolation of the polynucleotide, or to improve the introduction of the polynucleotide into cells. The use of cloning vectors, expression vectors, adapters, and linkers is well known in the art.

[0073] In some embodiments, the nucleic acid molecule is inserted into a vector that is capable of expressing the GPC3 CAR of the present disclosure when introduced into an appropriate cell. In some embodiments, the appropriate cell is a T cell.

[0074] Generation of IL-18-expressing GPC3 CAR-T cells Provided herein are methods for producing immune cells comprising a GPC3 CAR that expresses IL-18. In some embodiments, the immune cells into which the CAR is introduced are human immune cells. In some embodiments, the immune cells are autologous human immune cells. In some embodiments, the immune cells are allogeneic human immune cells. In some embodiments, the immune cells are CD4 +T cells (helper T cells, T H cells), CD8 + The immune cells 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.

[0075] In some embodiments, the present disclosure provides a method of producing engineered immune cells, the method comprising introducing into immune cells (i) a nucleic acid sequence encoding a GPC3 CAR comprising a GPC3 antigen-binding domain and a sequence encoding interleukin-18 (IL-18), or (ii) a vector comprising a nucleic acid encoding a GPC3 CAR comprising a GPC3 antigen-binding domain and a nucleic acid encoding IL-18. In some embodiments, the method of producing engineered immune cells of the present disclosure further comprises culturing the engineered immune cells in vitro for at least 2 days, 5 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days.

[0076] In some embodiments, the present disclosure provides a method of preparing an autologous engineered immune cell of the present disclosure, comprising providing or obtaining an analysis of binding of a GPC3 antigen-binding domain to T cells from a subject, and if binding is below a threshold, engineering immune cells from the subject to express a CAR comprising the GPC3 antigen-binding domain, wherein the CAR further expresses IL-18. In some embodiments, the method of producing autologous engineered immune cells of the present disclosure (e.g., GPC3 CAR-T cells expressing IL-18) further comprises culturing the autologous engineered immune cells in vitro for at least 2 days, 5 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days.

[0077] Any method known in the art for expressing a CAR in immune cells can be used in the context of the present disclosure. Various nucleic acid vectors for expression are known in the art, such as linear polynucleotides, polynucleotides bound to ionic or amphiphilic compounds, plasmids, or viral vectors, but the present disclosure is not limited thereto. In some embodiments, the vector for CAR expression in immune cells can be or include an autonomously replicating plasmid or virus or its derivatives. Viral vectors can include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, etc. In some embodiments, a lentiviral vector, which is a retroviral vector, can be used. In some embodiments, the vector is a non-plasmid and non-viral compound, such as a liposome.

[0078] The present disclosure encompasses the recognition that IL-18 co-expressing GPC3 CAR-T cells generated by the methods described herein may be therapeutically useful (e.g., for the treatment of cancer).

[0079] therapeutic use Provided herein is a method of treating a subject having a glypican-3-associated cancer, the method comprising administering to the subject a composition comprising or delivering immune cells comprising a GPC3 CAR that expresses IL-18.

[0080] "Glypican-3-associated cancer" refers to cancers characterized by cancer cells that have glypican-3 on their surface. GPC3 is a membrane-bound heparan sulfate proteoglycan that is overexpressed in approximately 70%-80% of hepatocellular carcinomas, but is generally not expressed in healthy tissue. In addition, GPC3 overexpression has been found in several tumors, most notably hepatocellular carcinoma, hepatoblastoma, germ cell tumors (e.g., yolk sac tumor, choriocarcinoma), Wilms' tumor, gastric carcinoma, non-small cell lung cancer, and thyroid cancer.

[0081] Interleukin-18 (IL-18) is a cytokine that enhances innate and adaptive immune responses and is produced by activated immune cells, such as monocytes, macrophages, dendritic cells (DCs), neutrophils, natural killer (NK) cells, T cells, and B cells. Various types of cancer produce IL-18, which induces cell migration, invasion, and proliferation, leading to increased metastasis and tumor growth. IL-18 transcription levels are known to be elevated in most types of cancer, including, but not limited to, cervical squamous cell carcinoma and adenocarcinoma, colon adenocarcinoma, glioblastoma, renal papillary cell carcinoma, acute myeloid leukemia, pancreatic adenocarcinoma, breast cancer, brain cancer, and pancreatic cancer.

[0082] Cancer can refer to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth can lead to the formation of malignant tumors that invade adjacent tissues and may also metastasize to distant parts of the body via the lymphatic system or bloodstream. Cancer or cancerous tissue can include tumors.

[0083] Cancers suitable for treatment with the methods of the present disclosure may 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, blood 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, cancers for treatment with the methods of the present disclosure may include, and may include, but are not limited to, carcinoma, lymphoma (e.g., Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. In some embodiments, the cancer 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 carcinoma, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer. In some embodiments, the cancer may be embryonal tumors (Wilms' tumor, hepatoblastoma, rhabdoid tumor, neuroblastoma), germ cell tumors (yolk sac tumor, immature teratoma, and embryonal carcinoma), carcinomas (hepatocellular carcinoma and lung squamous cell carcinoma), sarcomas (malignant rhabdoid tumor and RMS), or malignant melanoma. In some embodiments, the glypican-3-associated cancer is liver cancer.

[0084] Immune cells (e.g., GPC3 CAR-T cells expressing IL-18) can be administered to a patient in need thereof in a therapeutically effective amount. For example, a therapeutically effective amount of immune cells (e.g., GPC3 CAR-T cells expressing IL-18) can be administered in a therapeutically effective amount of at least about 10 4 cells, at least about 10 5 cells, at least about 10 6 cells, at least about 10 7 cells, at least about 10 8 cells, at least about 10 9 or at least about 10 10In some embodiments, the therapeutically effective amount of T cells can be about 10 4 cells, approximately 10 5 cells, approximately 10 6 cells, approximately 10 7 cells, approximately 10 8 cells, approximately 10 9 cells, or approximately 10 10 In some embodiments, the therapeutically effective amount of T cells is about 0.1 x 10 6 ~about 2×10 10 T cells (e.g., approximately 0.1 x 10 6 ~about 2×10 10 T cells, approximately 0.2 x 10 6 ~about 2×10 10 T cells, approximately 0.4 x 10 6 ~about 2×10 10 T cells, approximately 0.6 x 10 6 ~about 2×10 10 T cells, approximately 0.8 x 10 6 ~about 2×10 10 T cells, approximately 1.0 x 10 6 ~about 2×10 10 T cells, approximately 2.0 x 10 6 ~about 2×10 10 T cells, approximately 3.0 x 10 6 ~about 2×10 10 T cells, approximately 4.0 x 10 6 ~about 2×10 10 T cells, approximately 5.0 x 10 6 ~about 2×10 10 T cells, approximately 6.0 x 10 6 ~about 2×10 10 T cells, approximately 7.0 x 10 6 ~about 2×10 10 T cells, approximately 8.0 x 10 6 ~about 2×10 10 T cells, approximately 9.0 x 10 6 ~about 2×10 10 T cells, approximately 1.0 x 10 7 ~about 2×10 10 T cells, approximately 2.0 x 10 7 ~about 2×10 10 T cells, approximately 3.0 x 10 7~about 2×10 10 T cells, approximately 4.0 x 10 7 ~about 2×10 10 T cells, approximately 5.0 x 10 7 ~about 2×10 10 T cells, approximately 6.0 x 10 7 ~about 2×10 10 T cells, approximately 7.0 x 10 7 ~about 2×10 10 T cells, approximately 8.0 x 10 7 ~about 2×10 10 T cells, approximately 9.0 x 10 7 ~about 2×10 10 T cells, approximately 1.0 x 10 8 ~about 2×10 10 T cells, approximately 2.0 x 10 8 ~about 2×10 10 T cells, approximately 3.0 x 10 8 ~about 2×10 10 T cells, approximately 4.0 x 10 8 ~about 2×10 10 T cells, approximately 5.0 x 10 8 ~about 2×10 10 T cells, approximately 6.0 x 10 8 ~about 2×10 10 T cells, approximately 7.0 x 10 8 ~about 2×10 10 T cells, approximately 8.0 x 10 8 ~about 2×10 10 T cells, approximately 9.0 x 10 8 ~about 2×10 10 T cells, approximately 1.0 x 10 9 ~about 2×10 10 T cells, approximately 2.0 x 10 9 ~about 2×10 10 T cells, approximately 3.0 x 10 9 ~about 2×10 10 T cells, approximately 4.0 x 10 9 ~about 2×10 10 T cells, approximately 5.0 x 10 9 ~about 2×10 10 T cells, approximately 6.0 x 10 9 ~about 2×10 10 T cells, approximately 7.0 x 10 9 ~about 2×1010 T cells, approximately 8.0 x 10 9 ~about 2×10 10 T cells, approximately 9.0 x 10 9 ~about 2×10 10 T cells, or approximately 1.0 x 10 10 ~about 2×10 10 In some embodiments, the therapeutically effective amount of T cells is about 0.4 x 10 8 pieces, approximately 0.5×10 8 pieces, approximately 0.6×10 8 pieces, approximately 0.7×10 8 pieces, approximately 0.8×10 8 pieces, approximately 0.9×10 8 pieces, approximately 1.0×10 8 pieces, approximately 1.1×10 8 pieces, approximately 1.2×10 8 pieces, approximately 1.3×10 8 pieces, approximately 1.4×10 8 pieces, approximately 1.5×10 8 pieces, approximately 1.6×10 8 pieces, approximately 1.7×10 8 pieces, approximately 1.8×10 8 pieces, approximately 1.9×10 8 pieces, or approximately 2.0 x 10 8 These are T cells.

[0085] In some embodiments, the therapeutically effective amount of IL-18-expressing GPC3 CAR-T cells is about 2×10 6 cells / kg, approximately 3 x 10 6 cells / kg, approximately 4 x 10 6 cells / kg, approximately 5 x 10 6 cells / kg, approximately 6 x 10 6 cells / kg, approximately 7 x 10 6 cells / kg, approximately 8 x 10 6 cells / kg, approximately 9 x 10 6 cells / kg, approximately 1 x 10 7 cells / kg, approximately 2 x 10 7 cells / kg, approximately 3 x 10 7 cells / kg, approximately 4 x 10 7 cells / kg, approximately 5 x 10 7 cells / kg, approximately 6 x 107 cells / kg, approximately 7 x 10 7 cells / kg, approximately 8 x 10 7 cells / kg, or approximately 9 x 10 7 In some embodiments, a therapeutically effective amount of immune cells (e.g., GPC3 CAR-T cells expressing IL-18) is about 1 x 10 cells / kg of body weight. 6 ~about 2×10 6 From T cells up to approximately 1 × 10 10 In some embodiments, the therapeutically effective amount of T cells is about 1 x 10 per kg of body weight, up to a maximum dose of about 1 x 10 T cells. 6 or approximately 2 x 10 6 From T cells up to approximately 1 × 10 10 up to a maximum dose of 10 T cells.

[0086] The number of cells will depend on the end use for which the composition is intended, as will the type of cells contained therein. For example, in some embodiments, a population of T cells comprising a GPC3 CAR expressing IL-18 comprises more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, or more than 90% of such cells. In some embodiments, a population of T cells comprising a GPC3 CAR expressing IL-18 comprises more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, or more than 90% of such cells. A population of T cells containing a CAR may comprise about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to about 60%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, about 10% to about 15%, about 15% to about 90%, about 15% to about 80%, about 15% to about 70%, about 15% to about 60%, about 15% to about 50%, about 15% to about 40%, about 15% to about 30%, about 15% to about 20%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 5 ... Including 0%, about 20% to about 40%, about 20% to about 30%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, about 30% to about 60%, about 30% to about 50%, about 30% to about 40%, about 40% to about 90%, about 40% to about 80%, about 40% to about 70%, about 40% to about 60%, about 40% to about 50%, about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, about 50% to about 60%, about 60% to about 90%, about 60% to about 80%, about 60% to about 70%, about 70% to about 90%, about 70% to about 80%, or about 80% to about 90%. In some embodiments, the population of T cells for administration is in a volume of 1 liter or less. In some embodiments, the population of T cells for administration is in a volume of less than 500 ml, less than 250 ml, or less than 100 ml. In some embodiments, the desired density of T cells is typically less than 10 6 greater than 10 cells / ml, generally 10 7 greater than 10 cells / ml, generally 10 8 cells / ml or more. Clinically relevant numbers of immune cells are cumulatively greater than 10 7cells, 10 8 cells, 10 9 cells, 10 10 cells, 10 11 cells, or 10 12 The number of cells can be allocated to multiple infusions equal to or exceeding 100 cells.

[0087] In some embodiments, the composition may be administered parenterally to a patient. In some embodiments, the composition comprising or delivering T cells comprising a GPC3 CAR expressing IL-18 may be administered parenterally to a patient in one or more doses. In some embodiments, the composition comprising or delivering T cells comprising a GPC3 CAR expressing IL-18 may be administered parenterally to a patient once daily, once every 2-7 days, once a week, once every two weeks, once a month, once every three months, or once every six months.

[0088] In some embodiments, the present disclosure provides a method of inducing an immune response in a subject in need thereof, the method comprising administering to the subject a composition comprising or delivering T cells comprising an IL-18-expressing GPC3 CAR. In some embodiments, the T cells comprising an IL-18-expressing GPC3 CAR are autologous T cells. In some embodiments, the present disclosure provides a method of inducing an immune response in a subject in need thereof, the method comprising administering to the subject a composition comprising or delivering T cells comprising a nucleic acid and / or vector encoding an IL-18-expressing GPC3 CAR. In some embodiments, the T cells comprising a nucleic acid and / or vector encoding an IL-18-expressing GPC3 CAR are autologous T cells. In some embodiments, the subject has cancer or is at risk of developing cancer.

[0089] In some embodiments, the present disclosure provides a method of enhancing an immune response in a subject in need thereof, the method comprising administering to the subject a composition comprising or delivering T cells comprising an IL-18-expressing GPC3 CAR. In some embodiments, the T cells comprising an IL-18-expressing GPC3 CAR are autologous T cells. In some embodiments, the present disclosure provides a method of enhancing an immune response in a subject in need thereof, the method comprising administering to the subject a composition comprising or delivering T cells comprising a nucleic acid and / or vector encoding an IL-18-expressing GPC3 CAR. In some embodiments, the T cells comprising a nucleic acid and / or vector encoding an IL-18-expressing GPC3 CAR are autologous T cells. In some embodiments, the subject has cancer or is at risk of developing cancer.

[0090] In some embodiments, the disease suitable for treatment with the compositions and methods of the present disclosure is selected from a proliferative disease, such as a cancer or malignant tumor, or a precancerous condition. In some embodiments, the disease is associated with expression of GPC3. In some embodiments, the disease suitable for treatment with the compositions and methods of the present disclosure is cancer. In some embodiments, the cancer expresses the GPC3 antigen. In some embodiments, cancer cells have increased expression of the GPC3 antigen compared to non-cancerous cells from the subject. In some embodiments, GPC3 expression levels may be increased in subjects with cancer. In some embodiments, GPC3 expression levels may be undetectable in healthy subjects.

[0091] Pharmaceutical Compositions In some embodiments, the present disclosure provides a pharmaceutical composition comprising T cells comprising an IL-18-expressing GPC3 CAR and a pharmaceutically acceptable carrier. In some embodiments, the T cells comprising an IL-18-expressing GPC3 CAR are autologous T cells. In some embodiments, the present disclosure provides a pharmaceutical composition comprising T cells comprising a nucleic acid and / or vector encoding an IL-18-expressing GPC3 CAR and a pharmaceutically acceptable carrier. In some embodiments, the T cells comprising a nucleic acid and / or vector encoding an IL-18-expressing GPC3 CAR are autologous T cells. Compositions of the present disclosure include pharmaceutical compositions comprising T cells comprising an IL-18-expressing GPC3 CAR and / or a nucleic acid encoding an IL-18-expressing GPC3 CAR, obtained by the methods disclosed herein. In some embodiments, the pharmaceutical composition can comprise a buffer, a diluent, a solubilizer, an emulsifier, a preservative, an adjuvant, an excipient, or any combination thereof. In some embodiments, the compositions may also contain one or more additional therapeutically active agents, if desired.

[0092] In some embodiments, the T cells of the present disclosure are formulated by first harvesting the T cells from their culture medium, then washing and concentrating the cells in a medium and container system (a "pharmaceutically acceptable" carrier) suitable for administration in a therapeutically effective amount. A suitable infusion medium can be any isotonic medium formulation, typically saline, Normosol R (Abbott), or Plasma-Lyte A (Baxter), although 5% dextrose in water or Ringer's lactate solution can also be used. The infusion medium can be supplemented with human serum albumin.

[0093] In some embodiments, the compositions are formulated for parenteral administration. For example, the pharmaceutical compositions provided herein may be provided in a sterile injectable form (e.g., suitable for subcutaneous injection, hepatic artery infusion, or intravenous infusion). For example, in some embodiments, the pharmaceutical compositions are provided in a liquid form suitable for injection. In some embodiments, the pharmaceutical compositions are provided as a powder (e.g., lyophilized and / or sterilized) and can be reconstituted with an aqueous diluent (e.g., water, buffer, saline, etc.) prior to injection, optionally under high vacuum. In some embodiments, the pharmaceutical compositions are diluted and / or reconstituted with water, sodium chloride solution, sodium acetate solution, benzyl alcohol solution, phosphate buffered saline, etc. In some embodiments, the powder must be gently mixed with the aqueous diluent (e.g., not shaken).

[0094] In some embodiments, the IL-18-expressing GPC3 CAR of the present disclosure and / or T cells comprising a nucleic acid encoding an IL-18-expressing GPC3 CAR are formulated with a pharmaceutically acceptable parenteral vehicle. Examples of such vehicles include water, saline, Ringer's solution, dextrose solution, and 1-10% human serum albumin. Non-aqueous vehicles such as liposomes and fixed oils can also be used. The vehicle or lyophilized powder may contain additives to maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). In some embodiments, the formulation is sterilized by known or suitable techniques. Pharmaceutical compositions may additionally contain pharmaceutically acceptable excipients, which, as used herein, include any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersing or suspending aids, surface-active agents, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, as appropriate for the particular dosage form desired. Remington's *The Science and Practice of Pharmacy*, 21st Edition, A.R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006) discloses various excipients used in formulating pharmaceutical compositions and known techniques for their preparation. Except insofar as any conventional excipient vehicle is incompatible with a substance or its derivatives, for example, by producing any undesirable biological effects or otherwise interacting in a deleterious manner with any other component of the pharmaceutical composition, its use is contemplated within the scope of the present disclosure.

[0095] In some embodiments, compositions comprising a population of T cells comprising an IL-18-expressing GPC3 CAR of the present disclosure and / or a nucleic acid encoding an IL-18-expressing GPC3 CAR are stably formulated. In some embodiments, stable formulations of an IL-18-expressing GPC3 CAR of the present disclosure and / or a population of T cells comprising a nucleic acid encoding an IL-18-expressing GPC3 CAR may include preserved solutions and formulations containing saline or a phosphate buffer containing a selected salt and a preservative, and versatile preserved formulations suitable for pharmaceutical or veterinary use. The preserved formulation contains at least one known preservative, or optionally at least one selected from the group consisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate), alkyl parabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal, or mixtures thereof in an aqueous diluent. For example, 0.001 to 5%, or any range or value therein, including, but not limited to, 0.001, 0.003, 0.005, 0.009, 0.01, 0.02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 , 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.3, 4.5, 4.6, 4.7, 4.8, 4.9, or any range or value therein, can be used as known in the art.Non-limiting examples include preservative-free 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 ( For example, 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%), 0.0005 to 1.0% alkylparaben (for example, 0.00075, 0.0009, 0.001, 0.002, 0.005, 0.0075, 0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5, 0.75, 0.9, 1.0%), and the like.

[0096] In some embodiments, the pharmaceutical composition is provided in a form that can be refrigerated and / or frozen. In some embodiments, the pharmaceutical composition is provided in a form that cannot be refrigerated and / or frozen. In some embodiments, the reconstituted solution and / or liquid dosage form can be stored for a specified period of time after reconstitution (e.g., 2 hours, 12 hours, 24 hours, 2 days, 5 days, 7 days, 10 days, 2 weeks, 1 month, 2 months, or longer). In some embodiments, storing a composition containing an antibody agent for longer than the specified time period results in degradation of the antibody agent. The liquid dosage form and / or reconstituted solution may contain particulate matter and / or discoloration prior to administration. In some embodiments, the solution should not be used if it is discolored or cloudy and / or if particulate matter remains after filtration. General information on pharmaceutical formulation and / or manufacturing can be found, for example, in Remington: The Science and Practice of Pharmacy 21 st ed., Lippincott Williams & Wilkins, 2005.

[0097] In some embodiments, a pharmaceutical composition comprising an IL-18-expressing GPC3 CAR of the present disclosure and / or T cells comprising a nucleic acid encoding an IL-18-expressing GPC3 CAR may be contained in a container for storage or administration, such as a vial, a syringe (e.g., an IV syringe), or a bag (e.g., an IV bag). Pharmaceutical compositions according to the present disclosure may be prepared, packaged, and / or sold in bulk as a single unit dose and / or as multiple single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition comprising a predetermined amount of an active ingredient. The amount of the active ingredient generally equals the dosage of the active ingredient administered to a subject, and / or a convenient fraction of such a dosage, e.g., one-half or one-third of such a dosage.

[0098] kit The present disclosure further provides kits comprising one or more containers filled with at least one GPC3 CAR expressing IL-18 as described herein and / or a nucleic acid encoding a GPC3 CAR expressing IL-18. The kits may be used in any applicable method, including, for example, therapeutic methods, diagnostic methods, cell proliferation and / or isolation methods, etc. Optionally, associated with such containers may be a notice in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceutical or biological products, the notice reflecting (a) approval by the agency of manufacture, use, or sale for human administration, (b) instructions for use, or both.

[0099] In some embodiments, the kit may include one or more reagents for detection (e.g., detection of an IL-18-expressing GPC3 CAR and / or a nucleic acid encoding an IL-18-expressing GPC3 CAR). In some embodiments, the kit may include an IL-18-expressing GPC3 CAR and / or a nucleic acid encoding an IL-18-expressing GPC3 CAR in a detectable form (e.g., covalently associated with a detectable moiety or entity). In some embodiments, one or more IL-18-expressing GPC3 CARs and / or nucleic acids encoding an IL-18-expressing GPC3 CAR provided herein may be included in a kit used to treat a subject. In some embodiments, an IL-18-expressing GPC3 CAR and / or a nucleic acid encoding an IL-18-expressing GPC3 CAR provided herein may be included in a kit used to prepare autologous T cells expressing an IL-18-expressing GPC3 CAR.

[0100] In some embodiments, the kit may provide one, two, three, four, or more GPC3 antibody agents, each suitable for cloning into a CAR construct. In some embodiments, the kit may provide other reagents for assaying the binding affinity of GPC3 antibody agents and / or IL-18-expressing GPC3 CARs and / or IL-18-expressing GPC3 CAR T cells to T cells or GPC3 identified or isolated from a subject. In some embodiments, the kit may provide other reagents for assaying the functional avidity of antibody agents and / or IL-18-expressing GPC3 CARs and / or IL-18-expressing GPC3 CAR T cells to a subject's T cells.

[0101] Although several embodiments have been described, it will be understood that various modifications may be made without departing from the spirit and scope of the invention.

[0102] Exemplary embodiments: Embodiment 1. A chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain that specifically binds to glypican-3 (GPC3), a transmembrane domain, and an intracellular signaling domain; an exogenous nucleic acid comprising a sequence encoding interleukin-18; including immune cells.

[0103] Embodiment 2. The immune cell of embodiment 1, wherein the interleukin-18 is human interleukin-18.

[0104] Embodiment 3. The immune cell of embodiment 2, wherein the human interleukin-18 comprises a sequence that is at least 80% identical to SEQ ID NO: 11 or 12.

[0105] Embodiment 4. The immune cell of embodiment 3, wherein the human interleukin-18 comprises a sequence that is at least 90% identical to SEQ ID NO: 11 or 12.

[0106] Embodiment 5. The immune cell of embodiment 4, wherein the human interleukin-18 comprises a sequence that is at least 96% identical to SEQ ID NO: 11 or 12.

[0107] Embodiment 6 The immune cell of any one of embodiments 1 to 5, wherein the sequence encoding interleukin-18 further comprises a sequence encoding a secretory signal sequence.

[0108] Embodiment 7 The immune cell of embodiment 6, wherein the secretory signal sequence is an interleukin-2 secretory signal sequence.

[0109] Embodiment 8 The immune cell of embodiment 7, wherein the interleukin-2 secretory signal sequence comprises the sequence of SEQ ID NO: 13 or 14.

[0110] Embodiment 9 The immune cell of any one of embodiments 1 to 8, wherein the exogenous nucleic acid further comprises a promoter operably linked to a sequence encoding interleukin-18.

[0111] Embodiment 10. The immune cell of embodiment 9, wherein the promoter is a constitutive promoter.

[0112] Embodiment 11 The immune cell of embodiment 9, wherein the promoter is an inducible promoter.

[0113] Embodiment 12 The immune cell of embodiment 9, wherein the promoter is an NFAT promoter.

[0114] Embodiment 13 The immune cell of any one of embodiments 1 to 12, wherein the exogenous nucleic acid further comprises a sequence encoding a CAR.

[0115] Embodiment 14 The immune cell of embodiment 13, wherein the exogenous nucleic acid further comprises a promoter operably linked to the sequence encoding the CAR.

[0116] Embodiment 15. The immune cell of embodiment 14, wherein the promoter is a constitutive promoter.

[0117] Embodiment 16 The immune cell of embodiment 14, wherein the promoter is an inducible promoter.

[0118] Embodiment 17 The immune cell of embodiment 14, wherein the promoter is an NFAT promoter.

[0119] Embodiment 18. The immune cell of any one of embodiments 1 to 17, wherein the CAR is a single polypeptide.

[0120] Embodiment 19. The immune cell of any one of embodiments 1 to 17, wherein the CAR is composed of two polypeptides.

[0121] Embodiment 20. The extracellular antigen-binding domain comprises: a light chain variable domain comprising a CDR1 comprising SEQ ID NO: 1, a CDR2 comprising SEQ ID NO: 2, and a CDR3 comprising SEQ ID NO: 3; a heavy chain variable domain comprising CDR1 comprising SEQ ID NO: 4, CDR2 comprising SEQ ID NO: 5, and CDR3 comprising SEQ ID NO: 6; 20. The immune cell of any one of embodiments 1-19, comprising:

[0122] Embodiment 21 The immune cell of embodiment 20, wherein the light chain variable domain comprises a sequence that is at least 80% identical to SEQ ID NO: 10.

[0123] Embodiment 22 The immune cell of embodiment 21, wherein the light chain variable domain comprises a sequence that is at least 90% identical to SEQ ID NO: 10.

[0124] Embodiment 23 The immune cell of embodiment 22, wherein the light chain variable domain comprises a sequence that is at least 96% identical to SEQ ID NO: 10.

[0125] Embodiment 24 The immune cell of any one of embodiments 20-23, wherein the heavy chain variable domain comprises a sequence that is at least 80% identical to SEQ ID NO:8.

[0126] Embodiment 25. The immune cell of embodiment 24, wherein the heavy chain variable domain comprises a sequence that is at least 90% identical to SEQ ID NO:8.

[0127] Embodiment 26 The immune cell of embodiment 25, wherein the heavy chain variable domain comprises a sequence that is at least 96% identical to SEQ ID NO:8.

[0128] Embodiment 27 The immune cell of any one of embodiments 1 to 26, wherein the antigen-binding domain is humanized.

[0129] Embodiment 28 The immune cell of any one of embodiments 1 to 26, wherein the antigen-binding domain is human.

[0130] Embodiment 29 The immune cell of any one of embodiments 1 to 28, wherein the antigen-binding domain is an scFv.

[0131] Embodiment 30. The transmembrane domain is selected from the group consisting of 4-1BB / CD137, activating NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8 alpha, CD8 beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, ligand specifically binding to CD83, LIGHT, LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1), MHC class 1 molecule, NKG 29. The immune cell of any one of embodiments 1-29, wherein the transmembrane domain is selected from a protein selected from the group consisting of: 2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1), SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, and VLA-6.

[0132] Embodiment 31 The immune cell of embodiment 30, wherein the transmembrane domain is a transmembrane domain derived from CD8 alpha.

[0133] Embodiment 32. The intracellular signaling domain is selected from the group consisting of 4-1BB / CD137, activating NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8 alpha, CD8 beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRTAM, 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 Lyn, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, ligands that specifically bind to CD83, LIGHT, LTBR, ​​Ly9 (CD229), Ly108, lymphocyte function-associated antigen-1 (LFA-1), MHC class 1 molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SEL 32. The immune cell of any one of embodiments 1-31, comprising an intracellular signaling domain from a protein selected from the group consisting of PLG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1), SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, and VLA-6, or any combination thereof.

[0134] Embodiment 33 The immune cell of embodiment 32, wherein the intracellular signaling domain is derived from 4-1BB and CD3 zeta.

[0135] Embodiment 34 The immune cell of any one of embodiments 1 to 33, wherein the chimeric antigen receptor further comprises an additional antigen-binding domain.

[0136] Embodiment 35 The immune cell of embodiment 34, wherein the additional antigen-binding domain is an scFv.

[0137] Embodiment 36. The immune cell of any one of embodiments 1 to 35, which is a human immune cell.

[0138] Embodiment 37 The immune cell of embodiment 36, wherein the human immune cell is an autologous human immune cell.

[0139] Embodiment 38 The immune cell of embodiment 36, wherein the human immune cell is an allogeneic human immune cell.

[0140] Embodiment 39. The immune cell of any one of embodiments 1 to 38, which is a T cell.

[0141] Embodiment 40. The immune cell of any one of embodiments 1 to 38, which is an NK cell.

[0142] Embodiment 41. The immune cell of any one of embodiments 1 to 40, which secretes IL-18 encoded by an exogenous nucleic acid.

[0143] Embodiment 42. A pharmaceutical composition comprising the immune cells of any one of embodiments 1 to 41 and a pharmaceutically acceptable carrier.

[0144] Embodiment 43. A kit comprising the pharmaceutical composition of embodiment 42.

[0145] Embodiment 44. A method of treating a subject with a glypican-3-associated cancer, comprising administering to the subject an immune cell of any one of embodiments 1 to 41 or a pharmaceutical composition of embodiment 42.

[0146] Embodiment 45. A chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain that specifically binds to glypican-3 (GPC3), a transmembrane domain, and an intracellular signaling domain. and a sequence encoding Interleukin-18 coding sequence and A nucleic acid comprising:

[0147] Embodiment 46. The nucleic acid of embodiment 45, wherein the interleukin-18 is human interleukin-18.

[0148] Embodiment 47. The nucleic acid of embodiment 46, wherein the human interleukin-18 comprises a sequence that is at least 80% identical to SEQ ID NO: 11 or 12.

[0149] Embodiment 48. The nucleic acid of embodiment 47, wherein the human interleukin-18 comprises a sequence that is at least 90% identical to SEQ ID NO: 11 or 12.

[0150] Embodiment 49. The nucleic acid of embodiment 48, wherein the human interleukin-18 comprises a sequence that is at least 96% identical to SEQ ID NO: 11 or 12.

[0151] Embodiment 50. The nucleic acid of any one of embodiments 45 to 49, wherein the sequence encoding interleukin-18 further comprises a sequence encoding a secretory signal sequence.

[0152] Embodiment 51. The nucleic acid of embodiment 50, wherein the secretory signal sequence is an interleukin-2 secretory signal sequence.

[0153] Embodiment 52. The nucleic acid of embodiment 51, wherein the interleukin-2 secretory signal sequence comprises the sequence of SEQ ID NO: 13 or 14.

[0154] Embodiment 53. The nucleic acid of any one of embodiments 45 to 52, wherein the nucleic acid further comprises a promoter operably linked to the sequence encoding interleukin-18, wherein the promoter is positioned between the sequence encoding the CAR and the sequence encoding interleukin-18.

[0155] Embodiment 54. The nucleic acid of embodiment 53, wherein the promoter is a constitutive promoter.

[0156] Embodiment 55. The nucleic acid of embodiment 53, wherein the promoter is an inducible promoter.

[0157] Embodiment 56. The nucleic acid of embodiment 53, wherein the promoter is an NFAT promoter.

[0158] Embodiment 57. The nucleic acid of any one of embodiments 45 to 52, further comprising a sequence encoding a self-cleaving protein sequence located between the sequence encoding the CAR and the sequence encoding interleukin-18.

[0159] Embodiment 58 The nucleic acid of embodiment 57, wherein the self-cleaving protein sequence is a P2 self-cleaving protein sequence.

[0160] Embodiment 59. The nucleic acid of any one of embodiments 45 to 58, further comprising a poly(A) sequence located between the sequence encoding the CAR and the sequence encoding interleukin-18.

[0161] Embodiment 60. The nucleic acid of any one of embodiments 45 to 59, further comprising a promoter operably linked to the sequence encoding the CAR.

[0162] Embodiment 61. The nucleic acid of embodiment 60, wherein the promoter is a constitutive promoter.

[0163] Embodiment 62. The nucleic acid of embodiment 60, wherein the promoter is an inducible promoter.

[0164] Embodiment 63. The nucleic acid of embodiment 60, wherein the promoter is an NFAT promoter.

[0165] Embodiment 64. The nucleic acid of any one of embodiments 45 to 63, wherein the CAR is a single polypeptide.

[0166] Embodiment 65. The nucleic acid of any one of embodiments 45 to 63, wherein the CAR is composed of two polypeptides.

[0167] Embodiment 66. The extracellular antigen-binding domain comprises: a light chain variable domain comprising a CDR1 comprising SEQ ID NO: 1, a CDR2 comprising SEQ ID NO: 2, and a CDR3 comprising SEQ ID NO: 3; a heavy chain variable domain comprising CDR1 comprising SEQ ID NO: 4, CDR2 comprising SEQ ID NO: 5, and CDR3 comprising SEQ ID NO: 6; 66. The nucleic acid of any one of embodiments 45 to 65, comprising:

[0168] Embodiment 67. The nucleic acid of embodiment 66, wherein the light chain variable domain comprises a sequence that is at least 80% identical to SEQ ID NO: 10.

[0169] Embodiment 68. The nucleic acid of embodiment 67, wherein the light chain variable domain comprises a sequence that is at least 90% identical to SEQ ID NO: 10.

[0170] Embodiment 69. The nucleic acid of embodiment 68, wherein the light chain variable domain comprises a sequence that is at least 96% identical to SEQ ID NO: 10.

[0171] Embodiment 70. The nucleic acid of any one of embodiments 66-69, wherein the heavy chain variable domain comprises a sequence at least 80% identical to SEQ ID NO:8.

[0172] Embodiment 71. The nucleic acid of embodiment 70, wherein the heavy chain variable domain comprises a sequence that is at least 90% identical to SEQ ID NO:8.

[0173] Embodiment 72. The nucleic acid of embodiment 71, wherein the heavy chain variable domain comprises a sequence that is at least 96% identical to SEQ ID NO:8.

[0174] Embodiment 73. The nucleic acid of any one of embodiments 45 to 72, wherein the antigen-binding domain is humanized.

[0175] Embodiment 74. The nucleic acid of any one of embodiments 45 to 72, wherein the antigen-binding domain is human.

[0176] Embodiment 75. The nucleic acid of any one of embodiments 45 to 74, wherein the antigen-binding domain is an scFv.

[0177] Embodiment 76. The transmembrane domain is selected from the group consisting of 4-1BB / CD137, activating NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8 alpha, CD8 beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, ligand specifically binding to CD83, LIGHT, LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1), MHC class 1 molecule, NK 76. The nucleic acid of any one of embodiments 45-75, wherein the transmembrane domain is selected from a protein selected from the group consisting of G2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1), SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, and VLA-6.

[0178] Embodiment 77. The nucleic acid of embodiment 76, wherein the transmembrane domain is a transmembrane domain derived from CD8 alpha.

[0179] Embodiment 78. The intracellular signaling domain is selected from the group consisting of 4-1BB / CD137, activating NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8 alpha, CD8 beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRTAM, 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 costimulatory factor (ICOS), interleukin (IL-1), interleukin (IL-2R), interleukin (IL-7 ... Glin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, ligands that specifically bind to CD83, LIGHT, LTBR, ​​Ly9 (CD229), Ly108, lymphocyte function-associated antigen-1 (LFA-1), MHC class 1 molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SE 78. The nucleic acid of any one of embodiments 45-77, comprising an intracellular signaling domain from a protein selected from the group consisting of LPLG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1), SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, and VLA-6, or any combination thereof.

[0180] Embodiment 79. The nucleic acid of embodiment 78, wherein the intracellular signaling domain is derived from 4-1BB and CD3 zeta.

[0181] Embodiment 80. The nucleic acid of any one of embodiments 45 to 79, wherein the chimeric antigen receptor further comprises an additional antigen-binding domain.

[0182] Embodiment 81. The nucleic acid of embodiment 80, wherein the additional antigen-binding domain is an scFv.

[0183] Embodiment 82. A vector comprising the nucleic acid of any one of embodiments 45 to 81.

[0184] Embodiment 83. The vector of embodiment 82, which is a viral vector.

[0185] Embodiment 84. The vector of embodiment 83, wherein the viral vector is a lentiviral vector.

[0186] Embodiment 85. Introducing the nucleic acid of any one of embodiments 45 to 81 or the vector of any one of embodiments 82 to 84 into immune cells, thereby producing engineered immune cells. 1. A method for producing engineered immune cells, comprising:

[0187] Embodiment 86 The method of embodiment 85, further comprising culturing the engineered immune cells after the introducing step.

[0188] Embodiment 87. The method of embodiment 85 or 86, wherein the immune cell is a T cell.

[0189] Embodiment 88 The method of embodiment 85 or 86, wherein the immune cells are NK cells.

[0190] Embodiment 89. The method of any one of embodiments 85 to 88, further comprising the step of obtaining immune cells from the subject prior to the introducing step.

[0191] Embodiment 90 The method of embodiment 89, further comprising administering the engineered immune cells to a subject.

[0192] Embodiment 91. The method of embodiment 89 or 90, wherein the subject has been diagnosed with or identified as having a glypican-3 associated cancer.

[0193] Embodiment 92. An engineered immune cell produced by the method of any one of embodiments 85 to 89.

[0194] Embodiment 93. A pharmaceutical composition comprising the engineered immune cells of embodiment 92 and a pharmaceutically acceptable carrier.

[0195] Embodiment 94. A method of treating anti-glypican-3 associated cancer in a subject, comprising administering to the subject the engineered immune cells of embodiment 92 or the pharmaceutical composition of embodiment 93.

[0196] Embodiment 95. A chimeric antigen receptor (CAR) comprising an extracellular antigen-binding domain that specifically binds to glypican-3 (GPC3), a transmembrane domain, and an intracellular signaling domain. a first nucleic acid comprising a sequence encoding a second nucleic acid comprising a sequence encoding interleukin-18; A pair of nucleic acids, including:

[0197] Embodiment 96. The pair of nucleic acids of embodiment 95, wherein the interleukin-18 is human interleukin-18.

[0198] Embodiment 97. The pair of nucleic acids of embodiment 96, wherein the human interleukin-18 comprises a sequence that is at least 80% identical to SEQ ID NO: 11 or 12.

[0199] Embodiment 98. The pair of nucleic acids of embodiment 97, wherein the human interleukin-18 comprises a sequence that is at least 90% identical to SEQ ID NO: 11 or 12.

[0200] Embodiment 99. The pair of nucleic acids of embodiment 98, wherein the human interleukin-18 comprises a sequence that is at least 96% identical to SEQ ID NO: 11 or 12.

[0201] Embodiment 100. The nucleic acid pair of any one of embodiments 95 to 99, wherein the sequence encoding interleukin-18 further comprises a sequence encoding a secretory signal sequence.

[0202] Embodiment 101. The nucleic acid pair of embodiment 100, wherein the secretory signal sequence is an interleukin-2 secretory signal sequence.

[0203] Embodiment 102. The nucleic acid pair of embodiment 101, wherein the interleukin-2 secretory signal sequence comprises the sequence of SEQ ID NO: 13 or 14.

[0204] Embodiment 103. The pair of nucleic acids of any one of embodiments 95 to 102, wherein the second nucleic acid further comprises a promoter operably linked to the sequence encoding interleukin-18.

[0205] Embodiment 104. The pair of nucleic acids of embodiment 103, wherein the promoter is a constitutive promoter.

[0206] Embodiment 105. The nucleic acid pair of embodiment 103, wherein the promoter is an inducible promoter.

[0207] Embodiment 106. The pair of nucleic acids of embodiment 103, wherein the promoter is an NFAT promoter.

[0208] Embodiment 107. The pair of nucleic acids of any one of embodiments 95 to 106, wherein the first and / or second nucleic acid further comprises a poly(A) sequence.

[0209] Embodiment 108 The pair of nucleic acids of any one of embodiments 95 to 107, wherein the first nucleic acid further comprises a promoter operably linked to the sequence encoding the CAR.

[0210] Embodiment 109. The pair of nucleic acids of embodiment 108, wherein the promoter is a constitutive promoter.

[0211] Embodiment 110. The pair of nucleic acids of embodiment 108, wherein the promoter is an inducible promoter.

[0212] Embodiment 111. The pair of nucleic acids of embodiment 108, wherein the promoter is an NFAT promoter.

[0213] Embodiment 112. The pair of nucleic acids of any one of embodiments 95 to 111, wherein the CAR is a single polypeptide.

[0214] Embodiment 113. The pair of nucleic acids of any one of embodiments 95 to 111, wherein the CAR is composed of two polypeptides.

[0215] Embodiment 114. The extracellular antigen-binding domain comprises: a light chain variable domain comprising a CDR1 comprising SEQ ID NO: 1, a CDR2 comprising SEQ ID NO: 2, and a CDR3 comprising SEQ ID NO: 3; a heavy chain variable domain comprising CDR1 comprising SEQ ID NO: 4, CDR2 comprising SEQ ID NO: 5, and CDR3 comprising SEQ ID NO: 6; The nucleic acid pair of any one of embodiments 95 to 113, comprising:

[0216] Embodiment 115. The pair of nucleic acids of embodiment 114, wherein the light chain variable domain comprises a sequence that is at least 80% identical to SEQ ID NO: 10.

[0217] Embodiment 116. The pair of nucleic acids of embodiment 115, wherein the light chain variable domain comprises a sequence that is at least 90% identical to SEQ ID NO: 10.

[0218] Embodiment 117. The pair of nucleic acids of embodiment 116, wherein the light chain variable domain comprises a sequence that is at least 96% identical to SEQ ID NO: 10.

[0219] Embodiment 118. The pair of nucleic acids of any one of embodiments 114 to 117, wherein the heavy chain variable domain comprises a sequence that is at least 80% identical to SEQ ID NO:8.

[0220] Embodiment 119. The pair of nucleic acids of embodiment 118, wherein the heavy chain variable domain comprises a sequence that is at least 90% identical to SEQ ID NO:8.

[0221] Embodiment 120. The pair of nucleic acids of embodiment 119, wherein the heavy chain variable domain comprises a sequence that is at least 96% identical to SEQ ID NO:8.

[0222] Embodiment 121. The nucleic acid pair of any one of embodiments 95 to 120, wherein the antigen-binding domain is humanized.

[0223] Embodiment 122. The nucleic acid pair of any one of embodiments 95 to 120, wherein the antigen-binding domain is human.

[0224] Embodiment 123. The nucleic acid pair of any one of embodiments 95 to 122, wherein the antigen-binding domain is an scFv.

[0225] Embodiment 124. The transmembrane domain is selected from the group consisting of 4-1BB / CD137, activating NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8 alpha, CD8 beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, ligand specifically binding to CD83, LIGHT, LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1), MHC class 1 molecule, NKG2 C, a transmembrane domain selected from a protein selected from the group consisting of NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1), SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, and VLA-6.

[0226] Embodiment 125. The nucleic acid pair of embodiment 124, wherein the transmembrane domain is a transmembrane domain derived from CD8 alpha.

[0227] Embodiment 126. The intracellular signaling domain is selected from the group consisting of 4-1BB / CD137, activating NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8 alpha, CD8 beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRTAM, 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 Phosphorin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, ligands that specifically bind to CD83, LIGHT, LTBR, ​​Ly9 (CD229), Ly108, lymphocyte function-associated antigen-1 (LFA-1), MHC class 1 molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELP 126. The nucleic acid pair of any one of embodiments 95-125, comprising an intracellular signaling domain from a protein selected from the group consisting of LG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1), SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, and VLA-6, or any combination thereof.

[0228] Embodiment 127. The nucleic acid pair of embodiment 126, wherein the intracellular signaling domains are derived from 4-1BB and CD3 zeta.

[0229] Embodiment 128. The nucleic acid pair of any one of embodiments 95 to 127, wherein the chimeric antigen receptor further comprises an additional antigen-binding domain.

[0230] Embodiment 129. The nucleic acid pair of embodiment 128, wherein the additional antigen-binding domain is an scFv.

[0231] Embodiment 130. A pair of vectors, together comprising the pair of nucleic acids of any one of embodiments 95 to 129.

[0232] Embodiment 131. The vector pair of embodiment 130, which is a viral vector pair.

[0233] Embodiment 132. The pair of vectors of embodiment 131, wherein the pair of viral vectors is a pair of lentiviral vectors.

[0234] Embodiment 133. Introducing the pair of nucleic acids of any one of embodiments 95 to 129 or the pair of vectors of any one of embodiments 130 to 132 into immune cells, thereby producing engineered immune cells. 1. A method for producing engineered immune cells, comprising:

[0235] Embodiment 134 The method of embodiment 133, further comprising culturing the engineered immune cells after the introducing step.

[0236] Embodiment 135. The method of embodiment 133 or 134, wherein the immune cells are T cells.

[0237] Embodiment 136. The method of embodiment 133 or 134, wherein the immune cells are NK cells.

[0238] Embodiment 137. The method of any one of embodiments 133 to 136, further comprising the step of obtaining immune cells from the subject prior to the introducing step.

[0239] Embodiment 138 The method of embodiment 137, further comprising administering the engineered immune cells to a subject.

[0240] Embodiment 139. The method of embodiment 137 or 138, wherein the subject has been diagnosed with or identified as having a glypican-3 associated cancer.

[0241] Embodiment 140. An engineered immune cell produced by the method of any one of embodiments 133 to 137.

[0242] Embodiment 141. A pharmaceutical composition comprising the engineered immune cells of embodiment 140 and a pharmaceutically acceptable carrier.

[0243] Embodiment 142. A method for treating anti-glypican-3 associated cancer in a subject, comprising administering to the subject the engineered immune cells of embodiment 140 or the pharmaceutical composition of embodiment 141.

[0244] Embodiment 143. The method of any one of embodiments 44, 94, and 142, wherein the glypican-3 associated cancer is liver cancer.

[0245] Embodiment 144. The method of any one of embodiments 44, 94, 142, and 143, wherein the subject has previously been administered one or more additional anti-cancer therapies selected from the group consisting of ionizing radiation, chemotherapeutic agents, therapeutic antibodies, and checkpoint inhibitors.

[0246] Embodiment 145. The method of any one of embodiments 44, 94, 142, and 143, wherein the subject is further administered one or more additional anti-cancer therapies selected from the group consisting of ionizing radiation, chemotherapeutic agents, therapeutic antibodies, and checkpoint inhibitors.

[0247] Embodiment 146. The method of any one of embodiments 44, 94, and 142-145, wherein the subject has been identified or diagnosed as having a glypican-3 associated cancer. [Example]

[0248] The present disclosure is further described in the following examples, which do not limit the scope of the disclosure as claimed.

[0249] Example 1 Lentiviral Transfer Plasmids A DNA construct encoding a single-chain variable fragment (scFv) form of the humanized anti-GPC3 antibody (clone: ​​GC33) agent was generated using standard DNA cloning techniques known in the art by using an IDT vector to link the VL and VH regions (the sequences were designed to contain a VH-VL or VL-VH orientation). The lentiviral transfer plasmids used herein are shown in Table 3, and the nucleic acid sequence of huGC33 VH-VL is shown in Table 4.

[0250] IL-18-expressing GPC3 CARs were produced using the P2A self-cleaving peptide or the nuclear factor of activated T cells (NFAT)-IL-2 minimal promoter to express IL-18 only in activated T cells (Figure 1). The intracellular signaling domain of the IL-18-expressing GPC3 CAR contains the 4-1BB signaling domain followed by a 5-amino acid sequence. For the NFAT-IL-2 minimal promoter, a sequence from pGL3-NFAT luciferase was used. Sequence information for the NFAT-IL-2 minimal promoter-IL-18 and polyA-IL-18-NFAT-IL-2 minimal promoter is shown in Table 5.

[0251] [Table 3]

[0252] [Table 4]

[0253] [Table 5] TIFF0007778081000010.tif220156TIFF0007778081000011.tif233156

[0254] SEQ ID NO: 16 5 additional amino acids CGTTTCTCTGTTGTT SEQ ID NO: 17 4-1BB intracellular domain TIFF0007778081000012.tif19164 SEQ ID NO: 18 4-1BB intracellular domain with 5 additional amino acids (euBBz) TIFF0007778081000013.tif19163 SEQ ID NO: 19 CD3-Zeta TIFF0007778081000014.tif47162 SEQ ID NO: 20 P2A TIFF0007778081000015.tif12162 SEQ ID NO: 21 NFAT TIFF0007778081000016.tif40161 SEQ ID NO: 22 Poly A TIFF0007778081000017.tif33161

[0255] The lentiviral vector construct pELPS4-huGC33-euBBz was digested with EcoRV and SalI, and pELPS4 huGC33 HL-P2A-IL18 was inserted into the vector construct. Furthermore, after linearizing the constructs with SalI, the lentiviral constructs pELPS4-huGC33-euBBz, pELPS4-huGC33-euBBz-NFAT-IL18 and pELPS4-huGC33-euBBz-polyA-IL18-NFAT were subjected to infusion cloning. The results of DNA fragment purification are shown in Figures 2 and 3. The transducing units (TU / mL) of the lentiviruses were measured and are shown in Table 6.

[0256] [Table 6]

[0257] Example 2 huGC33-VHVL-P2A-IL18 and huGC33-VHVL-NFAT-IL18 in vitro Peripheral blood mononuclear cells (PBMCs) were cultured in cell culture medium containing 1 L of OpTmizer™ T-Cell Expansion Basal Medium, 25 mL of OpTmizer™ T-Cell Expansion Supplement, 50 mL of CTS™ Immune Cell SR, 10 mL of Pen-Strep (10,000 U / mL), and 10 mL of CTS™ GlutaMAX™-I Supplement to a cell density of 1 x 10 6 The culture medium was adjusted to 100 cells / mL, and IL-2 (400 IU / mL) was added during culture at 5% CO2 and 37°C.

[0258] PBMCs were then transduced with the lentiviral vectors, and cell growth and proliferation were measured from day 7 to day 14 of cell culture. Cells were then harvested on day 14 and used for further analysis.

[0259] The huGC33-VHVL-P2A-IL18 and huGC33-VHVL-NFAT-IL18 CAR-T constructs were compared in vitro. Cell growth of each group of CAR-T cells on day 14 of cell culture was compared by total fold expansion (Figure 4A). Cell proliferation and cell viability were compared from days 7 to 14. Nearly all conditions showed CAR-T cell viability above 90%, except for huGC33-VHVL-P2A-IL18 CAR-T cells, which showed cell viability below 90% on days 7, 9, and 11 (Figure 4B-4C). Target cells were harvested using a GPC3-positive cell line, and luciferase-based cytotoxicity was measured using CAR-T cells as effector cells. The cells were incubated in a 96-well white polystyrene microplate at an effector (E):target (T) ratio of 10:1, then incubated with Bright-Glo™ luciferase assay reagent for 5 minutes. Cytotoxicity was measured and quantified using a luminometer. The results show that in vitro killing activity was similar in all groups of CAR-T cells (Figure 4D). Furthermore, CAR expression was analyzed using flow cytometry on days 7, 9, 11, and 14 of cell culture (Figures 4E-4H).

[0260] Example 3 huGC33-VHVL-P2A-IL18 and huGC33-VHVL-NFAT-IL18 CAR-T cells in vivo Cancer cells derived from human hepatocellular carcinoma cell lines, Huh-7-GL, PLC / PRF / 5-GL (GPC3-positive cell line), and SK-Hep-1 (GPC3-negative cell line), were injected into NSG mice (2 × 10 6 The growth of liver tumors over time was monitored using a 100-well platelet count (100 cells / head). Animals were divided into groups according to the tumor size measured in each animal (Tables 7-10).

[0261] [Table 7]

[0262] Huh-7-GL cells were injected into NSG mice (2 × 106 After tumor development, mice were injected with huGC33-VHVL-P2A-IL18 CAR-T cells and huGC3-VHVL-NFAT-IL-18 CAR-T cells. All mice in the control group (naive group) that did not receive any injections died within 28 days of injection, only one mouse in the huGC33 VHVL group died 5 weeks after injection, and three mice in the huGC33-VHVL-P2A-IL18 group died. Meanwhile, all mice in the group injected with huGC33-VHVL-NFAT-IL18 CAR-T cells survived for at least 15 weeks after injection (Figure 5A).

[0263] Observation up to 15 weeks after GPC3 CAR-T cell injection showed that the group of mice injected with huGC33-VHVL-NFAT-IL18 showed the greatest increase in CAR-T cell levels in the blood (Figure 5B).

[0264] Fourteen days after injection of GPC3 CAR-T cells, blood serum was collected from the mice and analyzed for IL-18 using ELISA. The results showed that the concentration of IL-18 in serum reached approximately 20 pg / mL only in mice injected with huGC33-VHVL-NFAT-IL18 CAR-T cells (Figure 6).

[0265] Example 4 huGC33-VHVL-NFAT-IL18 and huGC33-VHVL-IL18-NFAT in vitro The huGC33-VHVL-NFAT-IL18 and huGC33-VHVL-IL18-NFAT CAR-T constructs were compared in vitro. Total fold expansion is compared for each CAR-T cell line on day 12 of cell culture (Figure 7A). Cell proliferation and viability were compared from days 5 to 12. At day 12, all CAR-T cells showed >90% viability (Figures 7B-7C). Target cells were harvested using a GPC3-positive cell line, and LDH-based cytotoxicity was measured using CAR-T cells as effector cells. The cells were incubated in a 96-well U-bottom plate at an effector (E):target (T) ratio of 10:1 and incubated with Cyto Tox96 reagent for 30 minutes. Cytotoxicity was measured and quantified using a microplate reader at a wavelength of 490 nm (Figure 7D). CAR expression was analyzed using flow cytometry on days 7, 9, and 12 (Figures 7E-7G). Furthermore, IL-18 concentration was analyzed using ELISA. The results show that huGC33 VHVL-NFAT-IL18 CAR-T cells exhibited the highest IL-18 expression (Figure 7H).

[0266] Example 5 huGC33-VHVL-NFAT-IL18 and huGC33-VHVL-IL18-NFAT CAR-T cells in vivo Huh-7-GL cells were cultured in NSG mice (2 × 10 6 The tumor size was approximately 330 mm. 3 (Table 8) or approximately 1100 mm 3After reaching one of the following markers (Table 9), mice were divided into groups receiving no treatment, 250,000 cells of CD19 CAR-T cells, 250,000 cells of huGC33 VHVL, 100,000 cells of huGC33 VHVL-NFAT-IL18, 250,000 cells of huGC33 VHVL-NFAT-IL18, 100,000 cells of huGC33 VHVL-IL18-NFAT, or 250,000 cells of huGC33 VHVL-IL18-NFAT CAR-T cells. huGC33-VHVL-NFAT-IL18 and huGC33-VHVL-IL18-NFAT CAR-T cells were then injected into each group of mice.

[0267] [Table 8]

[0268] [Table 9]

[0269] The result was that the tumor size was approximately 330 mm 3 The results show that mice reaching a tumor size of 100,000, untreated mice, and mice injected with CD19 CAR-T cells all died within 24 days of injection. Only two mice in the group injected with 100,000 huGC33-VHVL-NFAT-IL18 CAR-T cells died 49 days after injection, while three mice in the group injected with 250,000 huGC33-VHVL-IL18-NFAT CAR-T cells showed a slight reduction in tumor size but subsequently died due to tumor regrowth. Results from the group injected with 100,000 huGC33-VHVL-IL18-NFAT CAR-T cells indicated that the injection was not effective in reducing tumor size (Figure 8A). Observations up to 6 weeks after GPC3 CAR-T cell injection showed that only mice with reduced tumor size showed increased levels of CAR-T cells in the blood, which subsequently decreased after tumor size reduction (Figure 8B).

[0270] The tumor size is approximately 1100 mm 3 For the group of mice that reached 300 mm , only mice with huGC33-VHVL-NFAT-IL18 showed a reduction in tumor size by 49 days after injection, while all mice in the other groups died within 24 days of injection (Figure 9A). CAR-T cell levels in the blood decreased until tumor sizes reached approximately 300 mm . 3 The results showed that the serotonin concentration in the group of mice reaching 100 mg / kg / day was slightly lower than that in the group of mice reaching 100 mg / kg / day (Figure 9B).

[0271] Example 6. huGC33 VHVL-NFAT-IL18 Expression in Vivo by PLC / PRF / 5-GL and SK-Hep-1 Cell Lines Cancer cells derived from the PLC / PRF / 5-GL cell line, which exhibit low GPC3 expression, were injected into NSG mice (2 × 10 6 The growth of liver tumors over time was monitored using huGC33 VHVL-NFAT-IL18-injected mice (cells / head). The results showed that the group injected with huGC33 VHVL-NFAT-IL18 showed reduced tumor size compared with the untreated and CD19 groups, all of which died within 21 days of injection, indicating that huGC33 VHVL-NFAT-IL18 CAR T cells effectively reduced tumor size in GPC3-positive cancer cells (Figure 10A).

[0272] When huGC33 VHVL-NFAT-IL18 CAR T cells were injected into SK-Hep-1-GL cancer cells (a GPC3-negative cell line), the mice did not show a reduction in tumor size, and all mice died within 21 days of injection, indicating that the IL-18-expressing GPC3 CAR used here is specific for GPC3-expressing tumor cells (Figure 10B).

[0273] [Table 10]

[0274] These results indicate that huGC33-VHVL-NFAT-IL18 CAR-T cells are effective in maintaining effective concentrations of CAR-T cells in the serum while exhibiting higher survival rates.

Claims

1. an extracellular antigen-binding domain that specifically binds to glypican-3 (GPC3), the extracellular antigen-binding domain comprising a light chain variable domain represented by SEQ ID NO: 10 and a heavy chain variable domain represented by SEQ ID NO: 8; a transmembrane domain, and Intracellular signaling domains a chimeric antigen receptor (CAR) comprising: an exogenous nucleic acid comprising a sequence encoding interleukin-18 and an NFAT promoter; including immune cells.

2. The immune cell of claim 1, wherein the interleukin-18 is human interleukin-18.

3. The immune cell of claim 2, wherein the human interleukin-18 comprises a sequence that is at least 80%, 90%, or 96% identical to SEQ ID NO: 11 or 12.

4. The immune cell according to any one of claims 1 to 3, wherein the sequence encoding interleukin-18 further comprises a sequence encoding a secretory signal sequence.

5. The immune cell according to claim 4, wherein the secretory signal sequence is an interleukin-2 secretory signal sequence.

6. The immune cell of claim 5, wherein the interleukin-2 secretory signal sequence comprises the sequence of SEQ ID NO: 13 or 14.

7. The immune cell of any one of claims 1 to 6, wherein the NFAT promoter is operably linked to a sequence encoding interleukin-18.

8. The immune cell of any one of claims 1 to 7, wherein the antigen-binding domain is humanized.

9. The immune cell of any one of claims 1 to 8, wherein the antigen-binding domain is an scFv.

10. The transmembrane domain is selected from the group consisting of 4-1BB / CD137, activating NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8 alpha, CD8 beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, ligand specifically binding to CD83, LIGHT, LTBR, ​​Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1), MHC class 1 molecule, NK The immune cell of any one of claims 1 to 9, wherein the transmembrane domain is selected from a protein selected from the group consisting of G2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1), SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, and VLA-6.

11. The immune cell of claim 10, wherein the transmembrane domain is a transmembrane domain derived from CD8 alpha.

12. The intracellular signaling domain may be selected from the group consisting of 4-1BB / CD137, activating NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD30, CD4, CD40, CD49a, CD49D, and CD49. f, CD69, CD7, CD84, CD8, CD8 alpha, CD8 beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRTAM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrin , ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, ligand specifically binding to CD83, LIGHT, LTBR, ​​Ly9 (CD229), Ly108, lymphocyte function-associated antigen-1 (LFA-1), MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELP 12. The immune cell of any one of claims 1 to 11, comprising an intracellular signaling domain derived from a protein selected from the group consisting of LG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1), SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, and VLA-6, or any combination thereof.

13. The immune cell of claim 12, wherein the intracellular signaling domain is derived from 4-1BB and CD3 zeta.

14. The immune cell according to any one of claims 1 to 13, which is a human immune cell.

15. The immune cell according to any one of claims 1 to 14, which is a T cell or an NK cell.

16. The immune cell of any one of claims 1 to 15, which secretes the IL-18 encoded by the exogenous nucleic acid.

17. A pharmaceutical composition comprising the immune cells of any one of claims 1 to 16 and a pharmaceutically acceptable carrier.

18. 18. The pharmaceutical composition of claim 17 for use in the prevention or treatment of cancer.

19. The pharmaceutical composition of claim 18, wherein the cancer is a glypican-3 associated cancer.

20. 20. The pharmaceutical composition of claim 19, wherein the glypican-3 associated cancer is selected from the group consisting of liver cancer, hepatocellular carcinoma, hepatoblastoma, germ cell tumor, yolk sac tumor, choriocarcinoma, Wilms' tumor, gastric carcinoma, non-small cell lung cancer, and thyroid cancer.

21. A kit comprising the pharmaceutical composition of any one of claims 17 to 20.

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