Gpc3 car-t cell compositions and methods of making and using the same

Abstract

Provided are CAR-T compositions against GPC3, including chimeric receptors, and engineered immune cells against GPC3. The present disclosure also provides vectors, compositions, and methods of treatment using GPC3 antigen binding molecules and engineered immune cells. The GPC3 CAR compositions provided herein can be used to treat some cancers.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Application Serial No. 62 / 991,493, filed March 18, 2020; U.S. Provisional Application Serial No. 63 / 004,827, filed April 3, 2020; and U.S. Provisional Application Serial No. 63 / 043,237, filed June 24, 2020, each of which is incorporated herein by reference in its entirety. BACKGROUND

[0003] Cancer remains one of the leading causes of death worldwide. Recent statistics report that 13% of the world’s population dies from cancer. According to estimates by the International Agency for Research on Cancer (IARC), 14.1 million new cases of cancer and 8.2 million cancer deaths occurred worldwide in 2012. By 2030, the global burden is expected to grow to 21.7 million new cases and 13 million cancer deaths due to population growth and aging, as well as risk factors such as smoking, unhealthy diets, and lack of physical exercise. Moreover, the pain and medical expenses of cancer treatment lead to a decrease in the quality of life for cancer patients and their families.

[0004] Chimeric antigen receptor engineered T cells (CAR-T) have great therapeutic potential for treating diseases such as cancer. CAR-T therapy confers T cells with strong target affinity and signaling function. However, the efficacy of CAR-T therapy is impressive, but it is often accompanied by serious side effects, such as cytokine release syndrome (CRS). Therefore, there is still a need to develop CAR-T therapies and strategies with reduced side effects. SUMMARY

[0005] Provided herein are immune cells comprising a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor comprises an extracellular antigen binding domain that specifically binds glypican 3 (GPC3), a transmembrane domain, and an intracellular signaling domain. In some embodiments, the CAR is a single polypeptide. In some embodiments, the CAR comprises two polypeptides.

[0006] In some embodiments, 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; and a heavy chain variable domain comprising a CDR1 comprising SEQ ID NO: 4, a CDR2 comprising SEQ ID NO: 5, and a CDR3 comprising SEQ ID NO: 6. 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 antigen-binding domain is humanized. In some embodiments, the antigen-binding domain is human. In some embodiments, the antigen-binding domain is an scFv.

[0007] In some embodiments, the transmembrane domain comprises a transmembrane domain selected from a protein selected from the group consisting of 4-1BB / CD137, Activating NK cell receptor, Immunoglobulin, 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), Fcgamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, Inducible T cell costimulator (ICOS), Integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, Ligand that specifically binds CD83, LIGHT, LTBR, Ly9 (CD229), Lymphocyte function-associated antigen-1 (LFA-1), MHC class I molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, Programmed death-1 (PD-1), PSGL1, SELPLG (CD162), Signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1), SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, and VLA-6. In some embodiments, the transmembrane domain is a transmembrane domain from CD8 alpha.

[0008] In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain from a protein selected from the group consisting of 4-1BB / CD137, Activating NK Cell Receptor, Immunoglobulin, 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), Fcgamma Receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, Inducible T-cell Costimulator (ICOS), Integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, Ligand that Specifically Binds CD83, LIGHT, LTBR, Ly9 (CD229), Ly108, Lymphocyte Function-Associated Antigen-1 (LFA-1), MHC Class I 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, or any combination thereof. In some embodiments, the intracellular signaling domain is from 4-1BB and CD3 zeta.

[0009] 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. In some embodiments, the immune cell is a human immune cell. In some embodiments, the human immune cell is an autologous human immune cell. In some embodiments, the human immune cell is an allogeneic human immune cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is an NK cell.

[0010] Provided herein are pharmaceutical compositions comprising any of the immune cells described herein and a pharmaceutically acceptable carrier. Provided herein are kits comprising a pharmaceutical composition, wherein the pharmaceutical composition is any of the pharmaceutical compositions described herein.

[0011] Provided herein are methods for treating a subject having a glypican 3-associated cancer, the method comprising administering to the subject any of the immune cells or any of the pharmaceutical compositions.

[0012] Provided herein are nucleic acids encoding a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor comprises an extracellular antigen binding domain that specifically binds to glypican 3 (GPC3), a transmembrane domain, and an intracellular signaling domain.

[0013] In some embodiments, the CAR is a single polypeptide. In some embodiments, the CAR comprises two polypeptides.

[0014] In some embodiments, 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; and a heavy chain variable domain comprising a CDR1 comprising SEQ ID NO: 4, a CDR2 comprising SEQ ID NO: 5, and a CDR3 comprising SEQ ID NO: 6. 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 antigen-binding domain is humanized. In some embodiments, the antigen-binding domain is human. In some embodiments, the antigen-binding domain is an scFv.

[0015] In some embodiments, the transmembrane domain is a transmembrane domain selected from a protein selected from the group consisting of 4-1BB / CD137, Activating NK cell receptor, Immunoglobulin, 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), Fcgamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, Inducible T cell costimulator (ICOS), Integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, Ligand that specifically binds CD83, LIGHT, LTBR, Ly9 (CD229), Lymphocyte function-associated antigen-1 (LFA-1), MHC class I molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, Programmed death-1 (PD-1), PSGL1, SELPLG (CD162), Signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1), SLAMF4 (CD244), SLAMF6 (NTB-A), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, and VLA-6. In some embodiments, the transmembrane domain is a transmembrane domain from CD8 alpha.

[0016] In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain from a protein selected from the group consisting of 4-1BB / CD137, Activating NK Cell Receptor, Immunoglobulin, 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), Fcgamma Receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, Inducible T-cell Costimulator (ICOS), Integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, Ligand that Specifically Binds CD83, LIGHT, LTBR, Ly9 (CD229), Ly108, Lymphocyte Function-Associated Antigen-1 (LFA-1), MHC Class I 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, or any combination thereof. In some embodiments, the intracellular signaling domain is from 4-1BB and CD3 zeta.

[0017] 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.

[0018] Provided herein are vectors comprising any of the nucleic acids described herein. In some embodiments, the vector comprises a promoter operably linked to the nucleic acid. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a lentiviral vector.

[0019] Provided herein are methods for producing an engineered immune cell, the method comprising: introducing any of the nucleic acids described herein into an immune cell or vector described herein, thereby producing an engineered immune cell. In some embodiments, the engineered immune cell is cultured after the step of introducing. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is an NK cell. In some embodiments, the immune cell is obtained from a subject prior to the step of introducing. In some embodiments, the method further comprises administering the engineered immune cell to the subject. In some embodiments, the subject has been diagnosed or identified as having a glypican 3-associated cancer.

[0020] Provided herein are engineered immune cells produced by any of the methods described herein. In some embodiments, the pharmaceutical composition comprises any of the engineered immune cells described herein and a pharmaceutically acceptable carrier.

[0021] Provided herein are methods of treating a glypican 3-associated cancer in a subject, the method comprising administering an engineered immune cell or a pharmaceutical composition to the subject. In some embodiments, the glypican 3-associated cancer is a liver cancer. In some embodiments, the subject has been previously administered one or more additional anti-cancer therapies selected from the group consisting of: ionizing radiation, a chemotherapeutic agent, a therapeutic antibody, and a checkpoint inhibitor. In some embodiments, the is selected from the group consisting of: ionizing radiation, a chemotherapeutic agent, a therapeutic antibody, and a checkpoint inhibitor. In some embodiments, the subject has been identified or diagnosed as having a glypican 3-associated cancer. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 The structure of the scFv recombinant expression vector is shown.

[0023] Figure 2 The SDS-PAGE results of the muGC33 and huGC33 antibodies are shown.

[0024] Figure 3 The ELISA titer plots of the huGC33 antibody and muGC33 antibody are shown.

[0025] Figure 4 The ELISA titer plots of the huGC33 antibody are shown.

[0026] Figure 5 Enzymogram results for pELPS4-huGC33 VL-VH and pELPS4-huGC33 VH-VL post cloning are shown.

[0027] Figure 6A FIG. 4 is a graph showing cell growth of each CAR-T cell of untreated, truncated muGC33, muGC33, huGC33 VHVL, and huGC33 VLVH at day 12 of cell culture by total fold expansion comparison.

[0028] Figure 6B FIG. 5 is a bar graph comparing cell viability of each CAR-T cell of untreated, truncated muGC33, muGC33, huGC33 VHVL, and huGC33 VLVH from day 4 to day 12.

[0029] Figure 7A FIG. 6 shows FACS analysis results of untreated, truncated muGC33, muGC33, huGC33 VHVL, and huGC33 VLVH CAR-T cells at day 6.

[0030] Figure 7B FIG. 7 shows FACS analysis results of untreated, truncated muGC33, muGC33, huGC33 VHVL, and huGC33 VLVH CAR-T cells at day 9.

[0031] Figure 7C FIG. 8 shows FACS analysis results of untreated, truncated muGC33, muGC33, huGC33 VHVL, and huGC33 VLVH CAR-T cells at day 12.

[0032] Figure 8 FIG. 9 is a graph comparing cytotoxicity assay results of untreated, truncated muGC33, muGC33, huGC33 VHVL, and huGC33 VLVH CAR-T cells, where effector (E): target (T) cell ratio (E:T) can be 10:1, 3:1, 1:1, or 0.3:1. The results show that muGC33, huGC33 VHVL, and huGC33 VLVH CAR-T cells show similar in vitro killing activity, where untreated cells do not show in vitro killing activity.

[0033] Figure 9GPC3 expression in liver cancer cells is shown, where GPC3 expression was measured in cell lines HepG2, Hep3B, Huh-7, and SK-Hep-1. The results show that GPC3 is expressed in HepG2, Hep3B, and Huh-7 cell lines, but not in the SK-Hep-1 cell line.

[0034] Figure 10A FACS analysis results for the Huh-7 cell line are shown.

[0035] Figure 10B Results from luciferase assays from the Huh-7 cell line are shown, showing expression of luciferase GFP.

[0036] Figure 11 A set of graphs showing tumor growth in animal models after GPC3 CAR-T cell injection is shown. The results show that 20% of mice receiving huGC33 VHVL showed tumor growth after injection, while 40% of mice receiving huGC33 VLVH showed tumor growth after injection.

[0037] Figure 12 A graph showing the number of GPC3 CAR-T cells in the blood of animals over time after GPC3 CAR-T cell injection is shown. The results show that the total number of CAR-T cells in the blood peaked at day 14 after injection. DETAILED DESCRIPTION

[0038] The present disclosure describes T cells engineered with chimeric antigen receptors (CAR-T) including a GPC3 antigen binding domain, and methods of making and using the same.

[0039] Definitions:

[0040] About: When used herein to refer to a numerical value, the term "about" refers to a numerical value similar in context to the numerical value referred to. In general, one of skill in the art familiar with the context will appreciate the degree of relevant deviation encompassed by "about" in that context. For example, in some embodiments, the term "about" can encompass a range of values within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the value referred to.

[0041] Administration: As used herein, the term "administration" generally refers to the application of a composition to a subject or system to achieve the delivery of a medicament, which is the composition or is included in the composition. Those skilled in the art will appreciate the various routes by which administration to a subject, such as a human, may be used where appropriate. For example, in some embodiments, administration may be via the eye, oral, parenteral, topical, etc. In some specific embodiments, administration may be via the bronchus (e.g., via bronchial infusion), mouth, dermis (which may be or contain, e.g., one or more of topical to the dermis, intradermis, interdermis, transdermis, etc.), intestine, artery, intradermis, stomach, intramedullary, intramuscular, intranasal, intraperitoneal, intrasheath, intravenous, intravenous, intracardiac, intra-organ (e.g., intrahepatic), mucosa, nose, mouth, rectum, subcutaneous, sublingual, topical, trachea (e.g., via intratracheal infusion), vagina, vitreous body, 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 administration (e.g., multiple doses at intervals) and / or periodic administration (e.g., individual doses at common intervals). In some embodiments, administration may involve continuous administration (e.g., infusion) over at least a selected period of time.

[0042] Affinity: As known in the art, “affinity” is a measure of the tightness of binding between a particular ligand and its mate. Affinity can be measured in different ways. In some embodiments, affinity is measured by quantitative determination. In some such embodiments, the mate concentration may be fixed above the ligand concentration to simulate physiological conditions. Alternatively or additionally, in some embodiments, the mate concentration and / or ligand concentration may be different. In some such embodiments, affinity may be compared with reference values ​​under similar conditions (e.g., concentrations).

[0043] 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 covers any polypeptide or polypeptide complex that includes sufficient to confer a structural element of an immunoglobulin with specific binding. Exemplary antibody agents include, but are not limited to, monoclonal antibodies, polyclonal antibodies, and fragments thereof. In some embodiments, antibody agents may include one or more humanized, primate-derived, chimeric, etc., sequence elements, as known in the art. In many embodiments, the term "antibody agent" is used to refer to one or more of the known or developed constructs or forms in the art for utilizing the structural and functional characteristics of antibodies in alternative presentations. For example, in embodiments, the antibody agent used according to the present invention is selected from, but not limited to, intact IgA, IgG, IgE, or IgM antibodies; bispecific or multispecific antibodies (e.g., (etc.); antibody fragments, such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments and isolated CDRs or collections thereof; single-chain Fvs; peptide-Fc fusions; single-domain antibodies (e.g., shark single-domain antibodies, such as IgNAR or fragments thereof); cameloid antibodies; masking antibodies (e.g., );Small Modular ImmunoPharmaceuticals("SMIPs TM Single-chain or tandem double antibodies () );VHHs; Microantibodies; Ankylosing spondylogenetics or DART; TCR-like antibody; Microbial proteins; as well as In some embodiments, an antibody agent can lack covalent modifications (e.g., attached glycan) that it would have if naturally produced. In some embodiments, an antibody agent can contain covalent modifications (e.g., attached glycan, payload [e.g., detectable moiety, therapeutic moiety, 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 includes one or more structural elements identified by those skilled in the art as complementarity determining regions (CDRs); in some embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) that is substantially identical to a CDR found in a reference antibody. In some embodiments, an included CDR is substantially identical to a reference CDR in that it is identical in sequence or contains between 1 and 5 amino acid substitutions compared to the reference CDR. In some embodiments, an included CDR is substantially identical to a reference CDR in that it exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the reference CDR. In some embodiments, an included CDR is substantially identical to a reference CDR in that it exhibits at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity to the reference CDR. In some embodiments, an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is deleted, added, or substituted compared to the reference CDR, but the included CDR has an amino acid sequence that is otherwise identical to the amino acid sequence of the reference CDR. In some embodiments, an included CDR is substantially identical to a reference CDR in that between 1 and 5 amino acids within the included CDR are deleted, added, or substituted compared to the reference CDR, but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments, an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is substituted compared to the reference CDR, but the included CDR has an amino acid sequence that is otherwise identical to the amino acid sequence of the reference CDR. In some embodiments, an included CDR is substantially identical to a reference CDR in that between 1 and 5 amino acids within the included CDR are deleted, added, or substituted compared to the reference CDR, but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes structural elements identified by those skilled in the art as immunoglobulin variable domain. In some embodiments, an antibody agent is a polypeptide protein whose binding domain 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).

[0044] Antigen: As used herein, the term "antigen" refers to an agent that binds to an antibody agent. In some embodiments, an antigen binds to an antibody agent and can or can not induce a particular physiological response in an organism. Generally, an antigen can be or include any chemical entity, such as a small molecule, a nucleic acid, a polypeptide, a carbohydrate, a lipid, a polymer (including biological polymers [e.g., nucleic acid and / or amino acid polymers] and polymers other than biological polymers [e.g., polymers other than nucleic acid or amino acid polymers]), and the like. In some embodiments, an antigen is or comprises a polypeptide. In some embodiments, an antigen is or comprises a glycan. Those skilled in the art will appreciate that, generally, an antigen can be provided or used in isolated or pure form, or can be provided in crude material form (e.g., together with other materials, such as in a cell extract or other relatively crude preparation of a source containing the antigen). In certain embodiments, an antigen is present in a cellular environment (e.g., the antigen is expressed on the surface of a cell or is expressed in a cell). In some embodiments, an antigen is a recombinant antigen.

[0045] Antigen binding domain: As used herein, refers to an antibody agent or portion thereof that specifically binds to a target moiety or entity. Typically, the interaction between an antigen binding domain and its target is non-covalent. In some embodiments, a target moiety or entity can be of any chemical class, including, for example, a carbohydrate, a lipid, a nucleic acid, a metal, a polypeptide, or a small molecule. In some embodiments, an antigen binding domain can be or comprise a polypeptide (or complex thereof). In some embodiments, an antigen binding domain is a portion of a fusion polypeptide. In some embodiments, an antigen binding domain is a portion of a chimeric antigen receptor (CAR).

[0046] Associated: Two events or entities are “associated” with each other if the presence, extent, and / or form of one event or entity is related to the other event or entity, as this term is used herein. For example, a particular entity (e.g., a polypeptide, a genetic signature, a metabolite, a microbe, etc.) is considered to be associated with a particular disease, disorder, or condition if the presence, level, and / or form of the particular entity is correlated with the incidence of and / or susceptibility to the particular disease, disorder, or condition (e.g., within a relevant population). In some embodiments, two or more entities are physically “associated” with each other if they directly or indirectly interact with each other such that they have and maintain physical proximity to each other. In some embodiments, two or more entities that are physically associated with each other are covalently linked to each other; in some embodiments, two or more entities that are physically associated with each other are not covalently linked to each other but are non-covalently associated, e.g., through hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetic interactions, and combinations thereof.

[0047] Binding: It should be appreciated that, as used herein, the term “binding” generally refers to non-covalent association between two or more entities. “Direct” binding involves physical contact between entities or moieties; indirect binding involves physical interaction by way of physical contact with one or more intermediate entities. Binding between two or more entities can generally be assessed under any of a variety of circumstances, including studying the interacting entities or moieties in isolation or in more complex systems (e.g., when associated covalently or otherwise with carrier entities and / or in biological systems or cells).

[0048] 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, such that they exhibit an abnormal growth phenotype characterized by a significant loss of control of cell proliferation. In some embodiments, a tumor can be or comprise cells that are pre-cancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic. The present disclosure specifically identifies certain cancers to which the teachings thereof can be particularly relevant. In some embodiments, a relevant cancer can be characterized by a solid tumor. In some embodiments, a relevant cancer can be characterized by a hematological tumor. In general, examples of different types of cancers known in the art include, for example, hematopoietic cancers, including leukemias, lymphomas (Hodgkin’s lymphoma and non-Hodgkin’s lymphoma), myelomas, and myeloproliferative disorders; sarcomas, melanomas, adenomas, solid tissue carcinomas, squamous cell carcinomas of the mouth, throat, larynx, and lung, liver cancer, genitourinary tract cancers such as prostate cancer, cervical cancer, bladder cancer, uterine cancer, and endometrial cancer, and renal cell carcinoma, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, endocrine system cancer, thyroid cancer, parathyroid cancer, head and neck cancer, breast cancer, gastrointestinal cancer, and nervous system cancer, benign lesions such as papillomas, and the like.

[0049] 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 variable regions of the heavy and light chains, which are designated CDR1, CDR2, and CDR3 for each of the variable regions. A “set of CDRs” or “CDR set” refers to a set of three or six CDRs that occur in a single variable region that is capable of binding an antigen or in the CDRs of a cognate heavy and light chain variable region that is capable of binding an antigen. Certain systems for defining CDR boundaries have been established in the art (e.g., Kabat, Chothia, etc.); one of skill in the art understands the differences between these systems and is capable of understanding CDR boundaries to the extent required to understand and practice the claimed application.

[0050] Chemotherapeutic agent: As used herein, the term "chemotherapeutic agent" has its art- understood meaning and refers to one or more pro-apoptotic, cytostatic, and / or cytotoxic agents, including, for example, specifically agents used and / or recommended for the treatment of one or more diseases, disorders, or conditions associated with undesired cellular proliferation. In many embodiments, the chemotherapeutic agent can be used to treat cancer. In some embodiments, the chemotherapeutic agent can be or comprise one or more alkylating agents, one or more anthracycline antibiotics, one or more cytoskeletal disruptors (e.g., microtubule-targeting agents such as taxanes, maytansines, and analogs thereof), one or more epothilones, one or more histone deacetylase inhibitors (HDACs), one or more topoisomerase inhibitors (e.g., inhibitors of topoisomerase I and / or topoisomerase II), one or more kinase inhibitors, one or more nucleotide analogs or nucleotide precursor analogs, one or more peptide antibiotics, one or more platinum-based agents, one or more retinoids, one or more vinca alkaloids, and / or one or more analogs of the following (i.e., having related anti-proliferative activity).In some particular embodiments, the chemotherapeutic agent can be or comprise one or more of: Actinomycin, All-trans retinoic acid, Auiristatin, Azacitidine, Azathioprine, Bleomycin, Bortezomib, Carboplatin, Capecitabine, Cisplatin, Chlorambucil, Cyclophosphamide, Curcumin, Cytarabine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Epothilone, Etoposide, Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin, Imatinib, Irinotecan, Maytansine and / or analogs thereof (e.g. DM1), Mechlorethamine, Mercaptopurine, Methotrexate, Mitoxantrone, Maytansinoid, Oxaliplatin, Paclitaxel, Pemetrexed, Teniposide, Tioguanine, Topotecan, Valrubicin, Vinblastine, Vincristine, Vindesine, Vinorelbine, and combinations thereof. In some embodiments, the chemotherapeutic agent can be used in the context of an antibody-drug conjugate.In some embodiments, the chemotherapeutic agent is one found in an antibody-drug conjugate selected from the group consisting of D10-doxorubicin (hLLl-doxorubicin), hRS7-SN-38, hMN-14-SN-38, hLL2-SN-38, hA20-SN-38, hPAM4-SN-38, hLLl-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, hLLl-Pro-2-P-Dox, P4 / D10-doxorubicin, gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, glembatumomab 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, vorsetuzumab mafodotin, and lorvotuzumab mertansine.

[0051] Engineered: In general, the term“engineered” generally refers to aspects that have been manipulated by the hand of man. For example, when a polypeptide sequence has been artificially manipulated, the polypeptide is considered to be“engineered.” For example, in some embodiments of the application, an engineered polypeptide comprises a sequence that includes one or more amino acid mutations, deletions, and / or insertions that have been artificially introduced into a reference polypeptide sequence. In some embodiments, an engineered polypeptide includes a polypeptide that has been artificially fused (i.e., covalently linked) to one or more additional polypeptides to form a fusion polypeptide that does not naturally occur in vivo. Similarly, a cell or organism is considered to be“engineered” if it has been manipulated such that its genetic information is altered (e.g., new genetic material is introduced that was not previously present, such as by transformation, transfection, somatic hybridization, transfection, transduction, or other mechanism, or previously existing genetic material is altered or removed, such as by substitution or deletion mutation, or by a transfection protocol). As is conventional practice and understood by those skilled in the art, derivatives and / or progeny of an engineered polypeptide or cell are generally still referred to as“engineered,” even if the actual manipulation was performed on the original entity.

[0052] In vitro: As used herein, the term“in vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in a cell culture, etc., and not within a multicellular organism.

[0053] In vivo: As used herein, refers to events that occur within a multicellular organism such as a human and non-human animal. In the context of cell-based systems, the term can be used to refer to events that occur within a living cell (as opposed to, e.g., an in vitro system).

[0054] Isolated: As used herein, “isolated” refers to substances and / or entities that (1) have been separated from at least some of the components with which they were associated when initially produced, whether in nature or in an experimental setting; and / or (2) have been designed, produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities can be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they 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, a substance can still be considered “isolated” or even “pure” after being combined with certain other components, such as one or more carriers or excipients (e.g., buffers, solvents, water, etc.), as will be understood by those skilled in the art; in such embodiments, the percentage of isolation or purity of the substance is calculated without including such carriers or excipients. By way of example only, a naturally-occurring biopolymer such as a polypeptide or polynucleotide is considered “isolated” in some embodiments when a) it is not associated with some or all of the components that accompany it in its native state due to its source or derivation; b) it is substantially free of other polypeptides or nucleic acids of the same species as the species from which it is produced in nature; c) it is expressed or otherwise associated by components of a cell or other expression system other than the species from which it is produced in nature. Thus, for example, a polypeptide that is chemically synthesized or synthesized in a cellular system different from that in which the polypeptide is produced in nature is considered an “isolated” polypeptide in some embodiments. Alternatively or additionally, a polypeptide that has been subjected to one or more purification techniques can be considered an “isolated” polypeptide in some embodiments to the extent that a) it has been separated from other components with which it is associated in nature; and / or b) it has been separated from other components with which it is associated when initially produced.

[0055] Operably linked: As used herein, refers to a juxtaposition wherein the components are in a relationship permitting them to function in their intended manner. A control element that is “operably linked” with a functional element is associated in such a way as to effect expression and / or activity of the functional element under conditions compatible with the control element. In some embodiments, a control element that is “operably linked” is adjacent (e.g., covalently linked) to the coding element of interest; in some embodiments, the control element acts in trans or otherwise on the functional element of interest.

[0056] Pharmaceutical composition: As used herein, the term “pharmaceutical composition” refers to a composition formulated to allow the active agent to be administered to a human or animal subject. In some embodiments, the composition is suitable for administration to a human or animal subject. In some embodiments, the active agent is present in a unit dosage amount suitable for administration in a treatment regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.

[0057] Polypeptide: As used herein, the term “polypeptide” generally has the art-recognized meaning of a polymer of at least 3 amino acids. One of ordinary skill in the art will appreciate that the term “polypeptide” is intended to be generic enough to encompass polypeptides having the complete sequences recited herein, while also encompassing polypeptides that represent functional fragments of such complete polypeptides (e.g., fragments that retain at least one activity). Furthermore, one of ordinary skill in the art will appreciate that protein sequences can often be subjected to some substitutions without destroying activity. Thus, the related term “polypeptide” as used herein encompasses polypeptides that retain activity and share at least about 30% to 40% overall sequence identity, often greater than about 50%, 60%, 70%, or 80%, with another polypeptide of the same class, and often further include at least one region of higher identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%, of one or more highly conserved regions, often covering at least 3 to 4 and often up to 20 or more amino acids. The polypeptides can contain L-amino acids, D-amino acids, or both, and can contain any of a variety of amino acid modifications or analogs known in the art. Suitable modifications include, for example, terminal acetylation, amidation, methylation, etc. In some embodiments, the proteins can include natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof. The term “peptide” is often used to refer to polypeptides of less than about 100 amino acids in length, less than about 50 amino acids in length, less than about 20 amino acids in length, or less than about 10 amino acids in length. In some embodiments, the proteins are antibody agents, antibody fragments, biologically active portions thereof, and / or characteristic portions thereof.

[0058] Prevent or prevention: As used herein, when used in connection with the occurrence of a disease, disorder, and / or condition, means to reduce the risk of developing the disease, disorder, and / or condition and / or to delay the onset and / or severity of one or more features or symptoms of the disease, disorder, or condition. In some embodiments, prevention is so assessed on a population basis, with a pharmaceutical agent being deemed to “prevent” a particular disease, disorder, or condition if there is a statistically significant reduction in the development, frequency, and / or severity of one or more symptoms of the disease, disorder, or condition observed in a population susceptible to the disease, disorder, or condition.

[0059] Recombinant: As used herein, is intended to refer to polypeptides that are designed, engineered, prepared, expressed, produced, manufactured, and / or isolated by recombinant means, such as polypeptides expressed using a recombinant expression vector transfected into a host cell; polypeptides isolated from a recombinant combinatorial human polypeptide library; polypeptides isolated from an animal (e.g., mouse, rabbit, sheep, fish, etc.) that is transgenic or has otherwise been manipulated to express one or more genes or gene components that encode and / or direct expression of a polypeptide or one or more components, portions, elements, or domains thereof; and / or polypeptides prepared, expressed, produced, or isolated by any other method that involves splicing or ligating one or more selected nucleic acid sequence elements to one another, chemically synthesizing one or more selected sequence elements, and / or otherwise producing a nucleic acid that encodes and / or directs expression of a 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 by computer simulation. In some embodiments, one or more of such selected sequence elements are generated by mutagenesis (e.g., in vivo or in vitro) of known sequence elements, such as from a natural or synthetic source, e.g., in the germline of a source organism (e.g., human, mouse, etc.).

[0060] Specific binding: As used herein, the term "specific binding" refers to the ability to discriminate among possible binding partners in the context in which binding occurs. A binding agent that interacts with one particular target is said to "specifically bind" to that target when other potential targets are present. In some embodiments, specific binding is assessed by detecting or determining the extent of binding between a binding agent and its partner; in some embodiments, specific binding is assessed by detecting or determining the extent of dissociation of a binding agent-partner complex; in some embodiments, specific binding is assessed by detecting or determining the ability of a binding agent to compete for alternative interactions between its partner and another entity. In some embodiments, specific binding is assessed by performing such detection or determination over a range of concentrations.

[0061] Subject: As used herein, the term“subject” refers to an organism, typically a mammal (e.g., a human, including in some embodiments a prenatal human form). In some embodiments, a subject has a relevant disease, disorder, or condition. In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a subject exhibits one or more symptoms or characteristics of a disease, disorder, or condition. In some embodiments, a subject does not exhibit any symptoms or characteristics of a disease, disorder, or condition. In some embodiments, a subject refers to a human having one or more characteristics that are indicative of being susceptible to or at risk of having a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom a diagnosis and / or treatment is being made and / or has been made.

[0062] Therapeutic agent: As used herein, the phrase“therapeutic agent” generally refers to any agent that, when administered to an organism, elicits a desired pharmacological effect. In some embodiments, an agent is considered a therapeutic agent if it shows a statistically significant effect in an appropriate population. In some embodiments, an appropriate population can be a population of model organisms. In some embodiments, an appropriate population can be defined using various criteria, such as a particular age group, gender, genetic background, pre-existing clinical conditions, etc. In some embodiments, a therapeutic agent is any substance that can be used to relieve, alleviate, ameliorate, inhibit, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition. In some embodiments, a“therapeutic agent” is an agent that has been or needs to be approved by a governmental agency to be sold for administration to humans. In some embodiments, a“therapeutic agent” is an agent that requires a medical prescription to be administered to humans.

[0063] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" means an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder, and / or condition, in accordance with a therapeutic dosing regimen, to treat the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is an amount that reduces the incidence and / or severity of one or more symptoms of a disease, disorder, and / or condition, stabilizes one or more properties of a disease, disorder, and / or condition, and / or delays onset of one or more symptoms of a disease, disorder, and / or condition. It will be appreciated by one of ordinary skill in the art that the term "therapeutically effective amount" does not in fact require successful treatment in a particular individual. Rather, a therapeutically effective amount can be that which provides a particular, desired pharmacological response in a substantial number of subjects in need of such treatment when administered to a patient in need of such treatment. For example, in some embodiments, the term "therapeutically effective amount" refers to an amount that, when administered in the context of a therapy of the present application to an individual in need thereof, will block, stabilize, attenuate, or reverse a cancer-supporting process occurring in the individual or will enhance or increase a cancer-inhibiting process in the individual. In the context of cancer treatment, a "therapeutically effective amount" is an amount that, when administered to an individual diagnosed with cancer, will prevent, stabilize, inhibit, or reduce further progression of the individual's cancer. A particularly preferred "therapeutically effective amount" of a composition described herein reverses (in a therapeutic treatment) the development of a malignant tumor, such as a pancreatic cancer, or helps to achieve or prolong remission of a malignant tumor. A therapeutically effective amount administered to an individual to treat the individual's cancer can be the same as or different from a therapeutically effective amount administered to promote remission or inhibit metastasis. As with most cancer therapies, the treatment methods described herein are not to be construed as, limited to, or otherwise bound by "cure" of cancer; rather, the treatment methods involve the use of the described compositions to "treat" cancer, i.e., to achieve a desired or beneficial health change in an individual with cancer. Such benefits have been recognized by skilled healthcare providers in the field of oncology and include, but are not limited to, stabilization of patient condition, reduction in tumor size (tumor regression), improvement in life function (e.g., improvement in function of cancerous tissue or organ), reduction or inhibition of further metastasis, reduction in opportunistic infection, increased survivability, reduction in pain, improvement in motor function, improvement in cognitive function, improvement in energy feeling (vitality, reduction in malaise), improvement in health feeling, 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) can also be assessed at a molecular level by taking a sample of cancer cells from a site of a tumor, such as a pancreatic cancer (e.g., during a course of treatment) and testing the cancer cells for metabolic levels and signal markers of the cancer cells to monitor the status of the cancer cells, thereby verifying cancer cell regression to a less malignant phenotype.For example, an increase in an anti-angiogenic marker described herein, a decrease in any pro-angiogenic marker discussed above, a normalization of a metabolic pathway, an intercellular signaling pathway, or an intracellular signaling pathway that exhibits aberrant activity in a subject diagnosed with cancer (i.e., a change toward a state found in a normal subject not suffering from cancer), indicates that tumor regression is induced by use of the methods of the application. One of ordinary skill in the art will appreciate that, in some embodiments, a therapeutically effective amount can be formulated and / or administered in a single dose. In some embodiments, a therapeutically effective amount can be formulated and / or administered in multiple doses, for example as part of a dosing regimen.

[0064] Variant: As used herein, in the context of a molecule (e.g., a nucleic acid, a protein, or a small molecule), the term “variant” refers to a molecule that exhibits substantial structural identity to, but is structurally different from, a reference molecule, e.g., differs in the presence or absence of, or at the level of, one or more chemical moieties, as compared to the reference entity. In some embodiments, a variant is also functionally different from its reference molecule. In general, whether a particular molecule is properly considered a “variant” of a reference molecule depends on the degree of structural identity it shares with the reference molecule. As understood by one of skill in the art, any biological or chemical reference molecule has certain characteristic structural elements. By definition, a variant is a different molecule that shares one or more such characteristic structural elements, but differs from the reference molecule in at least one respect. To name just a few examples, a polypeptide can have a characteristic sequence element consisting of a plurality of amino acids that have specified positions relative to one another, and / or that constitute a particular structural motif and / or biological function, in linear or three-dimensional space; a nucleic acid can have a characteristic sequence element comprising a plurality of nucleotide residues that have specified positions relative to one another, in linear or three-dimensional space. In some embodiments, a variant polypeptide or nucleic acid can differ from a reference polypeptide or nucleic acid by 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 to 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.

[0065] 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, wherein 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 to which they are operatively linked. Such vectors are referred to herein as "expression vectors". Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures can be optionally employed in accordance with conventional methods in the art and as described in various general and more specific references which are cited herein and throughout the specification. See e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), which is incorporated herein by reference for any purpose.

[0066] Engineered immune cells

[0067] As used herein, "immune cell" refers to a cell of the immune system, which can be classified as a lymphocyte (e.g., T cell, B cell, and NK cell), neutrophil, and monocyte / macrophage. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is an NK cell. In some embodiments, the immune cell is an engineered immune cell, meaning that the immune cell has been genetically modified to express a non-naturally occurring protein (e.g., a chimeric antigen receptor) or to include an exogenous nucleic acid.

[0068] Immune cells (e.g., T cells) can be modified in one or more ways. Immune cells (e.g., T cells) can express at least one non-native molecule that is a receptor for an antigen present on the surface of one or more types of cells. In some embodiments, immune cells comprise immune cells (e.g., T cells) that are not found in nature as they are engineered to comprise or express at least one synthetic molecule that is not found in nature. In particular embodiments, immune cells (e.g., T cells) are engineered to express at least one chimeric antigen receptor (CAR), including CARs that target specific tumor antigens, such as glypican 3 (GPC3). In particular embodiments, immune cells can be T cells, e.g., CD4+ T cells, CD8+ T cells, Treg cells, Thl T cells, Th2 T cells, Thl 7 T cells, non-specific T cells, or a T cell population comprising a combination of any of the foregoing. Immune cells (e.g., T cells) engineered with chimeric antigen receptors (CAR T cells) have tremendous therapeutic potential for treating cancer. For CARs, the receptor can be programmed to recognize an antigen that, when bound, activates the immune cell 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 CD 19-targeted CAR-transduced T cells (CD 19-CAR T cells) against hematologic malignancies have shown the powerful effects of CAR T technology (Kochenderfer, J. N. et al. (2010) Blood 116:4099-4102; Porter, D. L. et al. (2011) N. Engl. J. Med. 365:725-733; Grupp, S. A. et al. (2013) N. Engl. J. Med. 368:1509-1518; Kochenderfer, J. N. et al. (2015) J. Clin. Oncol. 33:540-549; Brown, C. E. et al. (2016) N. Engl. J. Med. 375:2561-2569). The clinical success of CAR T is attributed, at least in part, to the fusion structure of the CAR, which is made by artificially combining a high-affinity antigen-binding domain with multiple signaling domains (Maus, M. V. et al. (2014) Blood 123:2625-2635; van der Stegen, S. J. et al. (2015) Nat. Rev. Drug Discov. 14:499-509).

[0069] 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 employs a transmembrane domain from a molecule such as CD8 and CD28, and the intracellular signaling domain employs an immunoreceptor tyrosine-based activation motif (e.g., intracellular signaling domains of CD3 zeta and costimulatory signaling molecules (e.g., CD28, CD137, and CD137 (4-1BB)).

[0070] As used herein, "single chain variable fragment, scFv" refers to an antibody fragment defined as a recombinant protein comprising a heavy chain variable domain (VH) and a light chain variable domain (VL) joined by a linker that binds the two domains together to form an antigen binding site.

[0071] In some embodiments, the transmembrane domain is a transmembrane domain from a protein selected from 4-1BB / CD137, an activating NK cell receptor, an immunoglobulin, 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, a cytokine receptor, DAP-10, DNAM1 (CD226), Fcgamma 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), integrins, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, a ligand that specifically binds with CD83, LIGHT, LTBR, Ly9 (CD229), Lymphocyte function-associated antigen-1 (LFA-1), MHC class I 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, a TNF receptor protein, TNFR2, TNFSF14, a Toll ligand receptor, TRANCE / RANKL, VLA1, and VLA-6.

[0072] In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain from a protein selected from the group consisting of 4-1BB / CD137, Activating NK Cell Receptor, Immunoglobulin, 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), Fcgamma Receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, Inducible T-Cell Costimulator (ICOS), Integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, Ligand that Specifically Binds CD83, LIGHT, LTBR, Ly9 (CD229), Lyl08, Lymphocyte Function-Associated Antigen-1 (LFA-1), MHC Class I molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, Programmed Death-1 (PD-1), PSGL1, SELPLG (CD 162), 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.

[0073] 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.

[0074] The immune cells (e.g., T cells) can be from any source known in the art. For example, the immune (e.g., T) cells can be differentiated in vitro from a population of hematopoietic stem cells, or the immune (e.g., T) cells can be obtained from a subject. The T cells can be obtained from peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, or a tumor. In addition, the immune (e.g., T) cells can be derived from one or more immune cell lines available in the art. In some embodiments, a number of techniques known to those of skill in the art can be used, such as FICOLL TM Isolation and / or apheresis obtains T cells from blood collected from a subject. Other methods of 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 WO 2015 / 120096) and International Application No. PCT / US2016 / 057983 (published as WO 2017 / 070395), each of which is incorporated herein by reference in its entirety.

[0075] In some embodiments, the immune cells are autologous T cells. In some embodiments, the immune cells are obtained from a subject that is not the patient. In some embodiments, the T cells used in the method of treatment are syngeneic (the donor and recipient are different but identical twins). In some embodiments, the T cells used in the method of treatment are allogeneic (from the same species but different donors) to the recipient subject. In some embodiments, the T cells are autologous stem cells (used in autologous stem cell therapy or 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 having a cancer or tumor.

[0076] The T cells can be engineered to express, for example, a chimeric antigen receptor (CAR). In some embodiments, the CAR-T cells can be engineered to express an extracellular single chain variable fragment (scFv). In some embodiments, the CAR is engineered such that the costimulatory domain is expressed 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 herein by reference in its entirety.

[0077] GPC3

[0078] Glypican 3 (GPC3) is a cell surface protein in humans encoded by the GPC3 gene and is a carcinoembryonic antigen that is re-expressed in tumor hepatocytes at high frequency. GPC3 is highly expressed in fetal liver, but not in normal adult liver tissue, but its expression is reactivated in hepatocellular carcinoma and is closely related to the development of liver cancer, in which the detection rate of GPC3 expression is relatively high in the early stage of liver cancer and increases as liver cancer develops. In addition, GPC3 is also expressed in tumors such as melanoma, ovarian clear cell carcinoma, yolk sac tumor, neuroblastoma, and other tumors. Given its high expression specificity in hepatocellular carcinoma, melanoma, and other tumors, GPC3 has become a useful immunohistochemical diagnostic test and a potential biomarker.

[0079] GPC3 is a member of the glypican family of proteoglycans that function in cell adhesion during organ formation either as extracellular matrix or as receptors for cell growth factors. The protein core of GPC3 comprises two subunits, as well as an N-terminal subunit and a C-terminal subunit. A glycosyl phosphatidylinositol (GPI) anchor is added to the serine at position 560 located on the carboxyl (C)-terminal side of GPC3. The GPI anchor plays a role in localizing GPC3 on the cell surface by covalently binding to cell membrane lipids. In addition, the serine at position 495 and the serine at position 509 of GPC3 are modified by heparan sulfate chains (HS chains), where HS chains are known to modulate various growth signaling pathways, such as Wnt signaling, FGF signaling, and BMP signaling transduction pathways. The growth signaling pathways involved are known to differ between cancer types. For example, in hepatocellular carcinoma (HCC), cells grow through stimulation of the Wnt signaling pathway.

[0080] GPC3 CAR

[0081] The present disclosure provides, at least in part, GPC3 CAR polypeptides. As used herein, “chimeric antigen receptor (CAR)” refers to a receptor that does not exist in nature and is capable of providing an immune effector cell with specificity for a particular antigen. In some embodiments, CAR refers to a receptor used to deliver the specificity of a monoclonal antibody agent to a T cell. Typically, 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.

[0082] 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 that results in T cell activation when the antigen binding antibody agent is bound.

[0083] [Table 1]

[0084] SEQ ID NO: Sequence 1 Light chain CDR1 RSSQSLVHSNGNTYLH 2 Light chain CDR2 KVSNRFS 3 Light chain CDR3 SQNTHVPPT 4 Heavy chain CDR1 DYEMH 5 Heavy chain CDR2 ALDPKTGDTAYSQKFKG 6 Heavy chain CDR3 FYSYTY

[0085] In some embodiments, the CAR polypeptide comprises: i) an extracellular antigen binding domain comprising a light chain variable domain 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: 10, and a heavy chain variable domain 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: 8; ii) a transmembrane domain; and iii) an intracellular signaling domain that results in T cell activation when the antigen binding antibody agent is bound.

[0086] 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 that results in T cell activation when the antigen binding antibody agent is bound.

[0087] [Table 2]

[0088]

[0089]

[0090] Nucleic acid

[0091] As used herein, “nucleic acid” is used to include any compound and / or substance comprising a polymer of nucleotides. In some embodiments, the polymer of nucleotides is referred to as a polynucleotide. Exemplary nucleic acids or polynucleotides can include, but are not limited to, ribonucleic acids (RNAs) and / or deoxyribonucleic acids (DNAs).

[0092] In some embodiments, the nucleic acid construct comprises a region encoding a GPC3 CAR. 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 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 that results in T cell activation when the antigen binding antibody agent is bound.

[0093] 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 that results in T cell activation when the antigen binding antibody agent is bound.

[0094] In some embodiments, the nucleic acid construct can be inserted into an expression vector or viral vector by methods known in the art, and the nucleic acid molecule can be operably linked to an expression control sequence. Non-limiting examples of expression vectors include plasmid vectors, transposon vectors, cosmid vectors, and viral vectors (e.g., any adenoviral vector (AV), cytomegalovirus (CMV) vector, simian virus (SV40) vector, adeno-associated virus (AAV) vector, lentiviral vector, and retroviral vector). In some embodiments, the expression vector is a viral vector. In some embodiments, the viral vector is a lentiviral vector.

[0095] Lentiviral vectors are derived from lentiviruses. Lentiviral vectors are based on single-stranded RNA lentiviruses, which are a subclass of retroviruses. They combine the advantages of moderate cloning capacity and stable gene expression, where they are able to transduce both dividing and non-dividing cells, including neurons. Upon infection, the lentiviral genome integrates the transgene into the host genome and facilitates long-term gene expression. Lentiviral vectors, such as HIV-based vectors, are examples of retroviral vectors for gene delivery. Unlike other retroviruses, HIV-based vectors are known to integrate their passenger genes into non-dividing cells, and thus are useful for treating persistent forms of disease.

[0096] Additional sequences can be added to such cloning and / or expression sequences to optimize their function in cloning and / or expression, to aid in isolation of the polynucleotide, or to improve the introduction of the polynucleotide into a cell. The use of cloning vectors, expression vectors, adaptors, and linkers are well known in the art.

[0097] In some embodiments, the nucleic acid molecule is inserted into a vector that is capable of expressing the GPC3 CAR of the disclosure when introduced into a suitable cell. In some embodiments, the suitable cell is a T cell.

[0098] Generation of GPC3 CAR-T cells

[0099] Provided herein are methods for generating an immune cell comprising a GPC3 CAR. In some embodiments, the immune cell into which the CAR is introduced is a human immune cell. In some embodiments, the immune cell is an autologous human immune cell. In some embodiments, the immune cell is an allogeneic human immune cell. In some embodiments, the immune cell is a CD4 + T cell (helper T cell, T H cell), a CD8 + T cell (cytotoxic T cell, CTL), a memory T cell, a regulatory T cell (Treg cell), an apoptotic T cell, but is not limited thereto. In some embodiments, the immune cell is an NK cell.

[0100] In some embodiments, the disclosure provides a method for generating an engineered immune cell comprising: (i) introducing into an immune cell a nucleic acid encoding a GPC3 CAR comprising a GPC3 antigen binding domain, or (ii) introducing into an immune cell a vector comprising a nucleic acid encoding a GPC3 CAR comprising a GPC3 antigen binding domain. In some embodiments, a method for generating an engineered immune cell of the disclosure further comprises culturing the engineered immune cell in vitro for at least 5 days, 7 days, 9 days, 10 days, 11 days, or 12 days.

[0101] In some embodiments, the disclosure provides a method for making an autologous engineered immune cell of the disclosure comprising: providing or obtaining an analysis of the binding of a GPC3 antigen binding domain to an immune cell from a subject; and if the binding is below a threshold, engineering an immune cell from the subject to express a CAR comprising the GPC3 antigen binding domain. In some embodiments, a method for generating an autologous engineered immune cell of the disclosure further comprises culturing the autologous engineered immune cell in vitro for at least 5 days, 7 days, 9 days, 10 days, 11 days, or 12 days.

[0102] Any method known in the art for expressing a CAR in an immune cell can be used in the context of the present disclosure. For example, there are various nucleic acid vectors known in the art for expression, such as linear polynucleotides, polynucleotides bound to ions or amphiphilic compounds, plasmids, or viral vectors, although the present disclosure is not limited thereto. In some embodiments, the vector for expressing a CAR in an immune cell can be or include an autonomously replicating plasmid or virus or derivative thereof. Viral vectors can include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like. In some embodiments, a lentiviral vector, which is a retroviral vector, can be used. In some embodiments, the vector is a non-plasmid non-viral compound, such as a liposome.

[0103] The present disclosure includes the recognition that GPC3 CAR-T cells produced by the methods described herein can be useful therapeutically (e.g., for treating cancer).

[0104] Therapeutic applications

[0105] Provided herein are methods for treating a subject having a glypican 3-associated cancer, wherein the method comprises administering to the subject a composition comprising or delivering a composition comprising immune cells comprising a GPC3 CAR.

[0106] A “glypican 3-associated cancer” is a cancer characterized by cancer cells that have glypican 3 present on their surface. GPC3, a membrane-bound heparan sulfate proteoglycan, is overexpressed in about 70-80% of hepatocellular carcinomas, but is generally not expressed in healthy tissue. Furthermore, GPC3 overexpression is found in several tumors, most notably in hepatocellular carcinoma, hepatoblastoma, germ cell tumors (e.g., yolk sac tumor, choriocarcinoma), Wilms tumor, gastric cancer, non-small cell lung cancer, and thyroid cancer.

[0107] Cancer can refer to a large class of diseases characterized by uncontrolled cell growth in the body. Unregulated cell division and growth results in the formation of malignant tumors, which invade neighboring tissues and can also metastasize to distant parts of the body through the lymphatic system or bloodstream. Cancer or cancer tissue can include a tumor.

[0108] Cancers suitable for treatment by the methods of the present disclosure can include, but are not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gall bladder cancer, gastrointestinal cancer, head and neck cancer, hematological cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, stomach cancer, thyroid cancer, pancreatic cancer, and prostate cancer. In some embodiments, cancers for treatment by the methods of the present disclosure can 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 can include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon carcinoma, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, leukemia and other lymphatic cancers, and various types of head and neck cancers.

[0109] In some embodiments, the cancer can be an embryonal tumor (nephroblastoma, hepatoblastoma, rhabdoid tumor, neuroblastoma), germ cell tumor (yolk sac tumor, immature teratoma, and embryonal carcinoma), carcinoma (hepatocellular carcinoma and lung squamous cell carcinoma), sarcoma (malignant rhabdoid tumor and RMS), or malignant melanoma. In some embodiments, the phosphatidylinositol glycan 3-associated cancer is liver cancer.

[0110] The immune cells (e.g., CAR-T cells) can be administered to a patient in need thereof in a therapeutically effective amount. For example, the therapeutically effective amount of immune cells (e.g., CAR-T cells) can be at least about 10 4 cells, at least about 10 5 cells, at least about 10 6 cells, at least about 10 7 cells, at least about 10 8 cells, at least about 10 9 cells, or at least about 10 10 cells. In some embodiments, the therapeutically effective amount of T cells is about 10 4 cells, about 10 5 cells, about 10 6 cells, about 10 7 cells, about 10 8 cells, about 10 9 cells, or about 10 10 cells. In some embodiments, the therapeutically effective amount of T cells is between about 0.1 x 10 6 and about 2 x 10 10 cells (e.g., about 0.1 x 10 6 and about 2 x 1010 about 0.2 x 10 6至 about 0.4 x 10 10 about 0.6 x 10 6 about 0.8 x 10 10 about 1.0 x 10 6 about 2 x 10 10 about 3 x 10 6 about 4 x 10 10 about 5 x 10 6 about 6 x 10 10 about 7 x 10 6 about 8 x 10 10 about 9 x 10 6 about 1.0 x 10 10 about 2 x 10 6 about 3 x 10 10 about 4 x 10 6 about 5 x 10 10 about 6 x 10 6 about 7 x 10 10 about 8 x 10 6 about 9 x 10 10 about 1.0 x 10 6 about 2 x 10 10 about 3 x 10 6 about 4 x 10 10 about 5 x 10 7 about 6 x 10 10 about 7 x 10 7至 about 8 x 10 10 about 9 x 10 7 about 1.0 x 10 10 about 2 x 10 7 about 3 x 10 10 about 4 x 10 7 about 5 x 10 10 about 6 x 10 7 about 7 x 10 10 about 8 x 10 7 about 9 x 10 10 about 1.0 x 10 7 about 2 x 10 10 about 3 x 10 7至 about 4 x 10 10 about 5 x 10 8Approximately 2×10 10 1 T cell, approximately 2.0 × 10 8 Approximately 2×10 10 1 T cell, approximately 3.0 × 10 8 Approximately 2×10 10 4.0 × 10 T cells 8 Approximately 2×10 10 5.0 × 10 T cells 8 Approximately 2×10 10 6.0 × 10 T cells 8 Approximately 2×10 10 7.0 × 10 T cells 8至 Approximately 2×10 10 8.0 × 10 T cells 8 Approximately 2×10 10 9.0 × 10 T cells 8 Approximately 2×10 10 One T cell, approximately 1.0 × 10 9 Approximately 2×10 10 1 T cell, approximately 2.0 × 10 9 Approximately 2×10 10 1 T cell, approximately 3.0 × 10 9 Approximately 2×10 10 4.0 × 10 T cells 9 Approximately 2×10 10 5.0 × 10 T cells 9至 Approximately 2×10 10 6.0 × 10 T cells 9 Approximately 2×10 10 7.0 × 10 T cells 9 Approximately 2×10 10 8.0 × 10 T cells 9 Approximately 2×10 10 9.0 × 10 T cells 9 Approximately 2×10 10 One T cell or approximately 1.0 × 10 10 Approximately 2×10 10 10 T cells. In some embodiments, the therapeutically effective dose of T cells is approximately 0.4 × 10⁻⁶ T cells. 8 Approximately 0.5 × 10 8 Approximately 0.6 × 10 8 Approximately 0.7 × 10 8 Approximately 0.8 × 10 8 Approximately 0.9 × 10 8 Approximately 1.0 × 10 8 Approximately 1.1 × 10 8about 1.2 x 10 8 about 1.3 x 10 8 about 1.4 x 10 8 about 1.5 x 10 8 about 1.6 x 10 8 about 1.7 x 10 8 about 1.8 x 10 8 about 1.9 x 10 8 about 2.0 x 10 8 T cells.

[0111] In some embodiments, the therapeutically effective amount of CAR T cells is about 2 x 10 6 cells / kg, about 3 x 10 6 cells / kg, about 4 x 10 6 cells / kg, about 5 x 10 6 cells / kg, about 6 x 10 6 cells / kg, about 7 x 10 6 cells / kg, about 8 x 10 6 cells / kg, about 9 x 10 6 cells / kg, about 1 x 10 7 cells / kg, about 2 x 10 7 cells / kg, about 3 x 10 7 cells / kg, about 4 x 10 7 cells / kg, about 5 x 10 7 cells / kg, about 6 x 10 7 cells / kg, about 7 x 10 7 cells / kg, about 8 x 10 7 cells / kg, or about 9 x 10 7 cells / kg. In some embodiments, the therapeutically effective amount of immune cells (e.g., CAR-T cells) is about 1 x 10 6 to about 2 x 10 6 cells / kg body weight, up to a maximum dose of about 1 x 10 10 T cells. In some embodiments, the therapeutically effective amount of T cells is about 1 x 10 6 or about 2 x 10 6 cells / kg body weight, up to a maximum dose of about 1 x 10 10 T cells.

[0112] The number of cells will depend on the end use of the composition, with the cell type included also depending on the end use of the composition. For example, in some embodiments, a population of T cells comprising a GPC3 CAR will contain greater than 10%, greater than 15%, greater than 20%, greater than 25%, greater than 30%, greater than 35%, greater than 40%, greater than 45%, greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, or greater than 90% of such cells. In some embodiments, a population of T cells comprising a GPC3 CAR will contain about 10% to about 90%, about 10% to about 80%, about 10% to about 70%, about 10% to 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 50%, 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% of such T cells. In some embodiments, the volume of a population of T cells for administration is 1 liter or less. In some embodiments, the volume of T cells for administration is less than 500 ml, less than 250 ml, or 100 ml or less. In some embodiments, the density of the desired T cells is typically greater than 10 6 cells / ml, typically greater than 10 7 cells / ml, typically 10 8 cells / ml or more. A clinically relevant number of immune cells can be apportioned into multiple infusions, cumulatively equaling or exceeding 10 7 cells, 10 8 cells, 10 9 cells, 10 10 cells, 10 11 cells, or 10 12 cells.

[0113] In some embodiments, the composition can be administered parenterally to the patient. In some embodiments, the composition comprising or delivering T cells comprising a GPC3 CAR can be administered parenterally to the patient in one or more administrations. In some embodiments, the composition comprising or delivering T cells comprising a GPC3 CAR can be administered parenterally to the patient once a day, once every 2 to 7 days, once a week, once every two weeks, once a month, once every three months, or once every 6 months.

[0114] 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 GPC3 CAR. In some embodiments, the T cells comprising a 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 a GPC3 CAR. In some embodiments, the T cells comprising a nucleic acid and / or vector encoding a GPC3 CAR are autologous T cells. In some embodiments, the subject has a cancer or is at risk of developing a cancer.

[0115] 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 GPC3 CAR. In some embodiments, the T cells comprising a 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 a GPC3 CAR. In some embodiments, the T cells comprising a nucleic acid and / or vector encoding a GPC3 CAR are autologous T cells. In some embodiments, the subject has a cancer or is at risk of developing a cancer.

[0116] 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 a 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 a cancer. In some embodiments, the cancer expresses a GPC3 antigen. In some embodiments, the cancer cells have increased expression of GPC3 antigen relative to non-cancerous cells from the subject. In some embodiments, the GPC3 expression level can be increased in a subject having a cancer. In some embodiments, the GPC3 expression level is not detectable in a healthy subject.

[0117] Pharmaceutical compositions

[0118] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a T cell and a pharmaceutically acceptable carrier, the T cell comprising a GPC3 CAR. In some embodiments, the T cell comprising a GPC3 CAR is an autologous T cell. In some embodiments, the present disclosure provides a pharmaceutical composition comprising a T cell and a pharmaceutically acceptable carrier, the T cell comprising a nucleic acid and / or a vector encoding a GPC3 CAR. In some embodiments, the T cell comprising a nucleic acid and / or a vector encoding a GPC3 CAR is an autologous T cell. Compositions of the present disclosure include pharmaceutical compositions comprising T cells comprising a GPC3 CAR and / or a nucleic acid encoding a GPC3 CAR obtained by the methods disclosed herein. In some embodiments, the pharmaceutical composition can include a buffer, a diluent, a solubilizer, an emulsifier, a preservative, an adjuvant, an excipient, or any combination thereof. In some embodiments, the composition can also contain one or more additional therapeutically active substances, if desired.

[0119] In some embodiments, T cells of the present disclosure are formulated by first harvesting them from their culture medium, then washing and concentrating the cells in a medium and container system suitable for administration in a therapeutically effective amount (“pharmaceutically acceptable” carrier). Suitable infusion media can be any isotonic media preparation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), but 5% dextrose in water or lactated Ringer’s can also be used. The infusion medium can be supplemented with human serum albumin.

[0120] In some embodiments, the compositions are formulated for parenteral administration. For example, the pharmaceutical compositions provided herein can be provided in sterile injectable form (e.g., a form suitable for subcutaneous injection, hepatic arterial infusion, or intravenous infusion). For example, in some embodiments, the pharmaceutical compositions are provided in a liquid dosage form suitable for injection. In some embodiments, the pharmaceutical compositions are provided as a powder (e.g., lyophilized and / or sterilized), optionally under vacuum, which can be reconstituted with an aqueous diluent (e.g., water, a buffer, a salt solution, etc.) prior to injection. In some embodiments, the pharmaceutical compositions are diluted and / or reconstituted in water, sodium chloride solution, sodium acetate solution, benzyl alcohol solution, phosphate buffered saline, etc. In some embodiments, the powder should be gently mixed with the aqueous diluent (e.g., not shaken).

[0121] In some embodiments, T cells comprising the GPC3 CAR of this disclosure and / or nucleic acids encoding the GPC3 CAR are formulated with pharmaceutically acceptable parenteral mediators. Examples of such mediators are water, saline, Ringer's solution, glucose solution, and 1% to 10% human serum albumin. Liposomes and non-aqueous mediators such as fixed oils may also be used. The mediator 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. The pharmaceutical composition may additionally contain pharmaceutically acceptable excipients, as used herein, including any and all solvents, dispersion media, diluents or other liquid media, dispersing or suspending agents, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, etc., suitable for the desired particular dosage form. Remington's *The Science and Practice of Pharmacy*, 21st edition, ARGennaro Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006) discloses various excipients for formulating pharmaceutical compositions and known techniques for their preparation. Use of any conventional excipient medium is contemplated within the scope of this disclosure unless it is incompatible with the substance or its derivatives, for example, producing any undesirable biological effects or otherwise interacting in a harmful manner with any other component of the pharmaceutical composition.

[0122] In some embodiments, the compositions of the present disclosure comprising a population of T cells comprising a GPC3 CAR and / or a nucleic acid encoding a GPC3 CAR are stably formulated. In some embodiments, the stable formulation of the population of T cells of the present disclosure comprising a GPC3 CAR and / or a nucleic acid encoding a GPC3 CAR can comprise a phosphate buffer containing saline or a selected salt, as well as a preservative solution and formulation containing a preservative, and a multipurpose preservative formulation suitable for pharmaceutical or veterinary use. The preservative formulation contains at least one known preservative or optionally selected from the group consisting of at least one 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 thiomersal, or mixtures thereof, in an aqueous diluent. Any suitable concentration or mixture can be used as known in the art, such as 0.001-5%, or any range or value therein, such as but not limited to 0.001, 0.003, 0.005, 0.009, 0.01, 0.02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.3, 4.5, 4.6, 4.7, 4.8, 4.9, or any range or value therein. Non-limiting examples include, no preservative, 0.1% to 2% m-cresol (e.g., 0.2%, 0.3%, 0.4%, 0.5%, 0.9%, 1.0%), 0.1% to 3% benzyl alcohol (e.g., 0.5%, 0.9%, 1.1%, 1.5%, 1.9%, 2.0%, 2.5%), 0.001% to 0.5% thiomersal (e.g., 0.005% to 0.01%), 0.001% to 2.0% phenol (e.g., 0.05%, 0.25%, 0.28%, 0.5%, 0.9%, 1.0%), 0.0005% to 1.0% alkyl paraben (e.g., 0.00075%, 0.0009%, 0.001%, 0.002%, 0.005%, 0.0075%, 0.009%, 0.01%, 0.02%, 0.05%, 0.075%, 0.09%, 0.1%, 0.2%, 0.3%, 0.5%, 0.75%, 0.9%, 1.0%), and the like.

[0123] In some embodiments, the pharmaceutical composition is provided in a refrigerated and / or frozen form. In some embodiments, the pharmaceutical composition is provided in a non-refrigerated and / or non-frozen form. In some embodiments, the reconstituted solution and / or liquid dosage form can be stored for a period of time (e.g., 2 hours, 12 hours, 24 hours, 2 days, 5 days, 7 days, 10 days, 2 weeks, one month, two months, or longer) after reconstitution. In some embodiments, storage of the composition comprising the antibody agent for longer than the specified time results in degradation of the antibody agent. The liquid dosage form and / or reconstituted solution can include particulate matter and / or discoloration prior to administration. In some embodiments, the solution should not be used if it is discolored or hazy and / or if particulate matter remains after filtration. General considerations in the formulation and / or manufacture of pharmaceutical agents can be found in, e.g., Remington’s The Science and Practice of Pharmacy, 21stEdition, Lippincott Williams & Wilkins, 2005.

[0124] In some embodiments, a pharmaceutical composition comprising a T cell of the disclosure comprising a GPC3 CAR and / or a nucleic acid encoding a GPC3 CAR can be included in a container for storage or administration, e.g., a vial, a syringe (e.g., an IV syringe), or a bag (e.g., an IV bag). Pharmaceutical compositions according to the present disclosure can be prepared, packaged, and / or sold in bulk, as single unit doses, and / or as a plurality of single unit doses. As used herein, a “unit dose” is a discrete amount of the pharmaceutical composition containing a predetermined quantity of an active ingredient. The quantity of the active ingredient is generally equal to a dose, and / or a convenient fraction of a dose, such as one-half or one-third of a dose.

[0125] Kit

[0126] The present disclosure also provides kits comprising one or more containers filled with at least one GPC3 CAR and / or nucleic acid encoding a GPC3 CAR as described herein. The kits can be used in any applicable method, including, e.g., therapeutic methods, diagnostic methods, cell proliferation and / or isolation methods, etc. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects (a) approval by the agency of the manufacture, use or sale for human administration, (b) directions for use, or both.

[0127] In some embodiments, a kit can include one or more reagents for detecting (e.g., detecting a GPC3 CAR and / or a nucleic acid encoding a GPC3 CAR). In some embodiments, a kit can include a GPC3 CAR and / or a nucleic acid encoding a GPC3 CAR in a detectable form (e.g., covalently bound to a detectable moiety or entity). In some embodiments, one or more GPC3 CARs and / or nucleic acids encoding a GPC3 CAR provided herein can be included in a kit for treating a subject. In some embodiments, a GPC3 CAR and / or a nucleic acid encoding a GPC3 CAR provided herein can be included in a kit for making autologous T cells expressing a GPC3 CAR.

[0128] In some embodiments, a kit can provide one, two, three, four or more GPC3 antibody agents, each of which is suitable for cloning into a CAR construct. In some embodiments, a kit can provide other reagents for assaying the binding affinity of a GPC3 antibody agent and / or a GPC3 CAR and / or a GPC3 CAR T cell to a T cell or GPC3 identified or isolated from a subject. In some embodiments, a kit can provide other reagents for assaying the functional affinity of an antibody agent and / or a GPC3 CAR and / or a GPC3 CAR T cell to a T cell of a subject.

[0129] Examples

[0130] The present disclosure is further described in the following examples, which do not limit the scope of the disclosure described in the claims.

[0131] Example 1 - Humanization of mouse GC33 (muGC33)

[0132] muGC33 VH humanization

[0133] To generate a humanized anti-GC33 antibody (huGC33), the complementarity determining regions (CDRs) of the heavy chain variable region (VH) were grafted into a human framework using a human VH framework containing sequences analogous to the mouse anti-GPC3 antibody (muGC33). Residues VH48, VH67, VH69, VH71, VH73, VH78, and VH93 were back-mutated to generate the huGC33 VH.

[0134] muGC33 VL humanization

[0135] To generate humanized anti-GC33 antibody (huGC33), the complementarity determining regions (CDRs) of the light chain variable region (VL) were grafted into a human framework using a human VL framework containing analogous sequences to the mouse anti-GPC3 antibody (muGC33). Residues VL36 and VL46 were back-mutated to generate huGC33 VL.

[0136] The humanized antibody huGC33 was designed to be produced in scFv format. Specifically, the antibody huGC33 scFv was prepared in scFv format of VL-(G4S)3-VH in pOptiVEC (Invitrogen) plasmid, which is a mammalian cell expression vector, conjugating Flag and 6X His tag to the c-terminus to prepare a gene (pOptiVEC huGC33 scFv). Figure 1 ) The plasmid introducing the gene was expressed in scFv format using Expi293 expression system (Invitrogen), and purified using Akta Pure purifier (GE healthcare) and HisTrap column (GE healthcare).

[0137] The antibody was added to the blotting sample buffer and incubated at 70°C for 10 minutes to produce a sample for analysis with SDS-PAGE. After running the sample, the final gel was analyzed with Chemidoc (bio-rad). Figure 2 ).

[0138] To analyze the binding affinity of muGC33 and huGC33 to the antigen, recombinant human glypican 3 protein (GPC3), GPC3 was diluted in 5X ELISA coating buffer, coated in a 96-well immunoplate, and incubated at 4°C overnight. Each well was treated with blocking solution and washed with PBST solution. Then, the antibodies muGC33 and huGC33 were added to each well and incubated at room temperature for 1 hour. After washing the plate, secondary antibody (monoclonal anti-FLAG M2-peroxidase (HRP)) was added to each well. The binding affinity was measured and quantified using a microplate reader at a wavelength of 450 nm. Figure 3 ). Then, the ELISA was repeated to calculate the binding affinity EC50 value of huGC33 scFv. Using the program Prism graphpad, the binding affinity EC50 value was calculated to be 1.401e-009 Figure 4 ).

[0139] Example 2 - Lentiviral transfer plasmid

[0140] The single chain variable fragment (scFv) format of the humanized anti-GC33 antibody agent was prepared Figure 1DNA constructs of huGC33-VHVL and huGC33-VLVH were generated by joining the VL and VH regions using standard DNA cloning techniques known in the art, where the sequences were designed to contain either a VH-VL orientation or a VL-VH orientation. The lentiviral transfer plasmids used herein are shown in Table 3, and the nucleic acid sequences of huGC33 VL-VH and huGC33 VH-VL are shown in Table 4.

[0141] [Table 3]

[0142]

[0143] [Table 4]

[0144]

[0145]

[0146] The lentiviral vector construct pELPS4-MVRL2H2-euBBZ was digested with BamHI and Nhel enzymes, and pELPS4 huGC33 VH-VL and pELPS4-huGC33 VL-VH were inserted into the vector construct. The results of the DNA fragment purification are shown in Table 2. Figure 5 The transduction units (TU / mL) of the lentivirus were also measured, as shown in Table 5.

[0147] [Table 5]

[0148]

[0149] Example 3 - huGC33-VHVL, huGC33-VLVH CAR-T in vitro

[0150] Peripheral blood mononuclear cells (PBMCs) were cultured in cell culture medium comprising 1 L OpTmizer TM T Cell Expansion Basal Medium, 25 mL OpTmizer TM T Cell Expansion Supplement, 50 mL CTS TM Immune Cell SR, 10 mL Penicillin-Streptomycin (10,000 U / mL), and 10 mL CTS TM GlutaMAX TM -I Supplement, where the cell density was adjusted to 1 x 10 6 cells / mL, and IL-2 (400 IU / mL) was added during the culture period at 5% CO2 and 37°C.

[0151] The immune cells were then transduced with the lentiviral vector, and cell growth and expansion were measured from day 5 to day 12 of the cell culture. The cells were then harvested at day 12 and used for further analysis.

[0152] In vitro comparison of huGC33-VHVL and huGC33-VLVH CAR-T constructs, wherein cell growth of each CAR-T cell at day 12 of cell culture was compared by total expansion fold. The results showed that untreated cells displayed 297.0 fold expansion, truncated muGC33 VHVL cells displayed 255.0 fold expansion, muGC33 VHVL cells displayed 192.0 fold expansion, huGC33 VHVH cells displayed 190.6 fold expansion, huGC33 VHVL cells displayed 137.8 fold expansion Figure 6A ) Cell expansion and cell viability from day 4 to day 12 was compared, although cell viability will vary for each group of CAR-T cells depending on the time at which the cells are harvested and measured, the results showed more than 84% viability in all groups of CAR-T cells Figure 6B ) and CAR expression was analyzed using flow cytometry at day 6, day 9 and day 12 of cell culture Figure 7A to 7C ) Additionally, the Huh-7 cell line (GPC3 positive cell line) was used to harvest target cells while CAR-T cells were used as effector cells to perform an LDH-based cytotoxicity assay. Cells were incubated in a 96-well U-bottom plate at an E:T (effector (E): target (T)) ratio = 10:1, then incubated with Cyto Tox 96 reagent for 30 minutes and cytotoxicity was measured and quantified using a microplate reader at a wavelength of 490 nm. Figure 8

[0153] Example 4 - Expression of GPC3 in hepatocarcinoma cells

[0154] To develop an animal model of hepatocarcinoma, a stable cell line expressing luciferase was generated. Three hepatocarcinoma cell lines expressing GPC3 and one hepatocarcinoma cell not expressing GPC3 were selected. GPC3 expression was determined using the four cell lines HepG2, Hep3B, Huh-7 and SK-Hep-1. The results showed that SK-Hep-1 did not show GPC3 expression, while HepG2, Hep3B and Huh-7 showed about 20% to 30% of GPC3 expression Figure 9

[0155] Example 5 - Development of a cell line expressing luciferase

[0156] Using the Huh-7 cell line expressing GPC3 in Example 4, a cell line expressing luciferase GFP was developed. Cells were transduced with a lentiviral vector and transduction efficiency was determined by GFP expression Figure 10A ) Then, cells transduced with the luciferase GFP gene were selectively isolated with puromycin and a luciferase function test was performed. The results showed that when the cells were reduced by about 50%, the RLU was also reduced by about 50% Figure 10B ​​). These results were used to develop an animal model of liver cancer.

[0157] Example 6 - GPC3 VH-VL CAR-T cells and GPC3 VL-VH CAR-T cells in vivo

[0158] Using the three cell lines in Example 4, cancer cells were injected into NSG mice (1 x 10 6 cells / head or 2 x 10 6 cells / head) and the growth of liver tumors was observed over time. The results showed that mice injected with Huh-7_Luc-GFP cells showed tumor growth in the shortest time.

[0159] Huh-7_Luc-GFP cells were injected into NSG mice (2 x 10 6 cells / head) and after the tumor size reached 150-200 mm 3 GPC3 VH-VL CAR-T cells and GPC3 VL-VH CAR-T cells were injected. All mice in the control group that did not receive any injection died after 25 days post-injection, while mice in the groups that received GPC3 CAR-T cell injection survived 25 days post-injection. Injection of 0.5 x 10 6 GPC3 VH-VL CAR-T cells showed that the tumor size increased then decreased 7 days post-injection, while injection of 0.5 x 10 6 GPC3 VL-VH CAR-T cells showed that the tumor size increased then decreased 7 to 10 days post-injection. Injection of 0.25 x 10 6 cells / head showed that 20% of the mice in the GPC3 VH-VL group showed an increase in tumor size, while 40% of the mice in the GPC3 VL-VH group showed an increase in tumor size Figure 11 ).

[0160] Observation of the mice 10 weeks post GPC3 CAR-T injection showed that the group of mice that received GPC3 VL-VH CAR-T cells (0.25 x 10 6 cells / head) showed 60% survival 21 days post-injection, while the group of mice that received GPC3 VH-VL CAR-T cells (0.25 x 10 6 cells / head) showed 80% survival 28 days post-injection. The group that received GPC3 VL-VH CAR-T cells (0.5 x 10 6 cells / head) showed 80% survival 56 days post-injection, while the group that received GPC3 VH-VL CAR-T cells (0.5 x 10 6 cells / head) showed 80% survival 66 days post-injection.

[0161] Blood analysis of mice showed that the number of CAR-T cells in the blood reached a peak at day 14 after injection, and the results of the two groups were similar. Figure 12

[0162] These results show that GPC3 VH-VL CAR-T cells are more effective than GPC3 VL-VH CAR-T cells in reducing tumor size, while showing a higher survival rate. SEQUENCE LISTING <110> EUTILEX CO., LTD. <120> GPC3 CAR-T cell compositions and methods of making and using the same <130> 47683-0036W01 <150> 62 / 991,493 <151> 2020-03-18 <150> 63 / 004,827 <151> 2020-04-03 <150> 63 / 043,237 <151> 2020-06-24 <160> 14 <170> PatentIn version 3.5 <210> 1 <211> 16 <212> PRT <213> Artificial <220> <223> Light chain CDR1 <400> 1 Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His 1 5 10 15 <210> 2 <211> 7 <212> PRT <213> Artificial <220> <223> Light chain CDR2 <400> 2 Lys Val Ser Asn Arg Phe Ser 1 5 ​<210> 3 <211> 9 <212> PRT <213> Artificial <220> <223> Light chain CDR3 <400> 3 Ser Gln Asn Thr His Val Pro Pro Thr 1 5 <210> 4 <211> 5 <212> PRT <213> Artificial <220> <223> Heavy chain CDR1 <400> 4 Asp Tyr Glu Met His 1 5 <210> 5 <211> 17 <212> PRT <213> Artificial <220> <223> Heavy chain CDR2 <400> 5 Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe Lys 1 5 10 15 Gly <210> 6 <211> 6 <212> PRT <213> Artificial <220> <223> Heavy chain CDR3 <400> 6 Phe Tyr Ser Tyr Thr Tyr 1 5 <210> 7 <211> 345 <212> DNA <213> Artificial <220> <223> huGC33 heavy chain variable region <400> 7 caagtgcaac tcgtacaatc aggtgctgaa gtcaaaaagc cgggagcctc tgttaaagtg 60 tcctgtaaag ccagcggcta cacctttacc gattatgaga tgcactgggt tcggcaggct 120 ccgggccaag gtctcgagtg gatcggggct cttgacccaa agacgggcga cacggcttat 180 tcacaaaaat tcaaaggtag ggctactctg actgccgata agtccaccag caccgcgtat 240 atggagctct ctagcttgcg aagcgaggac acggcggtgt actattgcac acgcttctat 300 agttacacat attggggtca aggcacgctt gtgaccgtgt ctagc 345 <210> 8 <211> 115 <212> PRT <213> Artificial <220> <223> huGC33 heavy chain variable region <400> 8 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gin Lys Phe 50 55 60 Lys Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gin Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 9 <211> 336 <212> DNA <213> Artificial <220> <223> huGC33 light chain variable region <400> 9 gacgtcgtta tgacacagag tcccctctcc ttgccggtga ccctgggtca gcctgcgtcc 60 atctcttgca gatcctccca gtctctggta cactccaacg gcaacacata cttgcactgg 120 taccaacaaa gacctggtca gtcaccgcga cttctcatat ataaagtttc caataggttc 180 agtggagtgc cagacaggtt cagtggttca ggatcaggca ctgatttcac gcttaaaatc 240 agtcgggttg aggcggagga cgtaggagtt tactattgca gccagaatac gcacgtgccg 300 cctacttttg gctctggaac caagttggaa ataaag 336 <210> 10 <211> 112 <212> PRT <213> Artificial <220> <223> huGC33 light chain variable region <400> 10 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95 Thr His Val Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 11 <211> 345 <212> DNA <213> Artificial <220> <223> muGC33 heavy chain variable region <400> 11 CAGGTTC AAC TGCAGCAGTC TGGGGCTGAG CTGGTGAGGC CTGGGGCTTC AGTGAAGCTG 60 TCCTGCAAGG CTTCGGGCTA CACATTTACT GACTATGAAA TGC ACTGGGT GAAGCAGACA 120 CCTGTGCA TG GCC TAA AATG GATTGGAGCT CTTGATCCTA AA ACTG GT A ACTGCCTAC 180 AGTCAGAAGT TCAAGGGCAA GGCCACACTG ACTGCAGACA AATCCTCCAG CACAGCCTAC 240 ATGGAGCTCC GCAGCCTGAC ATCTGAGGAC TCTGCCGTCT ATTACTGTAC AAGATTCTAC 300 TCCTATACTT ACTGGGGCCA AGGGACTCTG GTC ACTGTCTCT GCA 345 <210> 12 <211> 115 <212> PRT <213> Artificial <220> <223> muGC33 heavy chain variable region <400> 12 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Val Lys Gln Thr Pro Val His Gly Leu Lys Trp Ile 35 40 45 Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gin Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gin Gly Thr Leu Val Thr 100 105 110 Val Ser Ala 115 <210> 13 <211> 336 <212> DNA <213> Artificial <220> <223> muGC33 light chain variable region <400> 13 gatgttgtga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60 atctcttgca gatctagtca gagccttgta cacagtaatg gaaacaccta tttacattgg 120 tacctgcaga agccaggcca gtctccaaag ctcctgatct acaaagtttc caaccgattt 180 tctggggtcc cagacaggtt cagtggcagt ggatcaggga cagatttcac actcaagatc 240 agcagagtgg aggctgagga tctgggagtt tatttctgct ctcaaaatac acatgttcct 300 cctacgttcg gatcggggac caagctggaa ataaaa 336 <210> 14 <211> 112 <212> PRT <213> Artificial <220> <223> muGC33 light chain variable region <400> 14 Asp Val Val Met Thr Gin Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gin Ala Ser lie Ser Cys Arg Ser Ser Gin Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Leu Gin Lys Pro Gly Gin Ser 35 40 45 Pro Lys Leu Leu lie Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys lie 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Ser Gin Asn 85 90 95 Thr His Val Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu lie Lys 100 105 110

Claims

1. An immune cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an extracellular antigen-binding domain that specifically binds glypican 3 (GPC3), a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain comprises a heavy chain variable domain set forth in SEQ ID NO: 8 and a light chain variable domain set forth in SEQ ID NO:

10.

2. The immune cell of claim 1, wherein the CAR is a single polypeptide.

3. The immune cell of claim 1, wherein the CAR comprises two polypeptides.

4. The immune cell of any one of claims 1 to 3, wherein the extracellular antigen-binding domain comprises: a light chain variable domain comprising a CDR1 consisting of SEQ ID NO: 1, a CDR2 consisting of SEQ ID NO: 2, and a CDR3 consisting of SEQ ID NO: 3; and a heavy chain variable domain comprising a CDR1 consisting of SEQ ID NO: 4, a CDR2 consisting of SEQ ID NO: 5, and a CDR3 consisting of SEQ ID NO:

6. ​ ​ 5. The immune cell of claim 1, wherein the transmembrane domain comprises a transmembrane domain selected from a protein selected from the group consisting of 4-1BB / CD137, Activating NK Cell Receptor, Immunoglobulin, 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, 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 that Specifically Binds CD83, LIGHT, LTBR, Ly9 (CD229), Lymphocyte Function-Associated Antigen-1 (LFA-1), MHC Class I 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.

6. The immune cell of claim 5, wherein the transmembrane domain is a transmembrane domain from CD8 alpha.

7. The immune cell of claim 1, wherein the intracellular signaling domain comprises an intracellular signaling domain from a protein selected from the group consisting of 4-1BB / CD137, an activating NK cell receptor, an immunoglobulin, 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, CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, a cytokine receptor, DAP-10, DNAM1 (CD226), Fcgamma 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, a ligand that specifically binds CD83, LIGHT, LTBR, Ly9 (CD229), Ly108, Lymphocyte function-associated antigen-1 (LFA-1), MHC class I 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, or any combination thereof.

8. The immune cell of claim 7, wherein the intracellular signaling domain is from 4-1BB and CD3 zeta.

9. The immune cell of claim 1, wherein the chimeric antigen receptor further comprises an additional antigen binding domain.

10. The immune cell of claim 9, wherein the additional antigen binding domain is an scFv.

11. The immune cell of claim 1, wherein the immune cell is a human immune cell.

12. The immune cell of claim 11, wherein the human immune cell is an autologous human immune cell.

13. The immune cell of claim 11, wherein the human immune cell is an allogeneic human immune cell.

14. The immune cell of claim 1, wherein the immune cell is a T cell.

15. The immune cell of claim 1, wherein the immune cell is an NK cell.

16. A pharmaceutical composition comprising the immune cell of claim 1, and a pharmaceutically acceptable carrier.

17. A kit comprising the pharmaceutical composition of claim 16.

18. Use of the immune cell of claim 1 or the pharmaceutical composition of claim 16 in the manufacture of a medicament for treating a subject having a glypican 3-associated cancer.

19. A nucleic acid encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an extracellular antigen binding domain that specifically binds glypican 3 (GPC3), a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain comprises a heavy chain variable domain of SEQ ID NO: 8 and a light chain variable domain of SEQ ID NO:

10.

20. The nucleic acid of claim 19, wherein the CAR is a single polypeptide.

21. The nucleic acid of claim 19, wherein the CAR comprises two polypeptides.

22. The nucleic acid of claim 19, wherein the extracellular antigen binding domain comprises: a light chain variable domain comprising a CDR1 consisting of SEQ ID NO: 1, a CDR2 consisting of SEQ ID NO: 2, and a CDR3 consisting of SEQ ID NO: 3; and a heavy chain variable domain comprising a CDR1 consisting of SEQ ID NO: 4, a CDR2 consisting of SEQ ID NO: 5, and a CDR3 consisting of SEQ ID NO:

6. ​ ​ 23. The nucleic acid of claim 19, wherein the transmembrane domain is a transmembrane domain from a protein selected from the group consisting of 4-1BB / CD137, Activating NK Cell Receptor, Immunoglobulin, 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, CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, Cytokine Receptor, DAP-10, DNAM1 (CD226), Fcgamma Receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, Inducible T-Cell Costimulator (ICOS), Integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, Ligand that Specifically Binds CD83, LIGHT, LTBR, Ly9 (CD229), Lymphocyte Function-Associated Antigen-1 (LFA-1), MHC Class I 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.

24. The nucleic acid of claim 23, wherein the transmembrane domain is a transmembrane domain from CD8 alpha.

25. The nucleic acid of claim 19, wherein the intracellular signaling domain comprises an intracellular signaling domain from a protein selected from the group consisting of 4-1BB / CD137, an activating NK cell receptor, an immunoglobulin, 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, CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, a cytokine receptor, DAP-10, DNAM1 (CD226), Fcgamma 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, a ligand that specifically binds CD83, LIGHT, LTBR, Ly9 (CD229), Ly108, Lymphocyte function-associated antigen-1 (LFA-1), MHC class I 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, or any combination thereof.

26. The nucleic acid of claim 25, wherein the intracellular signaling domain is from 4-1BB and CD3 zeta.

27. The nucleic acid of claim 19, wherein the chimeric antigen receptor further comprises an additional antigen binding domain.

28. The nucleic acid of claim 27, wherein the additional antigen binding domain is an scFv.

29. A vector comprising the nucleic acid of claim 19.

30. The vector of claim 29, further comprising a promoter operably linked to the nucleic acid.

31. The vector of claim 30, wherein the promoter is a constitutive promoter.

32. The vector of claim 30, wherein the promoter is an inducible promoter.

33. The vector of any one of claims 29-32, wherein the vector is a viral vector.

34. The vector of claim 33, wherein the viral vector is a lentiviral vector.

35. A method for producing an engineered immune cell, the method comprising: introducing the nucleic acid of claim 19 or the vector of claim 29 into an immune cell, thereby producing an engineered immune cell.

36. The method of claim 35, further comprising culturing the engineered immune cell after the introducing step.

37. The method of claim 35, wherein the immune cell is a T cell.

38. The method of claim 35, wherein the immune cell is an NK cell.

39. The method of claim 35, further comprising obtaining the immune cell from a subject prior to the introducing step.

40. The method of claim 39, wherein the subject has been diagnosed or identified as having a glypican 3-associated cancer.

41. An engineered immune cell produced by the method of claim 35.

42. A pharmaceutical composition comprising the engineered immune cell of claim 41, and a pharmaceutically acceptable carrier.

43. Use of the engineered immune cell of claim 41 or the pharmaceutical composition of claim 42 for the manufacture of a medicament for treating a glypican 3-associated cancer in a subject.

44. The use of claim 18, wherein the glypican 3-associated cancer is a liver cancer.

45. The use of claim 43, wherein the glypican 3-associated cancer is a liver cancer.

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