Chimeric antigen receptor with 4-1BB costimulatory domain

Abstract

Provided are CAR-T compositions involving CARs, where the CAR comprises: (i) an extracellular domain that includes an antigen-binding domain; (ii) a transmembrane domain; and (iii) an intracellular domain that includes a co-stimulatory intracellular domain, where the co-stimulatory intracellular domain includes an intracellular signaling domain from 4-1BB / CD137 and five additional amino acids. The present disclosure also provides vectors, compositions, and methods of treatment using antigen-binding molecules and engineered immune cells comprising CARs. The CAR compositions provided herein can be used to treat certain cancers.

Description

[0001] Cross - Reference to Related Applications

[0002] This application is a PCT application claiming the priority and benefit of the following: U.S. Application No. 62 / 867,503, filed on June 27, 2019; International Application No. PCT / KR2019 / 010244, filed on August 12, 2019; U.S. Application No. 16 / 715,462, filed on December 16, 2019; U.S. Application No. 62 / 991,493, filed on March 18, 2020; U.S. Application No. 63 / 004,827, filed on April 3, 2020; U.S. Application No. 63 / 043,237, filed on June 24, 2020, the disclosure of each of which is incorporated herein by reference in its entirety. Background Art

[0003] Cancer remains one of the leading causes of death in the world. 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), in 2012, there were 14.1 million new cancer cases and 8.2 million cancer deaths worldwide. By 2030, due to population growth and aging, as well as exposure to risk factors such as smoking, unhealthy diet, and lack of physical activity, the global burden is expected to grow to 21.7 million new cancer cases and 13 million cancer deaths. Further, the pain and the medical costs of cancer treatment reduce the quality of life of both cancer patients and their families.

[0004] T cells engineered with chimeric antigen receptors (CAR-T) have great therapeutic potential in treating diseases such as cancer. CAR-T therapy endows T cells with strong target affinity and signal transduction functions. However, the impressive efficacy of CAR-T therapy is often accompanied by severe side effects, such as cytokine release syndrome (CRS). Therefore, there remains an unmet need for the development of CAR-T therapies and strategies with reduced side effects. Summary of the Invention

[0005] Provided herein are immune cells comprising a chimeric antigen receptor (CAR), wherein the (CAR) comprises: (a) an extracellular domain comprising an antigen-binding domain; (b) a transmembrane domain; and (c) an intracellular domain comprising a co-stimulatory intracellular domain, wherein the co-stimulatory intracellular domain comprises an intracellular signaling domain from 4-1BB / CD137 and five additional amino acids.

[0006] In some embodiments, the chimeric antigen receptor is a single polypeptide. In some embodiments, the chimeric antigen receptor comprises two polypeptides.

[0007] In some embodiments, the co-stimulatory intracellular domain comprises an intracellular signaling domain from 4-1BB / CD137 and five additional amino acids, wherein the five additional amino acids are encoded by SEQ ID NO:1. In some embodiments, the co-stimulatory intracellular domain comprises SEQ ID NO:2.

[0008] In some embodiments, the antigen-binding domain is humanized. In some embodiments, the antigen-binding domain is human. In some embodiments, the antigen-binding domain is a scFv. In some embodiments, the antigen-binding domain specifically binds an antigen associated with a disease. In some embodiments, the antigen-binding domain specifically binds a tumor antigen. In some embodiments, the antigen-binding domain specifically binds an antigen selected from the group consisting of: glypican-3 (GPC3), malignant variant receptor (MVR), and CD19.

[0009] In some embodiments, the transmembrane domain is a transmembrane domain selected from proteins selected from the group consisting of: 4-1BB / CD137, activated 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, CD3δ, CD3ε, CD3γ, CD3ζ, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8α, CD8β, CD96 (tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fcγ receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Igα (CD79a), IL-2Rβ, IL-2Rγ, IL-7Rα, inducible T cell co-stimulator (ICOS), integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, ligand specifically binding to CD83, LIGHT, LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1), MHC class 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 the transmembrane domain from CD8α. In some embodiments, the intracellular domain further comprises the intracellular domain from CD3ζ.

[0010] In some embodiments, the chimeric antigen receptor further comprises a signal peptide or a leader sequence. In some embodiments, the chimeric antigen receptor further comprises a hinge region. In some embodiments, the hinge region is a CD8α hinge. In some embodiments, the chimeric antigen receptor further comprises an additional antigen-binding domain. In some embodiments, the additional antigen-binding domain is a scFv.

[0011] 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 a NK cell.

[0012] Provided herein are nucleic acids encoding a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor comprises: (a) an extracellular domain comprising an antigen-binding domain; (b) a transmembrane domain; and (c) an intracellular domain comprising a co-stimulatory intracellular domain, wherein the co-stimulatory intracellular domain comprises an intracellular signaling domain from 4-1BB / CD137 and five additional amino acids.

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

[0014] In some embodiments, the co-stimulatory intracellular domain comprises an intracellular signaling domain from 4-1BB / CD137 and five additional amino acids, wherein the five additional amino acids are encoded by the nucleotide sequence of SEQ ID NO:1. In some embodiments, the co-stimulatory intracellular domain comprises SEQ ID NO:2.

[0015] 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 a scFv. In some embodiments, the antigen-binding domain specifically binds to a disease-associated antigen. In some embodiments, the antigen-binding domain specifically binds to a tumor antigen. In some embodiments, the antigen-binding domain specifically binds to an antigen selected from the group consisting of glypican-3 (GPC3), malignant variant receptor (MVR), and CD19.

[0016] In some embodiments, the transmembrane domain is a transmembrane domain selected from proteins selected from the group consisting of: 4-1BB / CD137, activated 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, CD3δ, CD3ε, CD3γ, CD3ζ, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8α, CD8β, CD96 (tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fcγ receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Igα (CD79a), IL-2Rβ, IL-2Rγ, IL-7Rα, inducible T cell co-stimulator (ICOS), integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, ligand specifically binding to CD83, LIGHT, LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1), MHC class 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 the transmembrane domain from CD8α.

[0017] In some embodiments, the intracellular domain further comprises the intracellular domain from CD3ζ. In some embodiments, the chimeric antigen receptor further comprises a signal peptide or a leader sequence. In some embodiments, the chimeric antigen receptor further comprises a hinge region. In some embodiments, the hinge region is the CD8α hinge.

[0018] The present disclosure provides a vector, which comprises any one of the nucleic acids described herein. In some embodiments, the vector further comprises a promoter operably linked to the nucleic acid. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a lentiviral vector.

[0019] The present disclosure provides a method for generating engineered immune cells, the method comprising: introducing any one of the nucleic acids described herein or any one of the vectors described herein into immune cells, thereby generating the engineered immune cells. In some embodiments, the method further comprises culturing the engineered immune cells after the introducing step. In some embodiments, the immune cells are T cells. In some embodiments, the immune cells are NK cells.

[0020] In some embodiments, the method further comprises obtaining the immune cells from a subject before the introducing step. In some embodiments, the method further comprises administering the engineered immune cells to the subject. In some embodiments, the subject has been diagnosed or identified as having cancer.

[0021] The present disclosure provides engineered immune cells generated by any one of the methods described herein.

[0022] The present disclosure provides a pharmaceutical composition, which comprises any one of the engineered immune cells described herein and a pharmaceutically acceptable carrier.

[0023] The present disclosure provides methods for treating cancer in a subject, the methods comprising administering to the subject any one of the engineered immune cells described herein or any one of the pharmaceutical compositions described herein. In some embodiments, the cancer is an anti-glypican-3 related cancer, an anti-CD19 related cancer, or an anti-MVR related cancer. In some embodiments, the cancer is carcinoma, lymphoma (e.g., Hodgkin's and non-Hodgkin's lymphomas), blastoma, sarcoma, leukemia, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, other lymphoproliferative disorders, and various types of head and neck cancers. In some embodiments, the subject has previously been administered one or more additional anti-cancer therapies selected from the group consisting of ionizing radiation, chemotherapeutic agents, therapeutic antibodies, and checkpoint inhibitors. In some embodiments, the subject has been identified or diagnosed as having the cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 FIG. shows a schematic diagram of an exemplary MVR CAR construct.

[0025] Figure 2 FIG. shows the exemplary enzyme mapping results after cloning of MVRL2H2-4-1BB.

[0026] Figure 3 FIG. shows the restriction enzyme digestion results of huGC33(VH-VL)-euBBz, where the expected sizes are marked on the DNA sequence ladder and the results are shown in the gel electrophoresis image.

[0027] Figure 4 FIG. shows the restriction enzyme digestion results of huGC33(VH-VL)-BBz, where the expected sizes are marked on the DNA sequence ladder and the results are shown in the gel electrophoresis image. Figure 5A FIG. is a graph showing the total in vitro expansion fold of CAR-T cells over the course of a day.

[0028] Figure 5B FIG. is a graph comparing the in vitro expansion fold of CAR-T cells.

[0029] Figure 5C FIG. is a graph showing the in vitro cell viability of CAR-T cells.

[0030] Figure 6 Analysis of CAR expression in T cells transduced with huGC33(VH-VL)-euBBz and huGC33(VH-VL)-BBz is shown.

[0031] Figure 7A Is a graph showing an LDH-based cytotoxicity assay using target cells from the Huh-7 cell line.

[0032] Figure 7B Is a graph showing an LDH-based cytotoxicity assay using target cells from the PLC / PRF / 5 cell line.

[0033] Figure 8 Is a set of graphs comparing the in vivo efficacy of huGC33(VH-VL)-euBBz and huGC33(VH-VL)-BBz CAR-T cells.

[0034] Figure 9A Is a graph showing CAR-T cell counts in a mouse model 5 weeks after injection of huGC33(VH-VL)-euBBz and huGC33(VH-VL)-BBz CAR-T cells.

[0035] Figure 9B Is a set of graphs comparing CAR-T cell counts in a mouse model 5 weeks after injection of huGC33(VH-VL)-euBBz and huGC33(VH-VL)-BBz CAR-T cells.

[0036] Figure 10 Shows the analysis of CAR-T cells in the blood, bone marrow, spleen, and liver of mice 5 weeks after injection of huGC33(VH-VL)-euBBz and huGC33(VH-VL)-BBz CAR-T cells using FACS staining.

[0037] Figure 11 A shows CAR expression in T cells transduced with CD19-BBz and CD19-euBBz.

[0038] Figure 11 B is a graph from a luciferase-based cytotoxicity assay showing the killing activity in T cells transduced with CD19-BBz and CD19-euBBz.

[0039] Figure 12 Shows the IVTS imaging results of the effects of CD19-BBz CAR-T cells and CD19-euBBz CAR-T cells using an animal model.

[0040] Figure 13It is a graph showing the photon values of cancer cells in an animal model after injection of CD19-BBz CAR-T cells and CD19-euBBz CAR-T cells.

[0041] Figure 14 A is a set of graphs showing the percentage of total CD19 CAR-T cells present in the blood using FACS after performing orbital blood collection in mice at intervals of 3 to 4 days.

[0042] Figure 14 B is a set of graphs showing the percentage of CD4 / CD8 CAR-T cells present in the blood using FACS after performing orbital blood collection in mice at intervals of 3 to 4 days.

[0043] Figure 14 C is a graph showing the number of total CD19 CAR-T present in the blood using FACS after performing orbital blood collection in mice at intervals of 3 to 4 days.

[0044] Figure 15 A shows a schematic diagram of an exemplary GPC3 CAR construct.

[0045] Figure 15 B shows a schematic diagram of an exemplary GPC3 CAR construct. Detailed Description

[0046] The present disclosure describes chimeric antigen receptors (CARs) comprising a 4-1BB co-stimulatory endodomain and methods for the preparation and use of said CARs.

[0047] Definitions

[0048] About: When used herein to refer to a value, the term "about" refers to a value similar to the referenced value in the context. Generally, one of ordinary skill in the art familiar with the context will understand the degree of relevant difference covered by "about" in said context. For example, in some embodiments, the term "about" may cover values within a range of 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the reference value.

[0049] Administration: As used herein, the term "administration" generally refers to the administration of a composition to a subject or system to effect delivery of the composition or an agent contained therein. One of ordinary skill in the art will recognize the various routes that can be used to administer to a subject, such as a human, in appropriate circumstances. For example, in some embodiments, administration can be ocular, oral, parenteral, topical, etc. In some specific embodiments, administration can be bronchial (e.g., by bronchial instillation), buccal, cutaneous (which can be or include, for example, one or more of topical to the dermis, intradermal, intercutaneous, transdermal, etc.), enteral, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a particular organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by endotracheal instillation), vaginal, intravitreal, etc. In some embodiments, administration can involve a single dose, multiple doses, or a fixed number of doses. In some embodiments, administration can involve dosing that is intermittent dosing (e.g., multiple doses separated in time) and / or periodic dosing (e.g., separate doses separated by a common time period). In some embodiments, administration can involve continuous dosing (e.g., perfusion) for at least a selected time period.

[0050] Affinity: As is known in the art, "affinity" is a measure of the strength with which a particular ligand binds its partner. Affinity can be measured in different ways. In some embodiments, affinity is measured by a quantitative assay. In some such embodiments, the concentration of the binding partner can be fixed at a level that exceeds the ligand concentration in order to mimic physiological conditions. Alternatively or additionally, in some embodiments, the concentration of the binding partner and / or the ligand concentration can vary. In some such embodiments, the affinity can be compared to a reference value under comparable conditions (e.g., concentration).

[0051] Antibody Agent: As used herein, the term "antibody agent" can refer to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex that contains immunoglobulin structural elements sufficient to confer specific binding. Exemplary antibody agents include, but are not limited to, monoclonal antibodies, polyclonal antibodies, and fragments thereof. In some embodiments, the antibody agent may contain one or more sequence elements known in the art such as humanized, primatized, chimeric, etc. In many embodiments, the term "antibody agent" is used to refer to one or more of the constructs or forms known in the art or developed that utilize antibody structural and functional features in alternative presentations. For example, in some embodiments, the antibody agent utilized according to the present invention is in a form selected from, but not limited to, the following: intact IgA antibody, IgG antibody, IgE antibody, or IgM antibody; bispecific antibody or multispecific antibody (e.g., etc.); antibody fragments such as Fab fragment, Fab' fragment, F(ab')2 fragment, Fd' fragment, Fd fragment, and isolated CDR or a collection thereof; single-chain Fv; polypeptide-Fc fusion; single-domain antibody (e.g., shark single-domain antibody such as IgNAR or a fragment thereof); cameloid antibody; masking antibody (e.g., ); small modular immunopharmaceutical (SMIPs TM ); single-chain diabody or tandem diabody VHH; minibody; ankyrin repeat protein or DART; TCR-like antibody; microprotein; and In some embodiments, an antibody agent may lack covalent modifications (e.g., attachment of glycans) that it would have when produced naturally. In some embodiments, an antibody agent may contain covalent modifications (e.g., attachment of glycans), payloads [e.g., detectable moieties, therapeutic moieties, catalytic moieties, etc.] or other side groups [e.g., polyethylene glycol, etc.]. In many embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence comprises one or more structural elements recognized by those skilled in the art as complementarity determining regions (CDRs). In some embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence comprises at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) that is substantially the same as the CDRs found in a reference antibody. In some embodiments, the included CDR is substantially the same as the reference CDR because the included CDR is identical in sequence or contains between 1 and 5 amino acid substitutions compared to the reference CDR. In some embodiments, the included CDR is substantially the same as the reference CDR because the included CDR exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the reference CDR. In some embodiments, the included CDR is substantially the same as the reference CDR because the included CDR exhibits at least 96%, 97%, 98%, 99% or 100% sequence identity with the reference CDR. In some embodiments, the included CDR is substantially the same as the reference CDR because 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 the same as the amino acid sequence of the reference CDR. In some embodiments, the included CDR is substantially the same as the reference CDR because 1-5 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 the same as the amino acid sequence of the reference CDR. In some embodiments, the included CDR is substantially the same as the reference CDR because 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 the same as the amino acid sequence of the reference CDR. In some embodiments, the included CDR is substantially the same as the reference CDR because 1-5 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 the same as the amino acid sequence of the reference CDR. In some embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence comprises structural elements recognized by those skilled in the art as immunoglobulin variable domains.In some embodiments, the antibody agent is a polypeptide having a binding domain that is homologous or substantially 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).

[0052] Antigen: As used herein, the term "antigen" can refer to an agent that binds to an antibody agent. In some embodiments, the antigen binds to the antibody agent and may or may not induce a specific physiological response in an organism. Generally, an antigen can be or comprise any chemical entity, such as a small molecule, nucleic acid, polypeptide, carbohydrate, lipid, polymer (including biopolymers [e.g., nucleic acid and / or amino acid polymers] and polymers other than biopolymers [e.g., polymers other than nucleic acid or amino acid polymers]), etc. In some embodiments, the antigen is or comprises a polypeptide. In some embodiments, the antigen is or includes a glycan. Those skilled in the art will appreciate that, generally, an antigen can be provided or used in an isolated or pure form, or alternatively can be provided in a crude form (e.g., together with other materials, such as in a cell extract or other relatively crude preparation of an extract containing the antigen).

[0053] In some certain embodiments, the antigen is present in a cellular environment (e.g., the antigen is expressed on the cell surface or in the cell). In some embodiments, the antigen is a recombinant antigen.

[0054] Antigen-binding domain: As used herein, the term "antigen-binding domain" can refer to an antibody agent or a portion thereof that specifically binds to a target moiety or entity. Generally, the interaction between the antigen-binding domain and its target is non-covalent. In some embodiments, the target moiety or entity can belong to any chemical class, including, for example, carbohydrates, lipids, nucleic acids, metals, polypeptides, or small molecules. In some embodiments, the antigen-binding domain can be or include a polypeptide (or a complex thereof). In some embodiments, the antigen-binding domain is part of a fusion polypeptide. In some embodiments, the antigen-binding domain is part of a chimeric antigen receptor (CAR).

[0055] Associated with: Two events or entities are "associated" with each other as the term is used herein if the existence, level, and / or form of one event or entity is related to the existence, level, and / or form of another event or entity. For example, if the existence, level, and / or form of a particular entity (e.g., a polypeptide, a genetic signature, a metabolite, a microorganism, etc.) is related to the incidence and / or susceptibility of a disease, disorder, or condition (e.g., across a relevant population), then the particular entity is considered to be associated with the particular disease, disorder, or condition. In some embodiments, two or more entities are "associated" physically with each other if they interact directly or indirectly such that they are in physical proximity to each other and / or remain in physical proximity to each other. In some embodiments, two or more entities that are associated physically with each other are covalently linked to each other; in some embodiments, two or more entities that are associated physically with each other are not covalently linked to each other but are non-covalently associated, for example, by hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0056] Binding: It should be understood that as used herein, the term "binding" generally refers to non-covalent association between or among two or more entities. "Direct" binding involves physical contact between entities or portions. Indirect binding involves physical interaction through physical contact with one or more intermediate entities. Binding between two or more entities can generally be evaluated in any of a variety of circumstances, including studying the interacting entities or portions in isolation or in the context of a more complex system (e.g., when covalently or otherwise associated with a carrier entity and / or in a biological system or cell).

[0057] 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 the cells exhibit an abnormal growth phenotype characterized by a significant loss of control of cell proliferation. In some embodiments, a tumor can be or include pre-cancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic cells. The present disclosure specifically identifies certain cancers to which its teachings may be particularly relevant. In some embodiments, the relevant cancer can be characterized by a solid tumor. In some embodiments, the relevant cancer can be characterized by a blood tumor.

[0058] Typically, examples of different types of cancers known in the art include, for example, hematopoietic system cancers, including leukemia, lymphoma (Hodgkin and non-Hodgkin), myeloma, and myeloproliferative disorders; sarcoma, melanoma, adenoma, solid tissue cancers, squamous cell carcinomas of the oral cavity, larynx, throat, and lungs, liver cancer, genitourinary system cancers such as prostate cancer, cervical cancer, bladder cancer, uterine cancer, and endometrial cancer, and renal cell cancer, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, endocrine system cancers, thyroid cancer, parathyroid cancer, head and neck cancers, breast cancer, gastrointestinal cancers, and nervous system cancers, benign lesions such as papilloma, and the like.

[0059] CDR: As used herein, "CDR" may refer to the complementarity determining regions within the variable regions of an antibody agent. There are three CDRs in each variable region of the heavy and light chains, designated CDR1, CDR2, and CDR3, respectively, for each variable region. "A set of CDRs" or "CDR set" refers to a set of three or six CDRs that occur in a single variable region capable of binding an antigen or the CDRs of homologous heavy and light chain variable regions capable of binding an antigen. Certain systems for defining CDR boundaries have been established in the art (e.g., Kabat, Chothia, etc.); those skilled in the art understand the differences between these systems and are able to understand CDR boundaries to the extent required for understanding and practicing the claimed invention.

[0060] Chemotherapeutic agent: As used herein, the term "chemotherapeutic agent" has its meaning as understood in the art and refers to one or more apoptosis-inducing agents, cell growth inhibitors, and / or cytotoxic agents, such as, specifically, agents that are used for and / or recommended for the treatment of one or more diseases, disorders, or conditions associated with undesired cell proliferation. In many embodiments, the chemotherapeutic agent can be used to treat cancer. In some embodiments, the chemotherapeutic agent can be or include one or more alkylating agents, one or more anthracyclines, one or more cytoskeleton disrupting agents (e.g., microtubule targeting agents such as taxanes, maytansine, and analogs thereof), one or more epothilones, one or more histone deacetylase inhibitors (HDACs), one or more topoisomerase inhibitors (e.g., inhibitors of topoisomerase I and / or topoisomerase II), one or more kinase inhibitors, one or more nucleotide analogs or nucleotide precursor analogs, one or more peptide antibiotics, one or more platinum-based agents, one or more retinoids, one or more vinca alkaloids, and / or one or more analogs (i.e., sharing related antiproliferative activity) of one or more of the following substances.In some specific embodiments, the chemotherapeutic agent can be or include one or more of the following: actinomycin, all-trans retinoic acid, auristatin, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, curcumin, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, gemcitabine, hydroxyurea, idarubicin, imatinib, irinotecan, maytansine and / or its analogs (e.g., DM1), mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, maytansinoid, oxaliplatin, paclitaxel, pemetrexed, teniposide, tioguanine, topotecan, valrubicin, vinblastine, vincristine, vindesine, vinorelbine.

[0061] 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 a chemotherapeutic agent found in an antibody-drug conjugate selected from the group consisting of: hLL1-doxorubicin, hRS7-SN-38, hMN-14-SN-38, hLL2-SN-38, hA20-SN-38, hPAM4-SN-38, hLL1-SN-38, hRS7-Pro-2-P-Dox, hMN-14-Pro-2-P-Dox, hLL2-Pro-2-P-Dox, hA20-Pro-2-P-Dox, hPAM4-Pro-2-P-Dox, hLL1-Pro-2-P-Dox, P4 / D10-doxorubicin, gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, glembatumumab vedotin, SAR3419, SAR566658, BIIB015, BT062, SGN-75, SGN-CD19A, AMG-172, AMG-595, BAY-94-9343, ASG-5ME, ASG-22ME, ASG-16M8F, MDX-1203, MLN-0264, anti-PSMA ADC, RG-7450, RG-7458, RG-7593, RG-7596, RG-7598, RG-7599, RG-7600, RG-7636, ABT-414, IMGN-853, IMGN-529, vorsetuzumab mafodotin, and lorvotuzumab mertansine.

[0062] Engineering: Generally, the term "engineering" can refer to aspects that are artificially manipulated. For example, when a polypeptide sequence is artificially manipulated, the polypeptide is considered "engineered". For example, in some embodiments of the present invention, an engineered polypeptide includes a sequence that contains one or more amino acid mutations, deletions, and / or insertions that have been artificially introduced into a reference polypeptide sequence. In some embodiments, an engineered polypeptide contains 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 occur naturally in vivo. Similarly, if a cell or organism has been manipulated such that its genetic information has been altered (e.g., new genetic material that was not previously present has been introduced, such as by transformation, mating, somatic hybridization, transfection, transduction, or other mechanisms, or existing genetic material has been altered or removed, such as by substitution or deletion mutations or by mating schemes), then the cell or organism is considered "engineered". As is common practice and understood by those skilled in the art, derivatives and / or progeny of engineered polypeptides or cells are generally still referred to as "engineered", even if the actual manipulation was performed on an existing entity.

[0063] Host cell: As used herein, the term "host cell" can refer to a cell of an organism that has been selected, modified, transformed, grown, used, or manipulated in any way to produce a material, such as the expression of a gene, DNA or RNA sequence, protein, or enzyme in the cell. Host cells can include immune cells, including but not limited to lymphocytes (e.g., T cells, B cells, and NK cells), neutrophils, and monocytes / macrophages.

[0064] 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 cell culture, etc.) rather than within a multicellular organism.

[0065] In vivo: As used herein, the term "in vivo" refers to events that occur within a multicellular organism such as a human or non-human animal. In the context of a cell-based system, the term can be used to refer to events that occur within a living cell (as opposed to an in vitro system, for example).

[0066] Isolated: As used herein, the term "isolated" can refer to a substance and / or entity that has been (1) separated from at least some of the components with which it was associated at the time of its initial production (whether in nature and / or in an experimental setting), and / or (2) a substance and / or entity that has been designed, produced, prepared, and / or manufactured by human means. The isolated substance and / or entity can be separated from the other components with which it was initially associated by 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%. In some embodiments, the isolated agent is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or greater than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. In some embodiments, as will be understood by one of ordinary skill in the art, a substance can still be considered "isolated" or even "pure" after being combined with certain other components such as, for example, one or more carriers or excipients (e.g., buffers, solvents, water, etc.); in such embodiments, the percentage of separation or purity of the substance is calculated without including such carriers or excipients. By way of example only, in some embodiments, a biopolymer that occurs in nature such as a polypeptide or polynucleotide is considered "isolated" if: a) due to its origin or source of derivation, it is not associated with some or all of the components that accompany it in its native state; b) the biopolymer is substantially free of other polypeptides or nucleic acids of the same species from the species that produces it in nature; c) it is expressed by or otherwise associated with components from a cell or other expression system that is not of the species that produces the biopolymer in nature. Thus, by way of example, in some embodiments, a polypeptide that is chemically synthesized or synthesized in a cell system different from the cell system that produces the polypeptide in nature is considered an "isolated" polypeptide. Alternatively or additionally, in some embodiments, a polypeptide that has been subjected to one or more purification techniques can be considered an "isolated" polypeptide to the extent that the polypeptide has been separated from: a) other components with which it was associated in nature; and / or b) other components with which it was associated at the time of its initial production.

[0067] Operably linked: As used herein, the term "operably linked" can refer to juxtaposition wherein the components described are in a relationship that permits the components to function in their intended manner. A control element "operably linked" to 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 "operably linked" to an encoding element of interest is contiguous (e.g., covalently linked). In some embodiments, the control element acts in trans or otherwise with respect to the functional element of interest.

[0068] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to a composition in which an active agent is formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the composition is suitable for administration to a human or animal subject. In some embodiments, the active agent is present in unit doses suitable for administration in a treatment regimen that has a statistically significant probability of achieving a predetermined therapeutic effect when administered to the relevant population.

[0069] Polypeptide: As used herein, the term "polypeptide" generally refers to its art-recognized meaning of a polymer of at least three amino acids. One of ordinary skill in the art will understand that the term "polypeptide" is intended to be general enough to encompass polypeptides having the full sequences recited herein, while also encompassing polypeptides representing functional fragments of such full polypeptides (e.g., fragments that retain at least one activity). Additionally, one of ordinary skill in the art will understand that protein sequences generally tolerate some substitutions without destroying activity. Thus, polypeptides that retain activity and share at least about 30%-40% overall sequence identity with another polypeptide of the same class, typically greater than about 50%, 60%, 70%, or 80%, and that generally further contain at least one region having higher identity, typically greater than 90% or even 95%, 96%, 97%, 98%, or 99%, in one or more highly conserved regions that generally span at least 3-4 and often up to 20 or more amino acids are encompassed within the related term "polypeptide" as used herein. Polypeptides can contain L-amino acids, D-amino acids, or both, and can contain any of the various amino acid modifications or analogs known in the art. Useful modifications include, for example, terminal acetylation, amidation, methylation, etc. In some embodiments, a protein can include natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to polypeptides having a length of less than about 100 amino acids, less than about 50 amino acids, less than about 20 amino acids, or less than about 10 amino acids. In some embodiments, a protein is an antibody agent, an antibody fragment, a bioactive portion thereof, and / or a characteristic portion thereof.

[0070] Prevention: As used herein, when used in connection with the occurrence of a disease, disorder, and / or condition, the term "prevent" or "prevention" can refer to reducing the risk of developing a disease, disorder, and / or condition and / or delaying the onset and / or severity of one or more characteristics or symptoms of a disease, disorder, or condition. In some embodiments, prevention is evaluated on a population basis such that if a statistically significant decrease in the development, frequency, and / or intensity of one or more symptoms of the disease, disorder, or condition is observed in a population susceptible to the disease, disorder, or condition, the agent is considered to "prevent" the particular disease, disorder, or condition.

[0071] Recombinant: As used herein, the term "recombinant" can refer to a polypeptide that is designed, engineered, prepared, expressed, produced, manufactured, and / or isolated by recombinant means, such as a polypeptide expressed using a recombinant expression vector transfected into a host cell; a polypeptide isolated from a recombinant, combinatorial human polypeptide library; a polypeptide isolated from an animal (e.g., mouse, rabbit, sheep, fish, etc.) that is transgenic or otherwise has been engineered to express one or more genes or genetic components that encode and / or direct the expression of a polypeptide or one or more of its components, parts, elements, or domains; and / or a polypeptide prepared, expressed, produced, or isolated by any other means that involves splicing or ligating selected nucleic acid sequence elements to each other, chemically synthesizing the selected sequence elements, and / or otherwise generating a nucleic acid that encodes and / or directs the expression of a polypeptide or one or more of its components, parts, elements, or domains. 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 on a computer. 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, e.g., from natural or synthetic sources, e.g., in the germline of a source organism of interest (e.g., human, mouse, etc.).

[0072] Specific binding: As used herein, the term "specific binding" can refer to the ability to distinguish possible binding partners in an environment where binding can occur. When other potential targets are present, a binding agent that interacts with a specific target is said to "specifically bind" to the target with which it interacts. In some embodiments, specific binding is evaluated by detecting or determining the degree of association between the binding agent and its partner. In some embodiments, specific binding is evaluated by detecting or determining the degree of dissociation of the binding agent-partner complex; in some embodiments, specific binding is evaluated by detecting or determining the ability of the binding agent to counter alternative interactions between its partner and another entity. In some embodiments, specific binding is evaluated by performing such detection or determination within a certain concentration range.

[0073] 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, the subject has a relevant disease, disorder, or condition. In some embodiments, the subject is predisposed to a disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject refers to a person having one or more characteristics predisposing to or at risk of having a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual to whom a diagnosis and / or therapy is being and / or has been administered.

[0074] Therapeutic agent: As used herein, the phrase "therapeutic agent" generally refers to any agent that elicits a desired pharmacological effect when administered to an organism. In some embodiments, an agent is considered a therapeutic agent if it exhibits a statistically significant effect across an appropriate population. In some embodiments, the appropriate population can be a population of model organisms. In some embodiments, the appropriate population can be defined by criteria such as certain age groups, genders, genetic backgrounds, pre-existing clinical conditions, etc. In some embodiments, a therapeutic agent is a substance that can be used to alleviate, improve, relieve, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition. In some embodiments, a "therapeutic agent" is an agent that has been or needs to be approved by a government agency for sale for administration to humans. In some embodiments, a "therapeutic agent" is an agent that requires a medical prescription for administration to humans.

[0075] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" means an amount sufficient to treat a disease, disorder, and / or condition when administered to a population having or susceptible to having the disease, disorder, and / or condition according to a therapeutic dosing regimen. In some embodiments, a therapeutically effective amount is an amount that reduces the incidence and / or severity of one or more symptoms of a disease, disorder, and / or condition, stabilizes one or more characteristics of a disease, disorder, and / or condition, and / or delays the onset of one or more symptoms of a disease, disorder, and / or condition. One of ordinary skill in the art will appreciate that the term "therapeutically effective amount" does not in fact require successful treatment of a particular individual. Instead, a therapeutically effective amount can be an amount that provides a particular desired pharmacological response in a large number of subjects when administered to patients in need of such treatment. For example, in some embodiments, the term "therapeutically effective amount" refers to an amount that, when administered to an individual in need in the context of the therapies of the present invention, will block, stabilize, attenuate, or reverse cancer-supporting processes occurring in the individual, or will enhance or increase cancer-suppressing processes 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 development of cancer in the individual. A particularly preferred "therapeutically effective amount" of the compositions described herein reverses (in a therapeutic treatment) the development of a malignancy such as pancreatic cancer, or helps to achieve or prolong remission of a malignancy. The therapeutically effective amount administered to an individual to treat the individual's cancer can be the same as or different from the therapeutically effective amount administered to promote remission or inhibit metastasis. Like most cancer therapies, the treatment methods described herein should not be construed as, limited to, or otherwise restricted to "curing" cancer; rather, the treatment methods involve the use of the described compositions for "treating" cancer, i.e., producing a desired or beneficial change in the health of an individual having cancer. Such benefits are recognized by skilled healthcare providers in the field of oncology and include, but are not limited to, patient condition stabilization, tumor size reduction (tumor regression), improvement in life functions (e.g., improvement in the function of a cancerous tissue or organ), reduction or inhibition of further metastasis, reduction of opportunistic infections, increased viability, pain reduction, improvement in motor function, improvement in cognitive function, improvement in energy perception (vitality, reduction of discomfort), improvement in well-being, restoration of normal appetite, restoration of healthy weight gain, and combinations thereof. Additionally, regression of a particular tumor in an individual (e.g., as a result of the treatment described herein) can also be evaluated by collecting a sample of cancer cells from the tumor site (e.g., during the course of treatment) such as pancreatic cancer and testing the levels of metabolic and signaling markers of the cancer cells to monitor the state of the cancer cells in order to verify the regression of the cancer cells to a less malignant phenotype at the molecular level.For example, tumor regression induced by the methods of the present invention will be indicated by finding a decrease in any of the pro-angiogenic markers discussed above, an increase in the anti-angiogenic markers described herein, normalization of a metabolic pathway, an intercellular signaling pathway, or an intracellular signaling pathway that exhibits abnormal activity in an individual diagnosed with cancer (i.e., a change to the state found in a normal individual without cancer). Those 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, e.g., as part of a dosing regimen.

[0076] Transfection: As used herein, the term "transfection" can refer to the introduction of exogenous nucleic acid into a cell using recombinant DNA techniques. As used herein, the term "transformation" can refer to the introduction of an exogenous gene, DNA, or RNA sequence into a host cell such that the host cell will express the introduced gene or sequence to produce the encoded protein or enzyme.

[0077] Transduction: As used herein, the term "transduction" can refer to the introduction of exogenous nucleic acid into a cell using a viral vector.

[0078] Variant: As used herein in the context of a molecule (e.g., a nucleic acid, protein, or small molecule), the term "variant" can refer to a molecule that shows significant structural identity to a reference entity but is structurally different from the reference molecule, e.g., in the presence or absence of one or more chemical moieties or at the level of the one or more chemical moieties. In some embodiments, the variant is also functionally different from its reference molecule. Generally, whether a particular molecule is properly regarded as a "variant" of a reference molecule is based on the degree of structural identity of the particular molecule to the reference molecule. As will be understood by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements. By definition, a variant is a distinct molecule that shares one or more such characteristic structural elements but differs from the reference molecule in at least one respect. To give just a few examples, a polypeptide can have a characteristic sequence element comprising a plurality of amino acids that have a specified position relative to each other in linear or three-dimensional space and / or contribute to a particular structural motif and / or biological function; a nucleic acid can have a characteristic sequence element comprising a plurality of nucleotide residues that have a specified position relative to each other in linear or three-dimensional space. In some embodiments, a variant polypeptide or nucleic acid can differ from a reference polypeptide or nucleic acid due to one or more differences in the amino acid or nucleotide sequence. In some embodiments, a variant polypeptide or nucleic acid shows an overall sequence identity to a reference polypeptide or nucleic acid of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. 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 of the biological activities of a reference polypeptide or nucleic acid.

[0079] Vector: As used herein, the term "vector" can refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. Vectors can encompass both non-viral vectors and viral vectors for introducing nucleic acids into cells in vitro, ex vivo, or in vivo.

[0080] A vector can be a replicon to which another DNA fragment is attached to amplify the attached fragment. The term "replicon" refers to any genetic element (e.g., plasmid, phage, cosmid, chromosome, or virus) capable of functioning as an autonomous unit of DNA replication in vivo.

[0081] Many vectors known in the art can be used to engineer nucleic acids, incorporate response elements and promoters into genes, etc. Preferred vectors include, but are not limited to, plasmids (e.g., PBR322 or pUC plasmid derivatives), modified viruses (e.g., adenovirus, retrovirus, adeno-associated virus, or herpes virus), or Bluescript vectors. For example, DNA fragments corresponding to response elements and promoters can be inserted into a suitable vector by combining the appropriate DNA fragment with a selected vector having complementary sticky ends. In some embodiments, the ends of the DNA molecule can be enzymatically modified, or any site can be created by ligating a nucleotide sequence to the DNA end via a linker. In some embodiments, the vector can be engineered to contain a selectable marker gene to screen for cells that have incorporated the marker into the cell genome. Such markers enable the identification and / or screening of host cells that express the protein encoded by the marker.

[0082] 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, into which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cells 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 the host cell when introduced into the host cell and thus replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes operably linked thereto. Such vectors are referred to herein as "expression vectors". Non-limiting examples of expression vectors and packaging constructs that can be used to deliver the chimeric antigen receptors described herein include retroviral vectors (e.g., SFG, pMX, pSAMEN, pMP71, pLXSN, pMSCV, pMSGV), lentiviral vectors (e.g., epHIV7, pLenO, pSIN, pSIEW, pELPS, pELNS, pHR), packaging constructs (psPAX2, pRDF, pEQ-PAM3(-E), pVSVg, pCL, pMEVSVg, pMD2G, pMDLg / p.RRE, pRSV.REV, pTSV.rev, pCHGP, pCMV-g, pCMV-Rev2, pCMVdR8.91, pGALY).

[0083] In some embodiments, the vector provides the necessary regulatory sequences (e.g., transcriptional and translational elements) to regulate the expression of the fusion protein in a suitable host cell. The regulatory sequences can include a promoter region, enhancer region, transcription termination site, ribosome binding site, start codon, splice signal, intron, polyadenylation signal, Shine / Dalgarno translation sequence, and Kozak consensus sequence. The regulatory sequences are selected in view of the host cell in which the fusion protein will be produced. In some embodiments, suitable bacterial promoters include, but are not limited to, phage λ pL or pR, T6, T7, T7 / lacO, lac, recA, gal, trp, ara, hut, and trp-lac. In some embodiments, suitable eukaryotic promoters include, but are not limited to, PRBI, GAPDH, metallothionein, thymidine kinase, viral LTR, cytomegalovirus, SV40, or tissue-specific or tumor-specific promoters such as alpha-fetoprotein, amylase, cathepsin E, M1 muscarinic receptor, or gamma-glutamyltransferase.

[0084] In some embodiments, additional vectors include lipid complexes (cationic liposome-DNA complexes), polyplexes (cationic polymer-DNA complexes), and protein-DNA complexes. In addition to the nucleic acid, the vector can also include one or more regulatory regions and / or selectable markers that can be used to select, measure, and monitor the results of nucleic acid delivery (e.g., delivery to certain tissues or duration of expression).

[0085] Vectors can be introduced into the desired host cells by methods known in the art, such as injection, transfection, electroporation, microinjection, transduction, cell fusion, lipofection, calcium phosphate precipitation (Graham, F. L. et al., Virology, 52:456 (1973), Chen and Okayama, Mol. Cell. Biol., 7:2745-2752 (1987)), liposome-mediated structured salt method (Wong, T. K. et al., Gene, 10:87 (1980), Nicolau and Sene, Biochim. Biophys. Acta, 721:185-190 (1982), Nicolau et al., Methods Enzymol., 149:157-176 (1987)), DEAE-dextran treatment (Gopal, Mol. Cell. Biol., 5:1188-1190 (1985)), gene bombardment using gene species or DNA vector transporters (see Wu et al., J. Biol. Chem., 267:963 (1992), Wu et al., J. Biol. Chem., 263:14621 (1988), Hartmut et al., Canadian Patent Application No. 2,012,311) (Yang et al., Proc. Natl. Acad. Sci., 87:9568-9572 (1990)).

[0086] In some embodiments, viral vectors have been widely used in gene transfer applications in cells as well as in live animal subjects. Viral vectors that can be used include, but are not limited to, adenovirus, retrovirus, vaccinia virus, poxvirus, adeno-associated virus, herpes simplex virus, lentivirus, baculovirus, Sendai virus, measles virus, simian virus 40, and Epstein-Barr

[0087] virus vectors. Non-viral vectors include plasmids, lipid complexes (cationic liposome-DNA complexes), polyplexes (cationic polymer-DNA complexes), and protein-DNA complexes. In addition to the nucleic acid, the vector can include one or more regulatory regions and / or selectable markers that can be used to screen, measure, and monitor the results of nucleic acid delivery (e.g., delivery to tissues or persistence of expression).

[0088] In some embodiments, polynucleotides can be introduced in vivo by lipofection. The use of liposomes for encapsulation and transfection of nucleic acids in vitro has increased. In some embodiments, synthetic cationic lipids designed to limit the difficulties and risks encountered with liposome-mediated transfection can be used to prepare liposomes for in vivo gene transfection (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413 (1987), Mackey et al., Proc. Natl. Acad. Sci. USA 85:8027 (1988), Ulmer et al., Science 259:1745 (1993)). In some embodiments, the use of cationic lipids can facilitate the encapsulation of negatively charged nucleic acids and can also facilitate fusion with negatively charged cell membranes (Feigner et al., Science 337:387 (1989)). Particularly useful lipid compounds and compositions for delivering nucleic acids are described in WO95 / 18863, WO96 / 17823, and U.S. 5,459,127, which are incorporated herein by reference in their entirety. In some embodiments, for tissues with cellular heterogeneity, such as the pancreas, liver, kidney, and brain, direct transfection of specific cell types is clearly particularly desirable. In some embodiments, lipids can be chemically conjugated to other molecules for targeting (Mackey et al., 1988). In some embodiments, targeting peptides such as hormones or neurotransmitters and protein or non-peptide molecules such as antibodies can be chemically conjugated to liposomes.

[0089] 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 the manufacturer's instructions or as commonly practiced in the art or as described herein. The foregoing techniques and procedures can generally be performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), which is incorporated herein by reference for any purpose.

[0090] Engineered immune cells

[0091] As used herein, "immune cell" refers to cells of the immune system, which can be classified as lymphocytes (e.g., T cells, B cells, and NK cells), neutrophils, and monocytes / macrophages. 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 a macrophage. 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., chimeric antigen receptor) or contains exogenous nucleic acid.

[0092] 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-natural molecule that is a receptor for an antigen present on the surface of one or more types of cells. In some embodiments, the immune cell comprises an immune cell (e.g., T cell) not found in nature because the immune cell has been engineered to include or express at least one synthetic molecule not found in nature. In specific 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), mutant variant receptor (MVR), HLA-DR (human leukocyte antigen-D related), or CD19. In specific embodiments, the immune cell can be a T cell, e.g., CD4 + T cell, CD8 +T cells, Treg cells, Th1 T cells, Th2 T cells, Th17 T cells, non-specific T cells, or a population of T cells comprising any combination of the foregoing. Engineered immune cells (e.g., T cells) with chimeric antigen receptors (CAR T cells) have great therapeutic potential in treating cancer. Using a CAR, the receptor can be programmed to recognize an antigen that, upon binding, activates the immune cell to kill cells expressing the antigen. Thus, immune cells expressing a CAR against an antigen expressed on tumor cells can target and kill the tumor cells. For example, recent clinical trials of CD19-targeted CAR-transduced T cells (CD19-CAR T cells) for hematological malignancies have demonstrated 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 at least partially attributed to the fusion structure of the CAR, which is formed 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).

[0093] A CAR comprises 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 transmembrane domains from molecules such as CD8 and CD28, and the intracellular signaling domain employs intracellular signaling domains based on immunoreceptor tyrosine-based activation motifs (e.g., CD3ζ) and costimulatory signaling molecules (e.g., CD28 and CD137 (4-1BB)).

[0094] As used herein, "single-chain variable fragment, scFv" refers to a fragment of an antibody that is a recombinant protein defined to include a heavy-chain variable domain (VH) and a light-chain variable domain (VL) linked by a linker that associates the two domains such that an antigen-binding site is formed.

[0095] In some embodiments, the transmembrane domain is the transmembrane domain from a protein selected from: 4-1BB / CD137, activated 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, CD3δ, CD3ε, CD3γ, CD3ζ, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8α, CD8β, CD96 (tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRTAM, cytokine receptor, DAP-10, DNAM1 (CD226), Fcγ receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Igα (CD79a), IL-2Rβ, IL-2Rγ, IL-7Rα, inducible T cell co-stimulator (ICOS), integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, ligand that specifically binds to CD83, LIGHT, LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1), MHC class I molecule, NKG2C, NKG2D, 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.

[0096] In some embodiments, the intracellular signaling domain comprises the intracellular signaling domain from a protein selected from the group consisting of: 4-1BB / CD137, activated 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, CD3δ, CD3ε, CD3γ, CD3ζ, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8α, CD8β, CD96 (tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRTAM, cytokine receptor, DAP-10, DNAM1 (CD226), Fcγ receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Igα (CD79a), IL-2Rβ, IL-2Rγ, IL-7Rα, inducible T cell co-stimulator (ICOS), integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, ligand that specifically binds to CD83, LIGHT, LTBR, Ly9 (CD229), Lyl08, lymphocyte function-associated antigen-1 (LFA-1), MHC class 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.

[0097] In some embodiments, the chimeric antigen receptor further comprises an additional antigen-binding domain. In some embodiments, the chimeric antigen receptor is a bispecific CAR (i.e., targeting two antigen-binding domains). In some embodiments, the chimeric antigen receptor is multivalent (i.e., targeting multiple antigen-binding domains). In some embodiments, the additional antigen-binding domain is a scFv.

[0098] Immune cells (e.g., T cells) can be from any source known in the art. For example, immune (e.g., T) cells can be differentiated in vitro from a population of hematopoietic stem cells, or immune (e.g., T) cells can be obtained from a subject. T cells can be obtained from peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, or a tumor. Additionally, immune (e.g., T) cells can be derived from one or more immune cell lines available in the art. In some embodiments, any number of techniques known to those of skill in the art (such as FICOLL TM separation and / or apheresis of blood components) can be used to obtain T cells from blood collected from a subject. Additional methods for isolating T cells for T cell therapy are disclosed in U.S. Patent Publication No. 2013 / 0287748. Other non-limiting examples can be found in International Application No. PCT / US2015 / 014520 (published as WO2015 / 120096) and International Application No. PCT / US2016 / 057983 (published as WO2017 / 070395), each of which is incorporated herein by reference in its entirety.

[0099] In some embodiments, the immune cells are autologous T cells. In some embodiments, the immune cells are obtained from a subject who is not the patient. In some embodiments, the T cells for a treatment method are syngeneic (the donor and recipient are different, but identical twins). In some embodiments, the T cells for a treatment method are allogeneic to the recipient subject (from the same species but different donors). In some embodiments, the T cells are autologous stem cells (for 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 with cancer or a tumor.

[0100] T cells can be engineered to express, for example, a chimeric antigen receptor (CAR). In some embodiments, CAR-T cells can be engineered to express an extracellular single-chain variable fragment (scFv). In some embodiments, the CAR is engineered such that the co-stimulatory 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, which are incorporated herein by reference in their entirety.

[0101] CAR construct

[0102] The present disclosure provides, at least in part, chimeric antigen receptor (CAR) polypeptides. As used herein, a "chimeric antigen receptor (CAR)" refers to a receptor that does not exist in nature and that can endow an immune effector cell with specificity for a particular antigen. In some embodiments, a CAR refers to a receptor for the specific delivery of a monoclonal antibody agent to a T cell. Generally, a CAR comprises an extracellular binding domain (ectodomain), a transmembrane domain, and an intracellular signaling domain (endodomain).

[0103] In some embodiments, to achieve robust immune (e.g., CAR-T) cell expansion, function, persistence, and anti-tumor activity, co-stimulatory signals can be provided by incorporating an intracellular signaling domain from an immune (e.g., T cell) cell co-stimulatory molecule into the CAR construct. In some embodiments, the choice and location of co-stimulatory domains within the CAR construct may affect immune (e.g., CAR-T) cell function and fate, and have different effects on immune (e.g., CAR-T) cell kinetics, cytotoxic function, and potential safety profiles. Non-limiting examples of co-stimulatory molecules include CD28, ICOS, CD27, 4-1BB / CD137, OX40, and CD40L.

[0104] As used herein, 4-1BB / CD137 is an activation-induced T cell co-stimulatory molecule that is expressed on a subset of resting CD8+ T cells and upregulated on both CD4+ and CD8+ T cells upon activation. In some embodiments, T cells expressing a CAR incorporating the 4-1BB / CD137 domain can express granzyme B, IFN-γ, TNF-α, GM-CSF, and the anti-apoptotic protein Bcl-XL (Zhong et al., Mol. Ther. 2010; 18:413-420), and a CAR incorporating the 4-1BB / CD137 co-stimulatory domain can exhibit longer CAR-T cell persistence (Zhao et al., Cancer Cell 2015; 28:415-428). In some embodiments, the intracellular domain of the chimeric receptor described herein comprises a 4-1BB signaling domain followed by a five amino acid sequence that can be further combined with any other desired extracellular domain, transmembrane domain, and / or intracellular domain useful in the context of the chimeric receptor.

[0105] In some embodiments, the CAR comprises: (a) an extracellular domain that includes an antigen-binding domain; (b) a transmembrane domain; and (c) an intracellular domain that includes a co-stimulatory intracellular domain, wherein the co-stimulatory intracellular domain includes an intracellular signaling domain from 4-1BB / CD137 and five additional amino acids. As contemplated herein, the CAR construct can include an extracellular domain involving any desired antigen-binding domain. In some embodiments, the co-stimulatory intracellular domain includes an intracellular signaling domain from 4-1BB / CD137 and five additional amino acids, wherein the five additional amino acids are encoded by SEQ ID NO:1. In some embodiments, the co-stimulatory intracellular domain includes SEQ ID NO:2. In some embodiments, the co-stimulatory intracellular domain includes a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:2 or 4.

[0106] SEQ ID NO:1 - Five additional amino acids (DNA sequence)

[0107]

[0108] SEQ ID NO:2 - 4-1BB co-stimulatory domain and five additional amino acids (DNA sequence)

[0109]

[0110] SEQ ID NO:3 - Five additional amino acids (amino acid sequence)

[0111]

[0112] SEQ ID NO:4 - 4-1BB co-stimulatory domain and five additional amino acids (amino acid sequence)

[0113]

[0114] In some embodiments, the extracellular binding domain of the CAR comprises an antigen-binding domain. In some embodiments, the antigen-binding domain specifically binds an antigen associated with a disease. In some embodiments, the antigen-binding domain specifically binds a tumor antigen. In some embodiments, the antigen-binding domain specifically binds any number of targets, including surface antigens, cytoplasmic antigens, or nuclear antigens. For example, the antigen-binding domain can bind BCMA, CD2, CD3, CD4, CD8, CD10, CD19, CD20, CD22, CD23, CD33, CD38, CD44, CD52, CD70, CD99, CD138, CD123, CD274, TIM-3, epidermal growth factor receptor family members (erb1, erb2, erb3, erb4, and their mutants), ephrin receptor family members (EphA1-10, EphB1-6), prostate-specific antigens (e.g., prostate stem cell antigen PSCA, prostate-specific membrane antigen PSMA), embryonic antigens (e.g., carcinoembryonic antigen CEA, fetal acetylcholine receptor), vascular endothelial growth factor family members (VEGFR1-3), epithelial cell adhesion molecule EpCAM, alpha-fetoprotein (AFP), mucin family members (e.g., MUC1, MUC16), follicle-stimulating hormone receptor (FSHR), human high-molecular-weight melanoma-associated antigen (HMW-MAA), folate-binding protein FBP, alpha-folate receptor, ligands of the NKG2D receptor, epithelial glycoprotein family members (e.g., EGP-2, EGP-4), disialogangliosides (e.g., GD2, GD3), carbonic anhydrase family members (e.g., CAIX), and carbohydrate antigen family members (e.g., Ley), including mutants of the named proteins and protein families. In some embodiments, the antigen-binding domain can bind an antibody or a fragment thereof that binds a cytoplasmic antigen or a nuclear antigen, such as the La / SSB antigen, members of the GTPase Rho family, members of the high-mobility group protein family, etc. Similarly, the antigen-binding domain can bind the alpha and beta or gamma and delta chains of a T cell receptor (TCR) or a fragment thereof. In some embodiments, the antigen-binding domain can bind a peptide presented by a human leukocyte antigen class (HLA) I and II protein complex.Examples include, but are not limited to, TCRs specific for peptides derived from proteins such as the EGFR family, survivin, sry-like high mobility group box (SOX) protein family, melanoma-associated antigens (e.g., the autoimmunogenic cancer / testis antigen NY-ESO-1, members of the melanoma antigen family A MAGEA, the antigen preferentially expressed in melanoma PRAME, gp100, MART-1), and leukemia-associated antigens (e.g., AML1-ETO, DEK-CAN, PML-RARα, Flt3-ITD, NPM1, AurA, Bcl-2, Bl-1, BMI1, BRAP, CML28, CML66, cyclin A, cyclin B1, cyclin E, CYP1B1, ETO / MTG8, G250 / CAIX, HOXA9, hTERT, Mcl-1, MAGE, mesothelin, mHAg, myeloperoxidase, MPP11, MUC1, NuSAP1, OFA / iLRP, PASD1, PRAME, proteinase 3, RAGE-1, RGS5, RHAMM, SSX2IP, survivin, Wilms tumor gene 1 (WT1)). The antigen-binding domain can bind to cytokine receptors (e.g., IL-13 receptor, IL-22 receptor), NKG2D receptors (e.g., ULBP1, ULBP2), EGFR family members, or autoreactive TCRs. In some embodiments, the antigen-binding domain specifically binds to a tumor antigen. Examples include, but are not limited to, glypican-3 (GPC), malignant variant receptor (MVR), HLA-DR (human leukocyte antigen-D related), AFP, CEA, CA-125, MUC-1, ETA, tyrosinase, MAGE, immature laminin receptor, TAG-72, HPV E6, HPV E7, BING-4, calcium-activated chloride channel 2, cyclin-B1, 9D7, Ep-CAM, EphA3, Her2 / neu, telomerase, mesothelin, SAP-1, survivin, NY-ESO-1 / LAGE-1, PRAME, SSX-2, BRCA1 / 2, CDK4, CML66, or CD19. 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, MVR, HLA-DR, or CD19.

[0115] In some embodiments, the transmembrane domain is a transmembrane domain from a protein selected from the following: 4-1BB / CD137, activated 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, CD3δ, CD3ε, CD3γ, CD3ζ, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8, CD8α, CD8β, CD96 (tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRTAM, cytokine receptor, DAP-10, DNAM1 (CD226), Fcγ receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Igα (CD79a), IL-2Rβ, IL-2Rγ, IL-7Rα, inducible T cell co-stimulator (ICOS), integrin, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, ligand that specifically binds to CD83, LIGHT, LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1), MHC class I molecule, NKG2C, NKG2D, 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 the transmembrane domain from CD8α. In some embodiments, the transmembrane domain comprises SEQ ID NO:5. In some embodiments, the transmembrane domain contains a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:5.

[0116] SEQ ID NO:5 - CD8 / Hinge / Transmembrane Domain

[0117]

[0118] In some embodiments, the intracellular domain further comprises the intracellular domain from CD3ζ. In some embodiments, the intracellular domain comprises SEQ ID NO:6. In some embodiments, the intracellular domain contains a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:6.

[0119] SEQ ID NO:6 - CD3ζ

[0120]

[0121] In some embodiments, the CAR further comprises a T2A self-cleaving peptide. In some embodiments, the CAR further comprises a signal peptide or a leader sequence. In some embodiments, the CAR further comprises a CD8α leader sequence. In some embodiments, the CAR further comprises a flag tag sequence. In some embodiments, the CAR further comprises a hinge region. In some embodiments, the hinge region is the CD8α hinge. In some embodiments, the CAR further comprises SEQ ID NO:7. In some embodiments, the CAR further comprises SEQ ID NO:8. In some embodiments, the extracellular domain contains a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:7 or 8.

[0122] SEQ ID NO:7 - CD8α leader sequence

[0123]

[0124] SEQ ID NO:8 - Flag tag sequence

[0125]

[0126] GPC3 CAR

[0127] Phosphatidylinositol glycan-3 (GPC3) is a cell surface protein encoded by the GPC3 gene in humans and is a carcinoembryonic antigen re-expressed in neoplastic hepatocytes at high frequency (Vidali et al., 2008, Journal of Hepatology (Jhepatol) 48:399-406). GPC3 is highly expressed in fetal liver and is not expressed in normal adult liver tissue, but its expression is reactivated in hepatocellular carcinoma and is closely related to the development of liver cancer, where the detection rate of GPC3 expression is relatively high in the early stage of liver cancer and increases with the development of liver cancer. Further, GPC3 is also expressed in tumors such as melanoma, ovarian clear cell carcinoma, yolk sac tumor, neuroblastoma and other tumors. Considering the high expression in hepatocellular carcinoma, melanoma and other tumors, GPC3 has become a useful immunohistochemical diagnostic test (Anatelli et al., 2008, American Journal of Clinical Pathology (Am J Clin Path) 130:219-223) and a potential biomarker (Aburatani, 2005, Journal of Gastroenterology (J Gastroenterol) 40.S16:1-6).

[0128] GPC3 is a member of the proteoglycan family that functions as an extracellular matrix or as a receptor for cell growth factors in cell adhesion during organogenesis. The proteonucleus of GPC3 consists of two subunits, an N-terminal subunit and a C-terminal subunit. A glycosylphosphatidylinositol (GPI) anchor is added to the serine at position 560 on the carboxyl (C) terminal side of GPC3. The GPI anchor plays a role in localizing GPC3 on the cell surface through covalent binding to cell membrane lipids. Similarly, the serine at position 495 and the serine at position 509 of GPC3 are modified by heparan sulfate chains (HS chains), and it is known that HS chains regulate multiple growth signal transduction pathways such as the Wnt signal, FGF signal and BMP signal transduction pathways. The growth signal transduction pathways involved are known to vary depending on the cancer type. For example, in hepatocellular carcinoma (HCC), cells grow through stimulation of the Wnt signaling pathway.

[0129] The present disclosure provides, at least in part, GPC3 CAR polypeptides. In some embodiments, the extracellular binding domain of GPC3 CAR includes 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 GPC3.

[0130] In some embodiments, the chimeric antigen receptor (CAR) polypeptide comprises: i) an extracellular antigen-binding domain, the extracellular antigen-binding domain including a light chain variable domain, the light chain variable domain including: a light chain CDR1 comprising SEQ ID NO:9; a light chain CDR2 comprising SEQ ID NO:10; and a light chain CDR3 comprising SEQ ID NO:11; and a heavy chain variable domain, the heavy chain variable domain including: a heavy chain CDR1 comprising SEQ ID NO:12; a heavy chain CDR2 comprising SEQ ID NO:13; and a heavy chain CDR3 comprising SEQ ID NO:14; ii) a transmembrane domain; and iii) an intracellular signaling domain that causes T cell activation when an antigen binds to an antibody agent.

[0131] Sequence SEQ ID NO: Light - chain CDR1 RSSQSLVHSNGNTYLH 9 Light - chain CDR2 KVSNRFS 10 Light - chain CDR3 SQNTHVPPT 11 Heavy - chain CDR1 DYEMH 12 Heavy - chain CDR2 ALDPKTGDTAYSQKFKG 13 Heavy - chain CDR3 FYSYTY 14

[0132] In some embodiments, the CAR polypeptide comprises: i) an extracellular antigen-binding domain, the extracellular antigen-binding domain including a light chain variable domain and a heavy chain variable domain, the light chain variable domain comprising a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:15, and the heavy chain variable domain comprising a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:16;

[0133] ii) a transmembrane domain; and iii) an intracellular signaling domain that causes T cell activation when an antigen binds to an antibody agent. In some embodiments, the CAR polypeptide comprises: i) an extracellular antigen-binding domain, the extracellular antigen-binding domain including a light chain variable domain and a heavy chain variable domain, the light chain variable domain comprising a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:17, and the heavy chain variable domain comprising a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:18; ii) a transmembrane domain; and

[0134] iii) an intracellular signaling domain that causes T cell activation when an antigen binds to an antibody agent.

[0135] In some embodiments, the CAR polypeptide comprises: i) an extracellular antigen-binding domain comprising a light chain variable domain comprising SEQ ID NO:15 and a heavy chain variable domain comprising SEQ ID NO:16; ii) a transmembrane domain; and iii) an intracellular signaling domain that causes T cell activation when an antigen binds to the antibody agent.

[0136] SEQ ID NO:15 - Human GC33 light chain variable region (amino acid sequence)

[0137]

[0138] SEQ ID NO:16 - Human GC33 heavy chain variable region (amino acid sequence)

[0139]

[0140] SEQ ID NO:17 - Human GC33 light chain variable region (DNA sequence)

[0141]

[0142] SEQ ID NO:18 - Human GC33 heavy chain variable region (DNA sequence)

[0143]

[0144] MVR CAR

[0145] Human leukocyte antigen-DR (HLA-DR) is a classical major histocompatibility complex class II molecule (Shackelford, D.A. et al., 1982 Immunol. Rev. 66:133-187). HLA-DR and its ligands (peptides nine amino acids or longer in length) constitute the ligands for the T cell receptor (TCR). HLA-DR molecules are upregulated in response to signaling. In the case of infection, peptides such as staphylococcal enterotoxin I peptides bind to DR molecules and are presented to the T cell receptor found on T helper cells. These cells then bind to antigens on the surface of B cells, thereby stimulating B cell proliferation.

[0146] The main function of HLA-DR is to present peptide antigens that may be foreign in origin to the immune system, to initiate or inhibit T (helper) cell responses that ultimately lead to the production of antibodies against the same peptide antigen. HLA-DR is an αβ heterodimeric cell surface receptor, each subunit of which contains two extracellular domains, a transmembrane domain, and a cytoplasmic tail region. Both the α-chain and the β-chain are anchored in the membrane. The N-terminal domain of the mature protein forms an α-helix that constitutes the exposed part of the binding groove, and the C-terminal cytoplasmic region interacts with the other chain, thereby forming a transmembrane β-sheet under the binding groove. Most of the peptide contact positions are in the first 80 residues of each chain.

[0147] HLA-DR has restricted expression on antigen-presenting cells (e.g., dendritic cells, macrophages, monocytes, and B cells). An increase in the abundance of HLA-DR "antigen" on the cell surface is usually a response to stimulation, and thus HLA-DR is also a marker of immune stimulation. Due to the high expression level of HLA-DR in B cell malignancies and its limited expression profile on normal cells, antibodies against HLA-DR have been developed and tested in preclinical and clinical studies for B cell malignancies. (Nagy, Z. A. et al., (2002) Nat. Med. 8:801 - 807; DeNardo, G. L. et al., (2005) Clin. Cancer Res. 11:7075s - 7079s; Ivanov, A. et al., (2009) J. Clin. Invest. 119:2143 - 2159; Lin, T. S. et al., (2009) Leuk. Lymphoma 50:1958 - 1963). In phase I / II trials, although the toxicity was not severe, further studies were stopped due to limited efficacy (Lin, T. S. et al., (2009) Leuk. Lymphoma 50:1958 - 1963).

[0148] As used herein, the malignant variant receptor (MVR) antibody agent recognizes the polymorphic region of HLA-DR (described in U.S. Patent Application Publication No. US 2016-0257762, which is incorporated herein by reference in its entirety). The present disclosure provides, at least in part, MVR CAR polypeptides. Figure 1 A schematic diagram of an exemplary MVR CAR construct according to the present disclosure is shown. In some embodiments, the extracellular domain of the CAR includes an antigen-binding domain. In some embodiments, the antigen-binding domain is or includes an antibody agent. In some embodiments, the antigen-binding domain is or includes an antibody agent that specifically binds HLA-DR.

[0149] In some embodiments, the CAR polypeptide comprises a single-chain variable fragment (scFv) form of an anti-MVR antibody agent. In some embodiments, the CAR polypeptide comprises SEQ ID NO:19. In some embodiments, the CAR comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:19.

[0150] In some embodiments, the CAR polypeptide comprises a single-chain variable fragment (scFv) form of an anti-MVR antibody agent. In some embodiments, the CAR polypeptide comprises SEQ ID NO:20. In some embodiments, the CAR comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:20.

[0151] SEQ ID NO:19 - MVRL2H2 (amino acid sequence)

[0152]

[0153] SEQ ID NO:20 - MVRL2H2 (DNA sequence)

[0154]

[0155] CD19 CAR

[0156] CD19 is a biomarker of normal and neoplastic B cells as well as follicular dendritic cells. CD19 plays a key role in establishing the intrinsic B cell signaling threshold by regulating both B cell receptor-dependent and -independent signaling. In addition, CD19 functions as the major signaling component of a multimolecular complex on the surface of mature B cells together with the complement receptor CD21 and the tetraspanin CD81 (TAPA-1) as well as CD225, where CD19 also plays a key role in maintaining the balance between humoral, antigen-induced responses and tolerance induction.

[0157] The present disclosure provides, at least in part, CD19 CAR polypeptides. In some embodiments, the extracellular domain of the CAR comprises an antigen-binding domain. In some embodiments, the antigen-binding domain is or comprises an antibody agent. In some embodiments, the antigen-binding domain is or comprises an antibody agent that specifically binds CD19.

[0158] In some embodiments, the CAR polypeptide comprises a single-chain variable fragment (scFv) form of an anti-CD19 antibody agent. In some embodiments, the CAR polypeptide comprises SEQ ID NO:21. In some embodiments, the CAR comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:21.

[0159] In some embodiments, the CAR polypeptide comprises a single-chain variable fragment (scFv) form of an anti-CD19 antibody agent. In some embodiments, the CAR polypeptide comprises SEQ ID NO:22. In some embodiments, the CAR comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:22.

[0160] SEQ ID NO:21 - CD19 (amino acid sequence)

[0161]

[0162] SEQ ID NO:22 - CD19 (DNA sequence)

[0163]

[0164] Nucleic acid

[0165] As used herein, "nucleic acid" is used to encompass any compound and / or substance that comprises a polynucleotide. Exemplary nucleic acids or polynucleotides can include, but are not limited to, ribonucleic acid (RNA) and / or deoxyribonucleic acid (DNA).

[0166] In some embodiments, the nucleic acid construct comprises a region encoding a CAR, wherein the CAR comprises: (a) an extracellular domain comprising an antigen-binding domain; (b) a transmembrane domain; and (c) an intracellular domain comprising an intracellular signaling domain from 4-1BB / CD137 and five additional amino acids. In some embodiments, the nucleic acid construct can be inserted into an expression vector or a 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 virus-derived vectors (e.g., any adenovirus-derived vector (AV), cytomegalovirus-derived (CMV) vector, simian virus-derived (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.

[0167] In some embodiments, the expression vector further comprises a promoter operably linked to the nucleic acid. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the expression vector comprises SEQ ID NO:23, 24, 25, 26, 27, and / or 28. In some embodiments, the expression vector comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:23, 24, 25, 26, 27, and / or 28.

[0168] SEQ ID NO:23 - EF1α - promoter

[0169]

[0170] SEQ ID NO:24 - U5 repeat

[0171]

[0172] SEQ ID NO:25 - Gag / Pol

[0173]

[0174] SEQ ID NO:26 - cPPT

[0175]

[0176] SEQ ID NO:27 - Woodchuck / PRE

[0177]

[0178] SEQ ID NO:28 - R region

[0179]

[0180] Lentiviral vectors are derived from lentiviruses. Lentiviral vectors are based on single - stranded RNA lentiviruses, which are a subclass of retroviruses. Lentiviral vectors combine the advantages of moderate cloning capacity with stable gene expression, where the lentiviral vectors are capable of transducing both dividing and non - dividing cells, including neurons. After infection, the lentiviral genome integrates the transgene into the host genome and promotes long - term gene expression. Lentiviral vectors such as HIV - based vectors are exemplary retroviral vectors for gene delivery. Different from other retroviruses, HIV - based vectors are known to incorporate their passenger genes into non - dividing cells and can thus be used to treat persistent disease forms.

[0181] Additional sequences can be added to such cloning and / or expression sequences to optimize their function in cloning and / or expression, to aid in the isolation of polynucleotides or to improve the introduction of polynucleotides into cells. The use of cloning vectors, expression vectors, adaptors, and linkers is well known in the art.

[0182] In some embodiments, a nucleic acid molecule is inserted into a vector that is capable of expressing the CARs of the present disclosure when introduced into engineered immune cells. In some embodiments, the engineered immune cells are T cells.

[0183] Generation of CAR - T cells

[0184] Methods for generating immune cells comprising CARs are provided herein. In some embodiments, the immune cells into which the CAR is introduced are human immune cells. In some embodiments, the immune cells are autologous human immune cells. In some embodiments, the immune cells are allogeneic human immune cells. In some embodiments, the immune cells are CD4 + T cells (helper T cells, Th cells), CD8 + T cells (cytotoxic T cells, CTLs), memory T cells, regulatory T cells (Treg cells), apoptotic T cells, but are not limited thereto. In some embodiments, the immune cells are NK cells.

[0185] In some embodiments, viral infection of immune cells can include transfecting host cells (e.g., 293T cells, PBMCs, Plat-GP cells, or PA317) with a CAR expression vector and a packaging plasmid to produce a recombinant virus and infecting the immune cells with the recombinant virus. The viral infection method can be performed by any method known in the art. In some embodiments, the transfer of the CAR expression vector to the immune cells can be confirmed by examining the expression of the CAR or by examining the expression of a marker gene inserted in the vector using flow cytometry, Northern blotting, Southern blotting, PCR (e.g., RT-PCR), ELISA, or Western blotting.

[0186] In some embodiments, the present disclosure provides methods of generating engineered immune cells, the methods comprising: introducing into the immune cells: (i) a nucleic acid encoding a CAR, wherein the CAR comprises: (a) an extracellular domain comprising an antigen-binding domain; (b) a transmembrane domain; and (c) an intracellular domain comprising a co-stimulatory endodomain, wherein the co-stimulatory endodomain comprises an intracellular signaling domain from 4-1BB / CD137 and five additional amino acids; or (ii) a vector comprising a nucleic acid encoding a CAR, wherein the CAR comprises: (a) an extracellular domain comprising an antigen-binding domain; (b) a transmembrane domain; and (c) an intracellular domain comprising a co-stimulatory endodomain, wherein the co-stimulatory endodomain comprises an intracellular signaling domain from 4-1BB / CD137 and five additional amino acids.

[0187] In some embodiments, to increase the immunological efficacy in the cytoplasmic signaling domain 4-1BB, 5 amino acids are added to the 4-1BB cytoplasmic domain used to generate the CAR as a co-stimulatory signaling factor. In some embodiments, the complete construct comprises: an antigen-binding domain, which is a scFv; an EF1-α promoter; a hinge region and a transmembrane domain of human CD8; and an intracellular signaling domain. Specifically, the intracellular signaling domain comprises a stimulatory domain and a co-stimulatory signaling domain. In some embodiments, the transmembrane domain may comprise one or more of the α, β, or ζ chains of the T cell receptor, or CD28, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154, but is not limited thereto. In some embodiments, the transmembrane domain comprises CD8. In some embodiments, the intracellular signaling domain comprises a co-stimulatory signaling domain in the CD3ζ primary signaling domain, and the co-stimulatory signaling domain is selected from CD28, 0X40, CD27, ICAM-1, ICOS (CD278), and 4-1BB / CD137. In some embodiments, the co-stimulatory domain comprises 4-1BB, to which 5 consecutive amino acids are added. In some embodiments, the transmembrane domain is linked to CD3ζ.

[0188] In some embodiments, a method of generating the engineered immune cells of the present disclosure further comprises culturing the engineered immune cells in vitro for at least 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, or 12 days.

[0189] In some embodiments, the method of generating engineered immune cells further comprises culturing the engineered immune cells after the introducing step. In some embodiments, the method of generating engineered immune cells further comprises obtaining the immune cells from a subject before the introducing step.

[0190] In the context of the present disclosure, any method known in the art for expressing CAR in immune cells can be used. For example, there are various nucleic acid vectors known in the art for expression, such as linear polynucleotides, polynucleotides complexed with ionic or amphiphilic compounds, plasmids, or viral vectors, even though the present disclosure is not limited thereto. In some embodiments, the vector for expressing CAR in immune cells may be or comprise an autonomously replicating plasmid or virus or its derivative. The viral vector may include but is not limited to an adenovirus vector, an adeno-associated virus vector, a retroviral vector, etc. In some embodiments, a lentiviral vector, which is a retroviral vector, can be used. In some embodiments, the vector is a non-plasmid and non-viral compound, such as a liposome.

[0191] The present disclosure encompasses the recognition that CAR-T cells generated by the methods described herein can be therapeutically useful (e.g., for treating cancer).

[0192] Therapeutic applications

[0193] Provided herein are methods of treating a subject having cancer or other malignancy, wherein the methods comprise administering to the subject a composition comprising or delivering an immune cell comprising a CAR. In some embodiments, the cancer is a glypican-3-related cancer. In some embodiments, the cancer is a CD19-related cancer. In some embodiments, the cancer is an MVR-related cancer.

[0194] Cancer can refer to a large group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth lead to the formation of malignant tumors that invade adjacent tissues and can also metastasize to distant parts of the body through the lymphatic system or bloodstream. Cancer or cancerous tissue can contain tumors.

[0195] “Glypican-3-related cancer” is a cancer characterized by the presence of glypican-3 on the surface of cancer cells. The membrane-bound heparan sulfate proteoglycan GPC3 is overexpressed in approximately 70% to 80% of hepatocellular carcinomas but is not normally expressed in healthy tissues. Additionally, GPC3 overexpression has been found in several types of tumors, such as, but not limited to, hepatocellular carcinoma, hepatoblastoma, germ cell tumors (e.g., yolk sac tumor, choriocarcinoma), Wilms tumor, gastric cancer, non-small cell lung cancer, and thyroid cancer.

[0196] “CD19-related cancer” is a cancer characterized by CD19 expression, wherein CD19 has been shown to play an important role in B cell development and maturation. CD19 expression is highly conserved in most B cell tumors. CD19 is expressed in most acute lymphoblastic leukemias (ALL), chronic lymphocytic leukemias (CLL), and B cell lymphomas.

[0197] "MVR-related cancer" is characterized by increased HLA-DR antigen expression in cancer cells relative to non-cancer cells from a subject. In some embodiments, cancers having higher HLA-DR antigen expression can include but are not limited to bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, blood cancer, laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, stomach cancer, thyroid cancer, pancreatic cancer, and prostate cancer. In some embodiments, diseases associated with HLA-DR expression can include but are not limited to atypical and / or non-classical cancers, malignancies, pre-cancerous conditions, or proliferative diseases expressing HLA-DR, or any combination thereof.

[0198] In some embodiments, cancers treated by the methods of the present disclosure can include but are not limited to carcinoma, lymphoma (e.g., Hodgkin lymphoma and non-Hodgkin lymphoma), blastoma, sarcoma, and leukemia. In some embodiments, cancers can include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, stomach cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, leukemia, and other lymphoproliferative disorders, as well as various types of head and neck cancer.

[0199] In some embodiments, the cancer suitable for treatment by the methods of the present disclosure is a blood cancer. In some embodiments, the blood cancer is leukemia. In some embodiments, the cancer is selected from the group consisting of: one or more acute leukemias, including but not limited to B-cell acute lymphoblastic leukemia (“BALL”), T-cell acute lymphoblastic leukemia (“TALL”), acute lymphoblastic leukemia (ALL); one or more chronic leukemias, including but not limited to chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL); additional blood cancers or blood disorders, including but not limited to B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative disorders, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myeloproliferative neoplastic syndromes, non-Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, and “preleukemia”, which are various collections of blood disorders united by ineffective production (or dysplasia) of myeloid blood cells.

[0200] In some embodiments, the cancer treated by the methods of the present disclosure is B-cell lymphoma (i.e., malignant lymphoma of B-cell origin). B-cell lymphoma includes Hodgkin lymphoma and non-Hodgkin lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, mucosa-associated lymphoid tissue lymphoma (MALT), chronic lymphocytic leukemia, mantle cell lymphoma (MCL), Burkitt's lymphoma, mediastinal large B-cell lymphoma, Waldenstrom macroglobulinemia, nodal marginal zone B-cell lymphoma (NMZL), splenic marginal zone lymphoma (SMZL), intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, and AIDS-related lymphoma, but is not particularly limited thereto, so long as the B-cell lymphoma is a lymphoma of B-cell origin.

[0201] 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 or at least about 10 10. In some embodiments, a therapeutically effective amount of T cells is about 10 4 cells, about 10 5 cells, about 10 6 cells, about 10 7 cells or about 10 8 cells. In some embodiments, a therapeutically effective amount of T cells ranges from about 0.4×10 8 to about 2×10 8 T cells. In some embodiments, a therapeutically effective amount of T cells is about 0.4×10 8 cells, about 0.5×10 8 cells, about 0.6×10 8 cells, about 0.7×10 8 cells, about 0.8×10 8 cells, about 0.9×10 8 cells, about 1.0×10 8 cells, about 1.1×10 8 cells, about 1.2×10 8 cells, about 1.3×10 8 cells, about 1.4×10 8 cells, about 1.5×10 8 cells, about 1.6×10 8 cells, about 1.7×10 8 cells, about 1.8×10 8 cells, about 1.9×10 8 or about 2.0×10 8 T cells.

[0202] In some embodiments, a therapeutically effective amount of CAR T cells is about 2×10 6 cells / kg, about 3×10 6 cells / kg, about 4×10 6 cells / kg, about 5×10 6 cells / kg, about 6×10 6 cells / kg, about 7×10 6 cells / kg, about 8×10 6 cells / kg, about 9×10 6 cells / kg, about 1×10 7 cells / kg, about 2×10 7 cells / kg, about 3×10 7 cells / kg, about 4×10 7 cells / kg, about 5×10 7 cells / kg, about 6×10 7 cells / kg, about 7×10 7cells / kg, about 8×10 7 cells / kg or about 9×10 7 cells / kg. In some embodiments, a therapeutically effective amount of immune cells (e.g., CAR-T cells) is between about 1×10 6 cells and about 2×10 6 T cells per kg body weight up to a maximum dose of about 1×10 8 T cells. In some embodiments, a therapeutically effective amount of T cells is about 1×10 6 cells or about 2×10 6 T cells per kg body weight up to a maximum dose of about 1×10 8 T cells.

[0203] The number of cells will depend on the intended final use of the composition, as will the type of cells included. For example, in some embodiments, a population of T cells comprising a CAR will contain greater than 10%, greater than 15%, greater than 20%, greater than 25%, greater than 30% or greater than 35% of such cells. In some embodiments, a population of T cells comprising a CAR will contain 10% to 50%, 15% to 45%, 20% to 40%, 25% to 35% or 20% to 30% of such T cells. In some embodiments, the volume of the population of T cells for administration is one liter or less. In some embodiments, the volume of the T cells for administration is less than 500 ml, less than 250 ml or 100 ml or less. In some embodiments, the desired density of the T cells is generally greater than 10 6 cells / ml, and generally greater than 10 7 cells / ml, typically 10 8 cells / ml or greater. A clinically relevant number of immune cells can be distributed in multiple infusions that cumulatively equal or exceed 10 7 cells, 10 8 cells, 10 9 cells, 10 10 cells, 10 11 cells or 10 12 cells.

[0204] In some embodiments, the composition can be administered parenterally to a patient. In some embodiments, a composition comprising or delivering a population of T cells comprising a CAR can be administered parenterally to a patient in one or more administrations. In some embodiments, a composition comprising or delivering a population of T cells comprising a CAR can be administered parenterally to a patient once daily, once every 2 to 7 days, once a week, once every two weeks, once a month, once every three months or once every 6 months.

[0205] In some embodiments, the present disclosure provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject engineered immune cells comprising a CAR, wherein the CAR comprises: (a) an extracellular domain comprising an antigen-binding domain; (b) a transmembrane domain; and (c) an intracellular domain comprising a co-stimulatory intracellular domain, wherein the co-stimulatory intracellular domain comprises an intracellular signaling domain from 4-1BB / CD137 and five additional amino acids.

[0206] In some embodiments, the subject has previously been administered one or more additional anti-cancer therapies selected from the group consisting of: ionizing radiation, chemotherapeutic agents, therapeutic antibodies, and checkpoint inhibitors. In some embodiments, the subject has been identified or diagnosed as having cancer.

[0207] Drug composition

[0208] In some embodiments, the present disclosure provides a drug composition comprising T cells comprising a CAR and a pharmaceutically acceptable carrier, wherein the CAR comprises: (a) an extracellular domain comprising an antigen-binding domain; (b) a transmembrane domain; and (c) an intracellular domain comprising a co-stimulatory intracellular domain, wherein the co-stimulatory intracellular domain comprises an intracellular signaling domain from 4-1BB / CD137 and five additional amino acids. In some embodiments, the T cells comprising a CAR are autologous T cells. In some embodiments, the drug composition may comprise a buffer, diluent, solubilizer, emulsifier, preservative, adjuvant, excipient, or any combination thereof. In some embodiments, if desired, the composition may also contain one or more additional therapeutically active substances.

[0209] In some embodiments, the T cells of the present disclosure are formulated by first harvesting the T cells from the culture medium and then washing and concentrating the cells in a medium and container system (a "pharmaceutically acceptable" carrier) suitable for administration in a therapeutically effective amount. Suitable infusion media can be any isotonic media formulation, typically saline, Normosol R (Abbott), or Plasma-Lyte A (Baxter), but 5% dextrose in water or lactated Ringer's can also be used. The infusion media can be supplemented with human serum albumin.

[0210] In some embodiments, the composition is formulated for parenteral administration. For example, the pharmaceutical compositions provided herein can be provided in a sterile injectable form (e.g., a form suitable for subcutaneous injection or intravenous infusion). For example, in some embodiments, the pharmaceutical composition is provided in a liquid dosage form suitable for injection. In some embodiments, the pharmaceutical composition is provided in powder form (e.g., lyophilized and / or sterilized), optionally under vacuum, which can be reconstituted with an aqueous diluent (e.g., water, buffer, salt solution, etc.) prior to injection. In some embodiments, the pharmaceutical composition is 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., without shaking).

[0211] In some embodiments, T cells of the present disclosure that include a CAR and / or a nucleic acid encoding the CAR are formulated with a pharmaceutically acceptable parenteral vehicle. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 1%-10% human serum albumin. Non-aqueous vehicles such as fixed oils and liposomes can also be used. The vehicle or lyophilized powder can contain additives that 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 can additionally include pharmaceutically acceptable excipients, as used herein, the excipients include any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like suitable for the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21st Edition, A.R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006) discloses various excipients for formulating pharmaceutical compositions and known techniques for their preparation. Unless any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effects or otherwise interacting harmfully with any other one or more components of the pharmaceutical composition, it is contemplated that it will be used within the scope of the present disclosure.

[0212] In some embodiments, compositions comprising T cell populations of the present disclosure that include a CAR and / or a nucleic acid encoding a CAR are stably formulated. In some embodiments, a stable formulation of a T cell population comprising a CAR and / or a nucleic acid encoding a CAR of the present disclosure may include a phosphate buffer containing saline or a selected salt, and a preservative solution and formulation containing a preservative and a multi-purpose preservative formulation suitable for pharmaceutical or veterinary use. In an aqueous diluent, the preservative formulation contains at least one known preservative or is optionally selected from the group consisting of: at least one phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrate, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate), alkyl parabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal, or mixtures thereof. Any suitable concentration or mixture known in the art may be used, 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 preservative-free, 0.1-2% m-cresol (e.g., 0.2, 0.3, 0.4, 0.5, 0.9, 1.0%), 0.1-3% benzyl alcohol (e.g., 0.5, 0.9, 1.1, 1.5, 1.9, 2.0, 2.5%), 0.001-0.5% thimerosal (e.g., 0.005, 0.01), 0.001-2.0% phenol (e.g., 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%), 0.0005-1.0% 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 so on.

[0213] In some embodiments, the pharmaceutical composition is provided in a form that can be refrigerated and / or frozen. In some embodiments, the pharmaceutical composition is provided in a form that cannot be refrigerated and / or frozen. In some embodiments, the reconstitution 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, storing the composition comprising the antibody agent for longer than a specified time results in degradation of the antibody agent. Prior to administration, the liquid dosage form and / or reconstitution solution can include particulate matter and / or discoloration. In some embodiments, if there is discoloration or turbidity and / or if particulate matter remains after filtration, the solution should not be used. General considerations in the compounding and / or manufacture of pharmaceutical agents can be found, for example, in Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins Publishers, 2005.

[0214] In some embodiments, a pharmaceutical composition comprising T cells comprising the CARs of the present disclosure and / or nucleic acids encoding the CARs can be contained in a container for storage or administration, such as a vial, a syringe (e.g., an intravenous syringe), or a bag (e.g., an intravenous infusion bag). The pharmaceutical composition according to the present disclosure can be prepared, packaged, and / or sold as a single unit dose and / or as a plurality of single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition that includes a predetermined amount of an active ingredient. The amount of the active ingredient is typically equal to the dose of the active ingredient to be administered to a subject and / or a convenient fraction of such a dose, such as half or one-third of such a dose.

[0215] Kit

[0216] The present disclosure further provides a kit that includes one or more containers filled with at least one CAR and / or nucleic acid encoding a CAR as described herein. The kit can be used in any applicable method, including, for example, therapeutic methods, diagnostic methods, cell proliferation and / or isolation methods, and the like. Optionally associated with such containers can be a notice in a form specified by a government agency regulating the manufacture, use, or sale of drugs or biological products, the notice reflecting (a) approval by the agency for manufacture, use, or sale for human administration, (b) instructions for use, or both.

[0217] In some embodiments, the kit can comprise one or more reagents for detecting (e.g., detecting the CAR and / or the nucleic acid encoding the CAR). In some embodiments, the kit can comprise the CAR and / or the nucleic acid encoding the CAR in a detectable form (e.g., covalently associated with a detectable moiety or entity). In some embodiments, one or more CARs and / or nucleic acids encoding the CARs as provided herein can be included in a kit for treating a subject. In some embodiments, the CARs and / or nucleic acids encoding the CARs as provided herein can be included in a kit for preparing autologous T cells expressing the CAR.

[0218] In some embodiments, the kit can provide one, two, three, four or more antigen - specific antibody agents, each of which is suitable for cloning into a CAR construct. In some embodiments, the kit can provide other reagents for determining the binding affinity of the antibody agent and / or the CAR and / or the CAR T cells for T cells identified or isolated from a subject. In some embodiments, the kit can provide other reagents for determining the functional affinity of the antibody agent and / or the CAR and / or the CAR T cells for the T cells of a subject.

[0219] Examples

[0220] The present disclosure is further described in the following examples, which do not limit the scope of the present disclosure described in the claims.

[0221] Example 1 - GPC3 Lentiviral Transfer Plasmid

[0222] Using standard DNA cloning techniques known in the art, a DNA construct encoding a single - chain variable fragment (scFv) form of a humanized anti - GC33 antibody agent ( Figure 2 ) was generated by ligating the VH and VL regions. The lentiviral transfer plasmids used herein are shown in Table 1.

[0223] Table 1.

[0224]

[0225] The huGC33 VH - VL - scFv was cloned into the lentiviral vector pELPS4 - MVRL2H2 - euBBz, where pELPS4 - MVRL2H2 - euBBz is a lentiviral vector containing the co - stimulatory domain 4 - 1BB and five additional amino acids. The lentiviral vector construct pELPS4 - huGC33 VH - VL was digested with restriction enzymes, and the Figure 3 restriction enzyme digestion results are shown therein.

[0226] To create the CAR construct without the need for five additional amino acids in the 4-1BB co-stimulatory domain, huGC33 VH-VL-scFv was cloned into the lentiviral vector pELPS2-CD19-BBz, where pELPS2-CD19-BBz is a lentiviral vector containing the co-stimulatory domain 4-1BB without the five additional amino acids. The lentiviral vector construct huGC33 (VH-VL)-BBz was digested with restriction enzymes, where Figure 4 the restriction enzyme digestion results are shown.

[0227] Example 2 - Pharmaceutical Composition of GPC3 CAR-T Cells

[0228] PBMC frozen tubes (5×10 7 cells / 1 mL / vial) were thawed and activated by placing them in a water bath for 2 - 3 minutes. 10 mL of CAR-T cell medium and 1 mL of PBMC were placed in a 50 mL conical tube and centrifuged at 1500 rpm for 5 minutes. The supernatant was removed, and the CAR-T cells were counted after resuspending 5 mL of the cells in fresh medium. Fresh cell medium was added to adjust the cell density to 1×10 6 cells / mL. 10 μL of T cell activation beads were added to every 1×10 6 cells, and the medium was supplemented with IL-2. The cell medium was cultured in a T75 flask in an incubator at 5% CO 2 2 and 37 °C.

[0229] Then, CAR-T cells were generated by spinoculating the activated T cells with the lentivirus encoding CAR. The activated PBMC from the cell culture were counted, and the cells were seeded in a 24-well plate in the presence of 500 μL of cell medium and the lentivirus. After spinoculation transduction, the transduced cells from 1 well were cultured in cell medium supplemented with IL-2.

[0230] The cultured CAR-T cells were counted every 2 - 3 days, and fresh medium and IL-2 were added after each count (Figure 5). On day 12, the cultured CAR-T cells were harvested and stored in a cryovial at -80 °C.

[0231] CAR expression was analyzed on day 12 of culture, and the results showed that the control group did not show expression, while the CAR-T cell group showed 54 - 66% CAR expression ( Figure 6 ).

[0232] Collect target cells (GPC3-positive cell line) and inoculate them in a 96-well U-bottom plate. Then add effector cells (CAR-T cells) to the wells at effector cell:target cell ratios of 10:1, 3:1, 1:1, and 0.3:1, and incubate at 37 °C for 24 hours. After incubation, add CytoTox96 reagent to each well, and quantify cytotoxicity by measuring the absorbance at 490 nm (Figure 7).

[0233] Example 3 - In Vivo huGC33(VH-VL)-BBz CAR-T Cells and huGC33(VH-VL)-euBBz CAR-T Cells

[0234] To verify and compare the efficacy of huGC33(VH-VL)-BBz CAR-T cells and huGC33(VH-VL)-euBBz CAR-T cells, Huh-7_Luf-GFP cells (2×10 6 cells / 200 μL / head) were injected into NSG mice (6 - 8 weeks old, male), and 35 days after injection, mice with a tumor size of approximately 200 mm 3 were divided into 5 groups, with 4 mice in each group. The control group received 5% HSA injection, while the other groups received CAR-T cell injection. Tumor growth was observed by measuring tumor size twice a week using TM900( Figure 8 ).

[0235] After CAR-T cell administration, orbital blood collection was performed on the mice once a week. For each 100 μL blood sample, it was centrifuged at 12,000 rpm for 10 minutes to confirm the proportion and cell count of CAR-T cells. Place 100 μL of blood in a FACS tube and perform live / dead cell staining using the Zombie NIR TM Fixable Viability Kit. After reaching a concentration of 0.1 μL / 100 μL PBS / tube, perform the staining at room temperature for 10 minutes. Add 25 μL of counting beads, 0.5 μL of CD45, 0.5 μL of CD8, 1.0 μL of CD45RO, 1.0 μL of CD62L, 1.0 μL of PD-1, 1.0 μL of Tim-3, 0.5 μL of CD4, 0.5 μL of CD69, and 0.0125 μL of Flag to 100 μL of FACS buffer and stain at room temperature for 30 minutes. After 30 minutes, add IX RBC lysis buffer and react at room temperature for 5 minutes. After centrifuging at 2,000 rpm for 4 minutes, discard all supernatants. Add 2 mL of FACS buffer to the tube and centrifuge at 2,000 rpm for 4 minutes, and perform analysis using FACSCelesta (Figure 9).

[0236] Six weeks after CAR-T injection, mouse spleens, livers, and bone marrows were collected to evaluate the ratio of huGC33(VH-VL)-BBzCAR-T cells to huGC33(VH-VL)-euBBz CAR-T cells. The tissue samples were processed and filtered through a 40 μm cell strainer, then centrifuged at 2,000 rpm for 5 minutes and all supernatants were discarded. 5 μL of IX ACK buffer was added and reacted for 10 minutes. Then, 10 mL of DPBS was added and centrifuged at 2,000 rpm for 5 minutes. FACS staining was performed as described above ( Figure 10 ).

[0237] Construction of Example 4 - CD19-euBBz CAR

[0238] To increase the immune efficacy of the cytoplasmic signaling domain 4-1BB, 5 amino acids were added as a co-stimulatory signal factor to the 4-1BB cytoplasmic domain used in CAR-T to newly construct a CAR expression vector (CD19-euBBz CAR). The completed construct included anti-CD19 which was an scFv, and the scFv contained an EF1α promoter, a hinge region, and a transmembrane domain of human CD8 as well as an intracellular signaling domain. Specifically, the intracellular signaling domain consisted of a stimulatory domain and a co-stimulatory signaling domain. The intracellular signaling domain was the co-stimulatory signaling domain 4-1BB, to which 5 consecutive amino acids were added and CD3ζ was linked to the amino acids. The finally produced CAR gene fragment was conjugated to an ELPS lentiviral expression vector cut with BamH I and Sal I. Additionally, cloning was performed using BamH I / Nhe I restriction enzymes to replace only the scFv part.

[0239] Example 5 - CD19-euBBz and CD19-BBz CAR-T Cells

[0240] The 293T cell culture used to produce recombinant lentivirus contained a medium including 10% FBS (Millipore, TMS-013-BKR) and high-glucose DMEM (Welgene, LM001-05) containing 1×P / S (Gibco, 15140-122). At 37 °C, 5% CO 2Before transduction in the incubator, 293T cells were incubated in DMEM medium containing 10% FBS for 24 hours. At the time of transfection the next day, the transfection reagent and the lentiviral plasmid were mixed in an appropriate ratio and incubated for 48 hours. Then the supernatant containing lentivirus was collected and centrifuged at 400 x g for 10 minutes. Additionally, the supernatant was filtered using a 0.45 μm syringe filter with a 50 mL syringe. The obtained supernatant was mixed with a lentivirus enrichment kit (Clontech, 631231) at a ratio of 3:1 and reacted at 4 °C for 24 to 48 hours. Then it was centrifuged at 4,000 rpm at 4 °C for 2 hours to obtain the virus, and the virus was resuspended in 0.5 mL RPMI (Wisent, LM001-01) without FBS to produce lentivirus.

[0241] To determine the transduction efficiency of mammalian cells, the transduction unit (TU / mL) of the actually transducing lentivirus was measured by counting the particles of the lentivirus using Jurkat cells. CAR expression could be determined by FACS. On the first day, Jurkat cells were seeded at 1 × 10 5 cells / 100 μL per well into a 96-well plate. On the second day, the lentivirus was serially diluted 1 / 3 in the 96-well plate and lentiviral transduction was performed on the already seeded Jurkat cells. At this time, the lentiviral transduction was further increased by introducing polybrene (Millipore) into the RPMI medium (10% FBS and 1 × P / S). After centrifugation at 1200 x g and 32 °C for 2 hours, the cells were incubated in a 37 °C 5% CO 2 incubator for 3 hours and only 100 μL RPMI was added per well. On the 5th day, the flag of the lentivirus infected into the cells was stained with anti-Flag-DYKDDDDK (Biolegend, catalog number 637310) to analyze the percentage of cells transduced by flow cytometry. Using this, the titer was calculated as described in Follenzi and Naldini, 2002 (Follenzi and Naldini, 2002).

[0242] FACS staining was performed to confirm the production ratio of the two types of CAR-T cells after 14 days of incubation. For each type of CAR-T cell, 2 × 10 5Cells were counted, and then 2 mL of FACS buffer was added, and centrifuged at 2,000 rpm for 5 minutes using a centrifuge (Thermo, ST16). After discarding the supernatant, 0.5 μL / tube of anti-CD8 APC (SKI, BioLegend, catalog number 344722), 0.5 μL / tube of anti-CD4 BV650 (RPA-T4, BioLegend, catalog number 300536), and 0.125 μL / tube of anti-flag PE (L5, BioLegend, catalog number 637310) were added, and stained for 30 minutes at room temperature. After adding 2 mL of FACS buffer and centrifuging at 2,000 rpm for 5 minutes, this process was repeated once. To stain live / dead cells, 1 μL / tube of 7-AAD (BioLegend, catalog number 420404) was added and the mixture was left at room temperature for 5 minutes, and then analyzed using FACS (BD, FACSCelesta).

[0243] Confirmation of the proportion of CD19 CAR-T cells produced using FACS staining confirmed that in the case of the improved construct CD19-euBBZ CAR-T cells, the cell ratios were CD4+ / CAR+ 29.4%, CD8+ / CAR+ 50.8%, and total CAR-T 80.2%. It was confirmed that for non-construct-improved CD19-BBz CAR-T cells, the cell ratio of CD4+ / CAR+ was 42.7%, the cell ratio of CD8+ / CAR+ was 29.3%, and the cell ratio of total CAR-T was 72.0%. Therefore, it was confirmed that the CAR expression of CD19-euBBz CAR-T cells was 8.2% higher than that of CD19-BBz CAR-T cells, and the CD8+ / CAR+ cells were 21.5% or approximately twice as many.( Figure 11 A)

[0244] Example 6 - Confirmation of the cytotoxicity of the produced CD19 CAR-T cells

[0245] To determine the cytotoxicity of the two types of CAR-T cells cultured for 14 days, CAR-T(E):LCL(T) was present in a 96-well white plate (Corning, catalog number 3917) at ratios of 30:1, 10:1, 3:1, and 1:1. First, the CAR-T cells were placed in the wells at 6×10 5 cells / 50 μL, 2×10 5 cells / 50 μL, 9×10 4 cells / 50 μL, and 2×10 4 cells / 50 μL respectively. Next, the target cell line, namely the CBK LCL-Luc cell line, was incubated at 37°C in CO 2Add 2×10 4 cells / 50 μL into the incubator (Mammert, INCO153med) and react for 4 hours. After 4 hours, add 100 μL Bright-Glo TM (Promega, catalog number E2620) into each well, and after 5 minutes, measure the relative light unit (RLU) value using a photometer (Thermo, Fluoroskan FL).

[0246] No difference in cytotoxicity was found between CAR-T cells introducing conventional 4-1BB and CAR-T cells introducing euBBz with 5 amino acids added to the 4-1BB domain.

[0247] The results showed that when the two kinds of CAR-T cells and the CBK LCL-Luc cell line were incubated together at a ratio of 30:1, the cytotoxicity was found to be about 80% after 4 hours, and when incubated at a ratio of 10:1, the cytotoxicity was about 50%. As the corresponding number of CAR-T cells incubated with cancer cells decreased by 3 times, the cytotoxicity decreased by about 3 times; in addition, when 5 amino acids were added to the 4-1BB domain in vitro, it was confirmed that this did not affect the in vitro cytotoxicity.( Figure 11 B)

[0248] Example 7 - Induction of subcutaneous animal model and CAR-T verification by automatic caliper and IVIS imaging

[0249] For experimental animals, NSG (NOD-scid IL2rγμL1) mice (The Jackson Laboratory) were used and managed under constant conditions in the animal nursery. The temperature was 23 ± 2 °C, and there was a 12-hour light / dark cycle and a humidity of 50 ± 10%; food and drink were provided ad libitum. In the efficacy experiment using CD19-euBBz CAR-T and adding five amino acids to the 4-1BB domain, the CBK LCL-Luc cell line was prepared at 2×10 6 cells / 100 μL DPBS / head and subcutaneously injected into 6-week-old female mice to induce a subcutaneous animal model. When the cancer size measured using an automatic caliper (Youngbio, TM900) reached between 50 and 100 mm 3 CD19-euBBz CAR-T cells and CD19-BBz CAR-T cells were at 2×10 6 cells / 100 μL DPBS / head (dose 1) and 6×10 6Cells / 100 μL DPBS / head (dose 2) were administered once via the tail vein to confirm efficacy. In all animal experiments, tumor size and viability were confirmed regularly.

[0250] More specifically, using an automated caliper and an IVIS imaging device (PerkinElmer, LunaIII), tumor size and photon values were measured every 3 days and 4 days after CAR-T administration ( Figure 12 、 13 ). In the case of using TM900, tumor size was determined after placing the device at the tumor site. When imaging and photon values were confirmed using the IVIS imaging device, 150 mg / kg XenoLight TM D-luciferin (PerkinElmer, catalog number 122799) was administered intraperitoneally to the mice first. After 15 minutes, inhaled anesthesia was induced using isoflurane, and after 5 minutes, the presence of cancer cells was imaged using the IVIS. After imaging, normalization was performed, and then the luciferase value (photon value) was confirmed and plotted. After constructing a subcutaneous animal model using the CBK LCL-Luc cell line, the effects of CD19-BBz CAR-T and CD19-euBBz CAR-T cells were compared using IVIS imaging. As Figure 12 shown, the effects could be confirmed within 1 week after administering the two types of CAR-T cells.

[0251] At 2×10 6 cells / 100 μL DPBS / head and 6×10 6 cells / 100 μL DPBS / head, in the experimental groups administered CD19-euBBz CAR-T cells, cancer cells were observed on IVIS imaging 1 week after administration. Additionally, in the group treated with CD19-BBz CAR-T, when 6×10 6 cells / 100 μL DPBS / head were administered, cancer cells were rarely observed by imaging within 1 week after administration. However, 1 week later, cancer cells were identified in the experimental group administered CD19-BBz CAR-T.

[0252] 1 week after CAR-T administration, as shown by the imaging, the luciferase values of the images taken in each subject were determined after the imaging process; luciferase values were confirmed only in the group administered CD19 CAR-T at 2×10 6 cells / 100 μL DPBS / head. When observed 3 weeks or more later, the tumors continued to grow in the mice not administered CAR-T cells, and no cancer cells were identified in the three experimental groups where the cancer cells had initially disappeared. However, 10 days later, those receiving 2×10 6The luciferase level in the group of CD19 CAR-T at 100 cells / 100 μL DPBS / head decreased, but the cancer cells did not completely disappear after 3 weeks. When 2×10 6 cells / 100 μL DPBS / head of CD19-euBBz CAR-T were administered, through experimental imaging of cancer cells, it was found that the efficacy was similar to that of the group treated with CD19-BBz CAR-T at 6×10 6 cells / 100 μL DPBS / head. The results showed that there was no difference in the in vitro cytotoxicity between CD19-euBBz CAR-T and CD19-BBz CAR-T, but it was confirmed that the efficacy was 5 times that of CD19-euBBz CAR-T in the animal model.( Figure 12 )

[0253] Example 8 - Confirmation of the ratio of CD19-euBBz CAR-T in an in vivo animal model

[0254] After administering CAR-T cells in a subcutaneous animal model to verify the efficacy of the improved construct CAR-T cells, the presence of CAR-T was confirmed from the mouse blood. More specifically, after the administration of CAR-T, orbital blood collection was performed on the mice at intervals of 3 days and 4 days. At each blood collection, 70 μL of blood was collected, and 60 μL of the blood was used to confirm the CAR-T cell ratio and cell count. The 60 μL of blood was placed in a 5 mL FACS tube, and Zombie Aqua BV510 (BioLegend, catalog number 423101) was used for live / dead cell staining. After reaching a concentration of 0.1 μL / 100 μL DPBS / tube, the staining was performed at room temperature for 10 minutes. Since beads (Molecularprobes, catalog number C36950) were counted, anti-CD45 FITC (HI30, BioLegend, catalog number 304006), anti-CD8 BV786 (SK-1, BioLegend, catalog number 344740), anti-CD4 BV650, and anti-flag PE were added and stained at room temperature for 30 minutes. Each antibody was mixed at 0.5 μL / 100 μL FACS buffer / tube, and 25 μL of counting beads was added thereto. After 30 minutes, 2 mL of 1X RBC lysis buffer (BioLegend, catalog number 422401) was added and reacted at

[0255] room temperature for 5 minutes. After centrifuging at 2,000 rpm for 5 minutes using a centrifuge, all the supernatants were discarded. 2 mL of FACS wash buffer was added to this tube, and centrifuged at 2,000 rpm for 5 minutes. This process was repeated once, and then 50 μL of FACS buffer was added and analyzed using FACS.

[0256] One week after CAR-T cell administration, in the CD19-euBBz CAR-T treatment group, approximately 20% CD19-euBBz CAR-T cells were confirmed in the blood at both 2×10 6 cells / 100 μL DPBS / head group and 6×10 6 cells / 100 μL DPBS / head group; however, in the group administered CD19-BBz CAR-T, only 5% CD19 CAR-T was confirmed when administered at 6×10 6 cells / 100 μL DPBS / head. Three days later, the number and proportion of CAR-T cells in the mice reached the maximum and decreased. Within one week, in the 3 experimental groups in which CAR-T cells were identified (CD19-euBBz CAR-T; 2×10 6 cells / 100 μL DPBS / head and 6×10 6 cells / 100 μL DPBS / head, CD19-BBz CAR-T; 6×10 6 cells / 100 μL DPBS / head), cancer cells were rapidly killed because the cancer cells might come into contact with a relatively large number of CAR-T cells before they proliferated in the mice. However, in the experimental group administered CD19-BBz CAR-T at 2×10 6 cells / 100 μL DPBS / head, the proportion and number of CAR-T cells reached the maximum at 2 weeks, and the CAR-T proportion was approximately 25%, where cancer cell proliferation was relatively good. After the CAR-T proportion initially increased and then decreased, CD19-euBBz CAR-T was more stable in number than CD19-BBz CAR-T. However, in the case of CD19-BBz CAR-T, the proportion of CAR-T cells increased and decreased at a slightly later time, and thus it took longer for the tumor to disappear in the mice. Therefore, as in the results of this experiment, the group administered CD19-euBBz CAR-T at 2×10 6 cells / 100 μL DPBS / head showed similar CAR-T levels and effects in the mice as the group administered CD19-BBz CAR-T at 6×10 6 cells / 100 μL DPBS / head, indicating that CD19-euBBz CAR-T has superior effects.( Figure 14 ) Sequence Listing <110> Eutilex Co., Ltd. <120> Chimeric Antigen Receptor with 4-1BB Costimulatory Domain <130> 47683-0044WO1 <150> 62 / 867,503 <151> June 27, 2019 <150> PCT / KR2019 / 010244 <151> August 12, 2019 <150> 16 / 715,462 <151> December 16, 2019 <150> 62 / 991,493 <151> March 18, 2020 <150> 63 / 004,827 <151> April 3, 2020 <150> 63 / 043,237 <151> June 24, 2020 <160> 28 <170> PatentIn version 3.5 <210> 1 <211> 15 <212> DNA <213> Artificial <220> <223> Synthetic oligonucleotide <400> 1 cgtttctctg ttgtt 15 <210> 2 <211> 141 <212> DNA <213> Artificial <220> <223> Synthetic oligonucleotide <400> 2 cgtttctctg ttgttaaacg gggcagaaag aaactcctgt atatattcaa acaaccattt 60 atgagaccag tacaaactac tcaagaggaa gatggctgta gctgccgatt tccagaagaa 120 gaagaaggag gatgtgaact g 141 <210> 3 <211> 5 <212> PRT <213> Artificial <220> <223> Synthetic polypeptide <400> 3 Arg Phe Ser Val Val 1 5 <210> 4 <211> 47 <212> PRT <213> Artificial <220> <223> Synthetic polypeptide <400> 4 Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe 1 5 10 15 Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly 20 25 30 Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 45 <210> 5 <211> 207 <212> DNA <213> Artificial <220> <223> CD8 / Hinge / Transmembrane domain <400> 5 accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgctag ccagcccctg 60 tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120 gacttcgcct gtgatatcta catctgggcg cccttggccg ggacttgtgg ggtccttctc 180 ctgtcactgg ttatcaccct ttactgc 207 <210> 6 <211> 339 <212> DNA <213> Artificial <220> <223> Synthetic oligonucleotide <400> 6 agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60 tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120 cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180 gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240 cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300 tacgacgccc ttcacatgca ggccctgccc cctcgctaa 339 <210> 7 <211> 69 <212> DNA <213> Artificial <220> <223> CD8α leader sequence <400> 7 ggatccatgg ccttaccagt gaccgccttg ctcctgccgc tggccttgct gctccacgcc 60 gccaggccg 69 <210> 8 <211> 24 <212> DNA <213> Artificial <220> <223> Flag tag sequence <400> 8 gactacaagg acgacgatga caag 24 <210> 9 <211> 16 <212> PRT <213> Artificial <220> <223> Light chain CDR1 <400> 9 Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu His 1 5 10 15 <210> 10 <211> 7 <212> PRT <213> Artificial <220> <223> Light chain CDR2 <400> 10 Lys Val Ser Asn Arg Phe Ser 1 5 <210> 11 <211> 9 <212> PRT <213> Artificial <220> <223> Light chain CDR3 <400> 11 Ser Gln Asn Thr His Val Pro Pro Thr 1 5 <210> 12 <211> 5 <212> PRT <213> Artificial <220> <223> Heavy chain CDR1 <400> 12 Asp Tyr Glu Met His 1 5 <210> 13 <211> 17 <212> PRT <213> Artificial <220> <223> Heavy chain CDR2 <400> 13 Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe Lys 1 5 10 15 Gly <210> 14 <211> 6 <212> PRT <213> Artificial <220> <223> Heavy chain CDR3 <400> 14 Phe Tyr Ser Tyr Thr Tyr 1 5 <210> 15 <211> 112 <212> PRT <213> Artificial <220> <223> Human GC33 light chain variable region <400> 15 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu His Trp Tyr Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn 85 90 95 Thr His Val Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 16 <211> 115 <212> PRT <213> Artificial <220> <223> Human GC33 heavy chain variable region <400> 16 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe 50 55 60 Lys Gly Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 <210> 17 <211> 336 <212> DNA <213> Artificial <220> <223> Human GC33 light chain variable region <400> 17 gacgtcgtta tgacacagag tcccctctcc ttgccggtga ccctgggtca gcctgcgtcc 60 atctcttgca gatcctccca gtctctggta cactccaacg gcaacacata cttgcactgg 120 taccaacaaa gacctggtca gtcaccgcga cttctcatat ataaagtttc caataggttc 180 agtggagtgc cagacaggtt cagtggttca ggatcaggca ctgatttcac gcttaaaatc 240 agtcgggttg aggcggagga cgtaggagtt tactattgca gccagaatac gcacgtgccg 300 cctacttttg gctctggaac caagttggaa ataaag 336 <210> 18 <211> 345 <212> DNA <213> Artificial <220> <223> Human GC33 heavy chain variable region <400> 18 caagtgcaac tcgtacaatc aggtgctgaa gtcaaaaagc cgggagcctc tgttaaagtg 60 tcctgtaaag ccagcggcta cacctttacc gattatgaga tgcactgggt tcggcaggct 120 ccgggccaag gtctggagtg gatcggggct cttgacccaa agacgggcga cacggcttat 180 tcacaaaaat tcaaaggtag ggctactctg actgccgata agtccaccag caccgcgtat 240 atggagctct ctagcttgcg aagcgaggac acggcggtgt actattgcac acgcttctat 300 agttacacat attggggtca aggcacgctt gtgaccgtgt ctagc 345 <210> 19 <211> 243 <212> PRT <213> Artificial <220> <223> Synthetic polypeptide <400> 19 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Asp His Ile Asn Asn Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Lys Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Thr Pro Phe 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu 115 120 125 Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys 130 135 140 Thr Val Ser Gly Phe Ser Leu Ser Arg Tyr Ser Val His Trp Ile Arg 145 150 155 160 Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu Gly Met Ile Trp Gly Gly 165 170 175 Gly Ser Thr Asp Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ser 180 185 190 Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys Leu Ser Ser Val Thr 195 200 205 Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asn Glu Gly Asp Thr 210 215 220 Thr Ala Gly Thr Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr 225 230 235 240 Val Ser Ser <210> 20 <211> 729 <212> DNA <213> Artificial <220> <223> Synthetic oligonucleotide <400> 20 gatattcaga tgacccagtc cccgagctcc ctgtccgcct ctgtgggcga tagggtcacc 60 atcacctgca aggccagtga ccacatcaac aactggctgg cctggtatca acagaaacca 120 ggaaaagctc cgaaactact gatcagcggc gccacctctc tggaaaccgg agtcccttct 180 cgcttctctg gttccggatc tgggaaggat tacactctga ccatcagcag tctgcagccg 240 gaagacttcg caacttatta ctgtcagcag tactggtcca cccccttcac cttcggacag 300 ggtaccaagg tggagatcaa aggcggaggc ggatctggcg gcggaggaag tggcggaggg 360 ggatctcagg tgcagctgca ggagtcgggc ccaggactgg tgaagccttc ggagaccctg 420 tccctcacct gcactgtctc tggtttctcc ctgagtcggt actctgtgca ttggatccgg 480 cagcccccag ggaagggact ggagtggctg gggatgatct ggggaggcgg cagcaccgac 540 tacaacagcg ccctgaagtc ccgactgacc atatcaaagg acaactccaa gaaccaggtg 600 tccttgaagc tgagctctgt gaccgctgcg gacacggccg tgtattactg tgcgagaaat 660 gagggcgata ccaccgccgg cacttggttt gcctattggg gccagggaac cctggtcacc 720 gtctcctca 729 <210> 21 <211> 242 <212> PRT <213> Artificial <220> <223> Synthetic polypeptide <400> 21 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu 115 120 125 Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys 130 135 140 Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg 145 150 155 160 Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser 165 170 175 Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile 180 185 190 Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln 195 200 205 Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly 210 215 220 Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val 225 230 235 240 Ser Ser <210> 22 <211> 726 <212> DNA <213> Artificial <220> <223> Synthetic oligonucleotide <400> 22 gacatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60 atcagttgca gggcaagtca ggacattagt aaatatttaa attggtatca gcagaaacca 120 gatggaactg ttaaactcct gatctaccat acatcaagat tacactcagg agtcccatca 180 aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240 gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 300 gggaccaagc tggagatcac aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc 360 ggatctgagg tgaaactgca ggagtcagga cctggcctgg tggcgccctc acagagcctg 420 tccgtcacat gcactgtctc aggggtctca ttacccgact atggtgtaag ctggattcgc 480 cagcctccac gaaagggtct ggagtggctg ggagtaatat ggggtagtga aaccacatac 540 tataattcag ctctcaaatc cagactgacc atcatcaagg acaactccaa gagccaagtt 600 ttcttaaaaa tgaacagtct gcaaactgat gacacagcca tttactactg tgccaaacat 660 tattactacg gtggtagcta tgctatggac tactggggcc aaggaacctc agtcaccgtc 720 tcctca 726 <210> 23 <211> 1184 <212> DNA <213> Artificial <220> <223> Synthetic oligonucleotide <400> 23 cgtgaggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt 60 tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg 120 aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa 180 gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtaa 240 gtgccgtgtg tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt 300 gaattacttc cacctggctg cagtacgtga ttcttgatcc cgagcttcgg gttggaagtg 360 ggtgggagag ttcgaggcct tgcgcttaag gagccccttc gcctcgtgct tgagttgagg 420 cctggcctgg gcgctggggc cgccgcgtgc gaatctggtg gcaccttcgc gcctgtctcg 480 ctgctttcga taagtctcta gccatttaaa atttttgatg acctgctgcg acgctttttt 540 tctggcaaga tagtcttgta aatgcgggcc aagatctgca cactggtatt tcggtttttg 600 gggccgcggg cggcgacggg gcccgtgcgt cccagcgcac atgttcggcg aggcggggcc 660 tgcgagcgcg gccaccgaga atcggacggg ggtagtctca agctggccgg cctgctctgg 720 tgcctggcct cgcgccgccg tgtatcgccc cgccctgggc ggcaaggctg gcccggtcgg 780 caccagttgc gtgagcggaa agatggccgc ttcccggccc tgctgcaggg agctcaaaat 840 ggaggacgcg gcgctcggga gagcgggcgg gtgagtcacc cacacaaagg aaaagggcct 900 ttccgtcctc agccgtcgct tcatgtgact ccactgagta ccgggcgccg tccaggcacc 960 tcgattagtt ctcgagcttt tggagtacgt cgtctttagg ttggggggag gggttttatg 1020 cgatggagtt tccccacact gagtgggtgg agactgaagt taggccagct tggcacttga 1080 tgtaattctc cttggaattt gccctttttg agtttggatc ttggttcatt ctcaagcctc 1140 agacagtggt tcaaagtttt tttcttccat ttcaggtgtc gtga 1184 <210> 24 <211> 84 <212> DNA <213> Artificial <220> <223> Synthetic oligonucleotide <400> 24 agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta 60 gtcagtgtgg aaaatctcta gcag 84 <210> 25 <211> 1377 <212> DNA <213> Artificial <220> <223> Synthetic oligonucleotide <400> 25 cgaacaggga cttgaaagcg aaagggaaac cagaggagct ctctcgacgc aggactcggc 60 ttgctgaagc gcgcacggca agaggcgagg ggcggcgact ggtgagtacg ccaaaaattt 120 tgactagcgg aggctagaag gagagagatg ggtgcgagag cgtcagtatt aagcggggga 180 gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat 240 gaattagatc gcgatgggaa aaaattcggt taaggccagg gggaaagaaa aaatataaat 240 taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt 300 taaaacatat agtatgggca agcagggagc tagaacgatt cgcagttaat cctggcctgt 300 tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag 360 tagaaacatc agaaggctgt agacaaatac tgggacagct acaaccatcc cttcagacag 360 gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa 420 gatcagaaga acttagatca ttatataata cagtagcaac cctctattgt gtgcatcaaa 420 ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa 480 ggatagagat aaaagacacc aaggaagctt tagacaagat agaggaagag caaaacaaaa 480 gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg 540 gtaagaccac cgcacagcaa gcggccgctg atcttcagac ctggaggagg agatatgagg 540 gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta 600 gacaattgga gaagtgaatt atataaatat aaagtagtaa aaattgaacc attaggagta 600 gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga 660 gcacccacca aggcaaagag aagagtggtg cagagagaaa aaagagcagt gggaatagga 660 gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg 720 gctttgttcc ttgggttctt gggagcagca ggaagcacta tgggcgcagc gtcaatgacg 720 ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg 780 ctgacggtac aggccagaca attattgtct ggtatagtgc agcagcagaa caatttgctg 780 agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc 840 agggctattg aggcgcaaca gcatctgttg caactcacag tctggggcat caagcagctc 840 caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg 900 caggcaagaa tcctggctgt ggaaagatac ctaaaggatc aacagctcct ggggatttgg 900 ggttgctctg gaaaactcat ttgcaccact gctgtgcctt ggaatgctag ttggagtaat 960 aaatctctgg aacagatttg gaatcacacg acctggatgg agtgggacag agaaattaac 1020 aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca agaaaagaat 1080 gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt taacataaca 1140 aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt aggtttaaga 1200 atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc accattatcg 1260 tttcagaccc acctcccaac cccgagggga cccgacaggc ccgaaggaat agaagaagaa 1320 ggtggagaga gagacagaga cagatccatt cgattagtga acggatctcg acggtat 1377 <210> 26 <211> 547 <212> DNA <213> Artificial <220> <223> Synthetic oligonucleotide <400> 26 tagactgtag cccaggaata tggcagctag attgtacaca tttagaagga aaagttatct 60 tggtagcagt tcatgtagcc agtggatata tagaagcaga agtaattcca gcagagacag 120 ggcaagaaac agcatacttc ctcttaaaat tagcaggaag atggccagta aaaacagtac 180 atacagacaa tggcagcaat ttcaccagta ctacagttaa ggccgcctgt tggtgggcgg 240 ggatcaagca ggaatttggc attccctaca atccccaaag tcaaggagta atagaatcta 300 tgaataaaga attaaagaaa attataggac aggtaagaga tcaggctgaa catcttaaga 360 cagcagtaca aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt 420 acagtgcagg ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac 480 aaaaacaaat tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccag 540 tttggct 547 <210> 27 <211> 591 <212> DNA <213> Artificial <220> <223> Synthetic oligonucleotide <400> 27 atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac tatgttgctc 60 cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt gcttcccgta 120 tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat gaggagttgt 180 ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca acccccactg 240 gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc cccctcccta 300 ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg gctcggctgt 360 tgggcactga caattccgtg gtgttgtcgg ggaagctgac gtcctttcca tggctgctcg 420 cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct tcggccctca 480 atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt ccgcgtcttc 540 gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcct g 591 <210> 28 <211> 98 <212> DNA <213> Artificial <220> <223> Synthetic oligonucleotide <400> 28 gggtctctct ggttagacca gatctgagcc tgggagctct ctggctaact agggaaccca 60 ctgcttaagc ctcaataaag cttgccttga gtgcttca 98

Claims

1. A nucleic acid encoding a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor comprises: (a) an extracellular domain comprising an antigen-binding domain that specifically binds to glypican-3 (GPC3); (b) a transmembrane domain; and (c) an intracellular domain comprising a co-stimulatory intracellular domain, wherein the co-stimulatory intracellular domain comprises an intracellular signaling domain from 4-1BB / CD137 and five additional amino acids; and (d) a signal peptide from CD8α; wherein the co-stimulatory intracellular domain comprises SEQ ID NO:4, wherein the antigen-binding domain that specifically binds to glypican-3 (GPC3) comprises a light chain variable domain shown by SEQ ID NO:15 and a heavy chain variable domain shown by SEQ ID NO:

16.

2. The nucleic acid according to claim 1, wherein the chimeric antigen receptor is a single polypeptide.

3. The nucleic acid according to claim 1, wherein the chimeric antigen receptor comprises two polypeptides.

4. The nucleic acid according to any one of claims 1 to 3, wherein the antigen-binding domain is humanized.

5. The nucleic acid according to claim 1, wherein the antigen-binding domain is human.

6. The nucleic acid according to claim 1, wherein the antigen-binding domain is a scFv.

7. The nucleic acid according to claim 1, wherein the antigen-binding domain specifically binds to a tumor antigen.

8. The nucleic acid according to claim 1, wherein the transmembrane domain is a transmembrane domain from CD8α.

9. The nucleic acid according to claim 1, wherein the intracellular domain further comprises an intracellular domain from CD3ζ.

10. The nucleic acid according to claim 1, wherein the chimeric antigen receptor further comprises a hinge region.

11. The nucleic acid according to claim 10, wherein the hinge region is a CD8α hinge.

12. A vector comprising the nucleic acid according to claim 1.

13. The vector according to claim 12, further comprising a promoter operably linked to the nucleic acid.

14. The vector according to claim 13, wherein the promoter is a constitutive promoter.

15. The vector according to claim 13, wherein the promoter is an inducible promoter.

16. The vector according to claim 12, wherein the vector is a viral vector.

17. The vector according to claim 16, wherein the viral vector is a lentiviral vector.

18. A method for generating engineered immune cells, the method comprises: introducing the nucleic acid according to any one of claims 1 to 11 or the vector according to any one of claims 12 to 17 into immune cells, thereby generating the engineered immune cells.

19. The method according to claim 18, further comprising culturing the engineered immune cells after the introducing step.

20. The method according to claim 18, wherein the immune cells are T cells.

21. The method according to claim 18, wherein the immune cell is a NK cell.

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